Oxygen,
which supports all aerobic lin class="Chemical">fe, is produced by photosynthetic
water oxidation in plants, algae, and cyanobacteria. The water-oxidation
reaction probably first appeared in nature ∼3 billion years
ago in the precursors to present-day cyanobacteria, although the exact
timing is not yet entirely clear.[1−5] A key component in the appearance of oxygenic photosynthesis was
a metal complex that could store oxidizing equivalents to facilitate
the four-electron oxidation of two water molecules to dioxygen, meanwhile
making the electrons available for the reductive carbon-fixing reactions
required for sustaining life.[6−8] Thismetal complex involved in
oxygenic photosynthesis, the oxygen-evolving complex (OEC), consists
of an oxo-bridged structure with four Mn atoms and one Ca atom. No
variations have been observed so far among oxygenic photosynthetic
organisms through higher plants and algae back to cyanobacteria, which
represents the earliest oxygenic photosynthetic organisms. Oxygen
itself is the byproduct of the photosynthetic water oxidation reaction
shown in eq 1:However,
it was thisoxygen that enabled oxygenic
life to evolve and that led to the current diverse and complex life
on earth by dramatically increasing the metabolic energy that became
available from aerobic respiration. Oxygen produced by this process
was also key for the development of the protective ozone layer, which
allowed life to transition from marine forms to terrestrial life.
The OEC is embedded in Photosystem II (PS II), a membrane pigment–protein
complex, where the primary charge separation by absorbed sunlight
energy and successive electron transn class="Chemical">fer occurs through several vectorially
arranged pigment molecules (Figure 1).[9] The electrons and protons produced in the water
oxidation reaction in PS II are ultimately used to store energy in
the form of ATP and to reduce CO2 to carbohydrates via
the Calvin–Benson cycle, and are the precursors for synthesis
of the biological molecules needed by the organism. Nature has thus
evolved an elegant way to store sunlight energy in the form of chemical
energy, and it is this form of energy created by photosynthesis that
all life depends on.
Figure 1
(a) Photosystem II structure from 1.9 Å data from
X-ray crystallography
showing the membrane spanning helices and the extrinsic polypeptides.
The location of the cofactors involved in charge separation and the
Mn4CaO5 cluster in the membrane are shown highlighted
against the polypeptide background. The Mn4CaO5 cluster is on the lumenal side of the membrane with the acceptor
quinones on the stromal cytoplasmic side of the membrane. (b) Cofactors
on both branches of PS II shown in detail. The distances between the
groups are also indicated. The figure was drawn using the coordinates
from the 1.9 Å structure. Adapted from ref (27). (c) S-state Kok scheme
for oxygen evolution along with proposed oxidation states of Mn in
the various intermediate S-states.
(a) Photosystem II structure from 1.9 Å data from
X-ray crystallography
showing the membrane spanning helices and the extrinsic polypeptides.
The location of the cofactors involved in charge separation and the
n class="Chemical">Mn4CaO5 cluster in the membrane are shown highlighted
against the polypeptide background. The Mn4CaO5 cluster is on the lumenal side of the membrane with the acceptor
quinones on the stromal cytoplasmic side of the membrane. (b) Cofactors
on both branches of PS II shown in detail. The distances between the
groups are also indicated. The figure was drawn using the coordinates
from the 1.9 Å structure. Adapted from ref (27). (c) S-state Kok scheme
for oxygen evolution along with proposed oxidation states of Mn in
the various intermediate S-states.
At present, the urgent need to develop renewable energy resources,
which are essentially carbon-neutral, has highlighted the importance
of learning how nature accomplishes tn class="Chemical">his process and how water is
oxidized to oxygen during photosynthesis.[10−12] This knowledge
could be critical for creating artificial photosynthetic devices.
One of the key questions is how nature manages the uphill photoinduced
water oxidation reaction by modulating the redox potential of each
of the steps in the four electron redox reaction.
In the Bioinorganic
Enzymology issue of Chemical Reviews in 1996, the
Mn cluster in PS II was reviewed.[13] In
the almost 18 years n class="Chemical">since then, there has been an enormous
amount of progress in the field with new studies in X-ray spectroscopy[14,15] and crystallography,[16−22] in addition to many other spectroscopic and biochemical studies,
that have had a profound effect on the understanding of the structure
and mechanism of photosynthetic water oxidation. In this review, we
summarize the current understanding of the structure of the Mn4CaO5 cluster, as well as the water oxidation reaction
based on the insights learned primarily from X-ray techniques since
that last review. More comprehensive surveys of the literature in
the field of oxygen evolution that emphasize other methods are provided
in several excellent reviews.[9,23−27]
Photosystem II
Electron Transfer Chain
The past
decade has seen impressive advances in the X-ray crystal structure
of membrane proteins involved in photosynthen class="Chemical">sis. Since the first X-ray
crystal structure of PS II from the thermophilic cyanobacteria at
∼3.8 Å was published in 2001,[16] the resolution of the structure has steadily improved.[17−20] The present X-ray crystal structure of PS II has been determined
up to a resolution of 1.9 Å,[21] and
the details of PS II are shown in Figure 1a.
This progress has had a major impact on the understanding of the structure
of PS II and its components and their function, as well as the mechanism
of light harvesting, charge separation, and catalysis.
The basic
structural motif of PS II is very n class="Chemical">similar to that determined for the
purple photosynthetic bacterial reaction centers,[28] albeit with the addition of the OEC. The crystallographic
asymmetric unit of PS II contains a dimer (700 kDa), and the two monomers
are related by a noncrystallographic 2-fold axis perpendicular to
the membrane plane. Each monomer consists of 17 or 18 membrane integral
subunits composed of 35–36 transmembrane helices and 3 peripheral
subunits. The monomer is characterized by pseudo-2-fold symmetry,
which rotates the D1, CP47, and PsbI subunits into the D2, CP43, and
PsbX subunits. Each monomer contains 35 chlorophyll a (Chl), 11–12
all-trans β-carotene molecules, 1 OEC (Mn4CaO5 cluster), 1 heme b, 1 heme c, 2 or 3 plastoquinones, 2 pheophytins,
1 nonhemeFe, and around 20–25 lipids.
Figure 1b shows the electron transfer chain
in PS II where the light harvesting, charge separation, charge stabilization,
and electron transfer take place. The P680 reaction center
in the D1 and D2 subunits is the primary electron donor that traps
the light energy delivered from the inner antenna subunits (CP43 and
CP47 subunits) or the outer antenna complexes (LHC1 and LHC2) of PS
II. The excited state of the primary donor P680·+ rapidly transfers the electron to ChlD1, pheophytin (PheoD1), and eventually to the acceptor, plastoquinone QA (a firmly bound plastoquinone), and subsequently to the final electron
acceptor plastoquinone QB, stabilizing the charge separated
state. Acceptor QB exits the pocket in PS II after accepting
two electrons accompanied by protonation as plastoquinol QH2(B) and is released from PS II into the membrane matrix for transfer
to the cytb6f complex, which mediates the electron between
PS II and PS I. On the donor end of PS II, the cationic radical P680·+ is reduced by a tyrosine residue, TyrZ (D1Tyr161), to generate a neutral tyrosine radical TyrZ• that acts as an oxidant for the water
oxidation process at the OEC.The OEC cycles through a series
of five intermediate S-states (S, i = 0–4), representing
the number of oxidizing equivalents stored on the OEC driven by the
energy of the four successive photons absorbed by the PS II reaction
center (Figure 1c).[29] When PS II is dark-adapted, it relaxes to the S1 state
(note that, although S0 is the most reduced state of the
OEC, the S0 state is oxidized by n class="Chemical">tyrosine D(YD+) during the dark adaptation and therefore the S1 state becomes the dark-stable state). Illumination of dark-adapted
PS II (the S1 state) with saturating flashes of visible
light leads to a maximum O2 yield after the third flash,
and then after every fourth flash. Each flash advances the oxidation
state of the OEC by removal of one electron, and the OEC acts like
a redox capacitor for the water oxidation reaction until the four
oxidizing equivalents are accumulated (S4-state). Once
four oxidizing equivalents are accumulated in the OEC (S4-state), a spontaneous reaction occurs that results in the oxidation
of water, the release of O2, and the formation of the S0-state. Thus, the Mn4CaO5 complex in
the OEC couples the four-electron oxidation of water with the one-electron
photochemistry occurring at the PS II reaction center by acting as
the locus of charge accumulation.[9]
During the reaction, the Mn cluster provides a high degree of redox
and chemical flexibility, while the protein residues are critical
for mediating the reaction by modulating the redox potentials and
providing pathways for electrons, protons, substrate n class="Chemical">H2O, and product O2.[30−32] PS II orchestrates the well-controlled
catalytic reaction at close to the thermodynamic potential and also
avoids releasing chemical intermediate species, such as superoxide
or peroxide during the water oxidation reaction, that can be detrimental
to the protein matrix.[33]
Oxygen-Evolving Complex and the Mn4Ca Cluster
The geometry of the Mn4CaO5 cluster in the
OEC (Figure 2) has been revealed
in the 1.9 Å crystal structure.[21] Prior
to the recent X-ray diffraction (XRD) studies, detailed extended X-ray
absorption fine structure (EXAFS) studies,[14,15,34] un class="Chemical">sing solutions,[35] oriented membranes,[36−40] single crystals,[41] and range-extended
methods,[42,43] have indicated that the Mn4CaO cluster in the dark stable S1 state consisted of three di-μ-oxo and one mono-μ-oxo-bridged
Mn–Mn interactions.[44] The presence
of Ca–Mn interactions is supported also by compelling evidence
from EXAFS measurements obtained at the Ca and Sr edges of Sr-substituted
PS II.[14,45,46] The EXAFS
studies showed that there are two Mn–Mn distances at ∼2.7
Å, one Mn–Mn distance at ∼2.8 Å, and one Mn–Mn
distance at ∼3.3 Å,[41,47] corresponding to the
di-μ-oxo and mono-μ-oxo bridges, whereas the XRD model[21] shows that the Mn–Mn distances are 2.8,
2.9, 3.0, and 3.3 Å. EXAFS studies have indicated four Ca–Mn
distances between 3.4 and 3.9 Å compared to XRD studies that
show three Ca–Mn at 3.4 Å and one at 3.8 Å. The combination
of polarized EXAFS data from single crystals of dimeric PS II with
XRD data[19] led to several possible models
for the Mn4CaO5 cluster.[41] The models were constructed by considering the numbers
of short and long Mn–Mn interactions and the changes in the
amplitudes (dichroism). However, the crystal symmetry, the orientation
of proteins within the unit cell, and the presence of the noncrystallographic
C2-axis created limitations for uniquely solving the structure,
and therefore multiple models exist. From the set of model structures
that were derived, three were considered as most likely structures,
of which one model is shown in Figure 3a that
is almost a mirror image of the 1.9 Å XRD structure. There are
differences in the distances between the XRD structure and the EXAFS
derived structure as shown in Figure 3. This
is likely caused by the X-ray-induced radiation damage during X-ray
diffraction data collection and will be discussed in section 2.3.
Figure 2
Structure of the Mn4CaO5 cluster
with the
residues that have been identified as ligands of Mn (crimson) and
Ca (green) and four water molecules (in red) from the 1.9 Å X-ray
crystal structure of PS II.[21] Oxygen
is shown in yellow.
Figure 3
Structural models for
the Mn4CaO5 cluster
from (a) the polarized EXAFS and Sr EXAFS studies and (b) the 1.9
Å resolution XRD study.[21] The Mn–Mn
and Mn–O/N ligand distances from each of these studies are
summarized below the respective structural model. Mn atoms are depicted
in red, and Ca atoms are depicted in green. The O atoms are in gray
in (a) and in yellow in (b). Reproduced with permission from ref (70). Copyright 2013 American
Society for Biochemistry and Molecular Biology.
Structure of the Mn4CaO5 cluster
with the
ren class="Chemical">sidues that have been identified as ligands of Mn (crimson) and
Ca (green) and four water molecules (in red) from the 1.9 Å X-ray
crystal structure of PS II.[21] Oxygen
is shown in yellow.
Structural models for
the Mn4CaO5 cluster
from (a) the polarized EXAFS and n class="Chemical">Sr EXAFS studies and (b) the 1.9
Å resolution XRD study.[21] The Mn–Mn
and Mn–O/N ligand distances from each of these studies are
summarized below the respective structural model. Mn atoms are depicted
in red, and Ca atoms are depicted in green. The O atoms are in gray
in (a) and in yellow in (b). Reproduced with permission from ref (70). Copyright 2013 American
Society for Biochemistry and Molecular Biology.
In the 1.9 Å crystal structural model (Figure 3), the metal ions in the cluster are bridged by
5 n class="Chemical">oxygens,
whose positions were modeled based on omit maps. The distance Mn1–Mn3
of 3.3 Å and the corresponding Mn–O distances of 2.4 and
2.6 Å are much too long for bridging via a di-μ-oxo bridge,
giving rise to the distorted nature of the cubane core of the cluster.
Also the Mn–O distances in the μ-oxo bridges connecting
Mn4 with the 3MnCa-core are elongated (2.4 and 2.5 Å) compared
to the expected values for these distances in di-μ-oxo-bridges,
probably due to the nondistinct or unclear position of O5 in the electron
density. Ca is connected to the cluster via interactions with three
of the bridging oxygens, leading to Mn–Ca distances of 3.3,
3.4, and 3.5 Å to the Mn in the core and 3.8 Å to the more
isolated Mn4. There are some differences in the distances obtained
from EXAFS studies and those observed in the 1.9 Å crystal structure.
Some elongation is observed in the average Mn–O interactions
(EXAFS: 1.9 Å, XRD: 2.2 Å) as well as the di-μ-oxo
bridged Mn–Mn interactions (EXAFS: 2.7–2.8 Å, XRD:
2.8–3.0 Å). This is likely due to Mn reduction in the
crystal structure during XRD data collection as described later in
this section. Additionally, electron paramagnetic resonance/electron
nuclear double resonance (EPR/ENDOR) studies suggest that Mn1 is 5-coordinate
rather than 6-coordinate as shown in the crystal structure model.[24]
There are seven ligands directly ligated
to the Mn4CaO5 cluster in the 1.9 Å crystal
structure: n class="Chemical">six carboxyl
ligands (aspartate and glutamate) and one imidazole ligand (histidine)
(see Figure 2 for ligand environment and numbering).
These ligands are from side chains from two domains of the D1 subunit,
the interhelical CD luminal loop (residues Asp170 and Glu189) and
the C-terminal region (between residues His332 and Ala344), and one
domain of CP43—the large helical EF luminal loop. Most of the
ligands are arranged in a bidentate fashion bridging two metals. Three
of them, D1-Glu333, D1-Asp342, and CP43-Glu354, form di-μ-oxo
bridges between Mn (Mn1–Mn2 (Asp342), Mn2–Mn3 (CP43-Glu354),
and Mn3–Mn4 (Glu333)). D1-Asp170 and the C-terminal carboxylate
group of D1-Ala344 bridge Ca with Mn4 and Mn2, respectively, and D1-Glu189
provides monodentate ligation to Mn1. The only His providing direct
Mn ligation is D1-His332, binding Mn1. The second His in close proximity
to the Mn4CaO5 cluster is D1-His337, which is
too far for direct ligation of a Mn but is in hydrogen-bonding distance
to the bridging oxygen involved in di-μ-oxo bridges between
Mn1–Mn2 and Mn2–Mn3. In a similar manner, CP43-Arg357
ligates one of the oxygen bridges connecting Mn3–Mn4. Umena
et al. located four water molecules bound to Mn/Ca in the electron
density, in addition to the μ-oxo bridges and protein ligands.[21] Two of the water molecules, W1 and W2, are bound
to Mn4; the other two, W3 and W4, are bound to Ca. With the proposed
ligation scheme, all Mn are fully coordinated, having six ligands
each, and the Ca has seven ligands.
The differences observed
in the atomic distances in the crystal
structure and EXAFS data are likely due to a certain extent of reduction
of Mn that occurs during the XRD data collection. Such changes are
often seen in redox-active n class="Chemical">metalloproteins. The effects of X-ray-induced
perturbations in PS II are summarized in the following section.
X-ray Radiation Damage to the Metal Cluster
One inherent problem of X-ray-related techniques for studying metalloproteins
like PS II is the high radiation senn class="Chemical">sitivity of the redox-active metal
cluster.[48] In the case of PS II, specific
radiation damage to the metal catalytic center occurs with relatively
low X-ray dose even at cryogenic temperature, resulting in reduction
of Mn and elongation of atomic distances, and destruction of the cluster
is observed under conditions used for XRD.[48] This makes it challenging to derive the structure of the intact
S1 and the other intermediate states by XRD, as a high
dose is required to obtain the crystal structure. The issue of radiation
damage from X-rays to biological systems in crystallography is well-known,[49−53] and it is a problem not only for PS II but also for other proteins
that contain redox-active metals.
A quantitative study, using
low-dose X-ray absorption near edge structure (n class="Chemical">XANES) measurements
of PS II at 10 K after applying various doses under conditions similar
to the ones used for X-ray diffraction measurements, showed that the
oxidation state of Mn in the OEC changes from MnIIIMnIV to MnII with increasing dose.[48] The percentage of MnII formed under different
dose and temperature conditions was used as an estimate of damage
(Figure 4a). From this study it was derived
that under the conditions used for the earlier crystallographic studies
at least 70–80% of the Mn was reduced to MnII. On
the other hand, the radiation dose was significantly reduced in the
1.9 Å structure, and the degree of Mn reduction is estimated
to be ∼25%. The reduction of Mn is accompanied by structural
changes (disruption of μ-oxo bonds and changes of bond lengths)
as indicated by Mn-EXAFS measurements under different dose conditions
(Figure 4b).[48,54]
Figure 4
(a) Radiation
damage to PS II, measured as the amount of MnII detected,
is plotted as a function of incoming X-ray dose
from PS II solutions (dashed lines) and crystals (solid lines). The
data at two different X-ray energies (13.3 keV, where XRD experiments
are conducted, and 6.6 keV) and also at two different temperatures,
100 and 10 K, are shown. The damage is mitigated by lower temperatures.
The dose used for the 1.9 Å crystal structure[21] corresponds to only ∼25% damage, whereas the dose
used for the earlier crystal structures[18,19] is in excess
of 70%. (b) EXAFS Fourier transforms (FTs) of PS II subjected to various
degree, 5, 10, 25, and 70%, of damage compared to an FT collected
with no damage (black). The decrease in intensity of the second and
third FT peaks corresponds to losing the Mn–Mn and Mn–Ca
distances and disruption of the bridged cluster. The first FT peak
is mostly from the Mn–O bridging atoms and moves to longer
distances; in the FT at 70% damage, only this one FT peak at longer
distance is visible, which corresponds to that from Mn–O in
the Mn(II) hexa-aquo species. (c) EXAFS FTs of PS II in S1, S0, and 25% reduced data. Figure 4a is adapted from
ref (48).
(a) Radiation
damage to PS II, measured as the amount of MnII detected,
is plotted as a function of incoming X-ray dose
from PS II solutions (dashed lines) and crystals (solid lines). The
data at two different X-ray energies (13.3 keV, where XRD experiments
are conducted, and 6.6 keV) and also at two difn class="Chemical">ferent temperatures,
100 and 10 K, are shown. The damage is mitigated by lower temperatures.
The dose used for the 1.9 Å crystal structure[21] corresponds to only ∼25% damage, whereas the dose
used for the earlier crystal structures[18,19] is in excess
of 70%. (b) EXAFS Fourier transforms (FTs) of PS II subjected to various
degree, 5, 10, 25, and 70%, of damage compared to an FT collected
with no damage (black). The decrease in intensity of the second and
third FT peaks corresponds to losing the Mn–Mn and Mn–Ca
distances and disruption of the bridged cluster. The first FT peak
is mostly from the Mn–O bridging atoms and moves to longer
distances; in the FT at 70% damage, only this one FT peak at longer
distance is visible, which corresponds to that from Mn–O in
the Mn(II) hexa-aquo species. (c) EXAFS FTs of PS II in S1, S0, and 25% reduced data. Figure 4a is adapted from
ref (48).
The origin of the elongation of the atomic distances
in the crystal
structure has been discussed by several groups; one possibility is
that the originally S1 state is reduced to the S0 state by the X-n class="Species">rays through the catalytic pathway.[55] Other possibilities are the presence of pre-S0 states such as S–1, S–2, and
S–3 states in the crystal structure based on the
atomic distances.[55,56] As shown in Figure 4c, the 25% radiation-damaged PS II EXAFS spectrum is substantially
different from the intact S0 state spectrum. Also, 5–10%
X-ray-reduced PS II spectra do not match with the intact S1 state spectrum. This implies that the reduction of the metal center
by X-rays does not necessarily go through the catalytic pathway, as
has also been shown for the MnFe ribonucleotide reductase.[57]
The degree of radiation damage in conventional
crystallography
can be decreased dramatically with the use of multiple crystals. Collecting
data under liquid helium flow, instead of liquid n class="Chemical">nitrogen, could also
help in reducing the effect. The use of X-ray-free electron lasers
(XFELs) is also changing the way crystallography is being done on
redox-active metalloenzymes[58−60] including PS II[61,62] as described in section 6.
Structural Changes of the Mn4CaO5 Cluster
during the Catalytic Cycle
Dark Stable S1 State Structure
The geometry of the Mn4CaO5 cluster in the
native dark S1 state has been the subject of intense study
by spectroscopic methods, mostly electron paramagnetic resonance (EPR)
and X-ray spectroscopy over the last three decades, and by the very
promin class="Chemical">sing X-ray crystallography studies over the last decade. The
first indication of at least two di-μ-oxo bridges with Mn–Mn
distance of 2.7 Å was established in the 1980s,[63,64] followed by the finding of a mono-μ-oxo Mn–Mn distance
of 3.3 Å[65,66] and the orientation dependence
of the Mn–Mn vectors.[36,37,39,67,68] This was followed by EXAFS studies that established that Ca is part
of the Mn cluster,[66,69] leading to, at that time, a very
novel idea for a heteronuclear Mn4Ca cluster. The determination
that there are three Mn–Mn distances between 2.7 and 2.8 Å,
and the subsequent polarized EXAFS of single crystals (Figure 5),[41] dramatically reduced
the number of possibilities for the geometry of the Mn4Ca cluster with open-cubane-like structures being proposed; one of
them, model III, is shown in Figure 3a. The
synthesis of inorganic complexes with features similar to those present
in the OEC has been important for this evolving denouement of the
structure of the Mn4Ca cluster.
Figure 5
Fourier transforms of
the EXAFS spectra from oriented PS II crystals
on the left, with the e-vector of the X-rays parallel to the a, b, and c axes of the
PS II crystal lattice shown on the right. The dichroism of the FT
peaks is very clear and shows the asymmetric nature of the Mn–Mn
and Mn–Ca vectors. This information was used to derive three
possible structural models, and one of them (model III) is shown in
Figure 3a. The modified version of the structural
model based on the result of the 1.9 Å crystal structure[21] is shown in Figure 6.
Adapted from ref (41).
Fourier transforms of
the EXAFS spectra from oriented PS II crystals
on the left, with the e-vector of the X-rays parallel to the a, b, and c axes of the
PS II crystal lattice shown on the right. The dichroism of the FT
peaks is very clear and shon class="Chemical">ws the asymmetric nature of the Mn–Mn
and Mn–Ca vectors. This information was used to derive three
possible structural models, and one of them (model III) is shown in
Figure 3a. The modified version of the structural
model based on the result of the 1.9 Å crystal structure[21] is shown in Figure 6.
Adapted from ref (41).
Figure 6
Spectroscopic model based on data from
polarized Mn EXAFS, Sr EXAFS,
and EPR data, starting from the geometry of the 1.9 Å crystal
structure. This structure is also similar to that proposed on the
basis of DFT calculations. The major difference between this model
and the 1.9 Å structure shown in Figure 3b is the asymmetric placement of the bridging O between Mn4 and Mn1,
leading to an open-structure compared to a closed-structure from XRD
data. Adapted from ref (80).
The most recent 1.9 Å XRD
structure has provided much needed
clarity to the geometry and a consensus among EPR,[24] EXAFS,[70] theoretical methods,[71] and the XRD structure[21] is getting close. On the basis of the geometry of the OEC from the
crystal structure and the spectroscopic information from EPR and the
polarized EXAFS of crystals of PS II, a posn class="Chemical">sible intact S1 state structure has been proposed as shown in Figure 6. Single-crystal EXAFS on the S1 state has suggested
an open-cubane motif where there are three Mn–Mn interactions
at ∼2.7–2.8 Å and one Mn–Mn interaction
at ∼3.2 Å as described previously. The EPR results also
show that the Mn1, which is ligated to His332, is a 5-coordinated
Mn(III) in the S2 state. Therefore, it is likely that the
Mn1–O5 bond shown in the crystal structure is not present,
leading to an open-cubane structure instead of a closed-cubane structure.
A similar model geometry was suggested by Siegbahn using density functional
theory (DFT) calculations.[71]We
use the Sstructural
model shown inFigure6to further discuss the structural changes in other S-states
in the following sections.
Spectroscopic model based on data from
polarized Mn EXAFS, Sr EXAFS,
and EPR data, starting from the geometry of the 1.9 Å crystal
structure. Tn class="Chemical">his structure is also similar to that proposed on the
basis of DFT calculations. The major difference between this model
and the 1.9 Å structure shown in Figure 3b is the asymmetric placement of the bridging O between Mn4 and Mn1,
leading to an open-structure compared to a closed-structure from XRD
data. Adapted from ref (80).
Structural
Changes of the Mn4CaO5 Cluster
Structural
changes of the Mn4CaO5 cluster have been studied
primarily by EXAFS methods,
un class="Chemical">sing native PS II as well as Sr EXAFS using Sr-substituted PS II
instead of Ca. The S-state preparation for EXAFS studies has benefitted
from using the oscillation of g = 2 multiline EPR
spectrum (MLS) amplitude as a function of flash number. The pure S-state
spectra were all extracted from deconvolutions that were based on
fits to the EPR spectrum amplitude oscillations as shown in Figure 7.
Figure 7
EXAFS data from the intermediate S-states is derived from
PS II
samples given 1–3 flashes. The samples do not advance completely
to the next state because of misses and double hits, and hence, to
obtain the pure S-state spectra, one needs to deconvolute the spectra.
This is an important aspect of the spectroscopic data. This is normally
accomplished by using the g = 2 multiline EPR signal
from the S2 state. A typical EPR spectrum given 0–4
flashes (0F–4F) is shown in (a), with the oscillation in the
intensity as a function of flashes, the best fit is shown in (b),
and the calculated S-states are shown in (c). Using this matrix, one
can then deconvolute the EXAFS spectra to obtain the pure S-state
spectra in the S1, S2, S3, and S0 states.
EXAFS data from the intermediate S-states is derived from
PS II
samples given 1–3 flashes. The samples do not advance completely
to the next state because of misses and double hits, and hence, to
obtain the pure S-state spectra, one needs to deconvolute the spectra.
This is an important aspect of the spectroscopic data. Tn class="Chemical">his is normally
accomplished by using the g = 2 multiline EPR signal
from the S2 state. A typical EPR spectrum given 0–4
flashes (0F–4F) is shown in (a), with the oscillation in the
intensity as a function of flashes, the best fit is shown in (b),
and the calculated S-states are shown in (c). Using this matrix, one
can then deconvolute the EXAFS spectra to obtain the pure S-state
spectra in the S1, S2, S3, and S0 states.
The EXAFS spectra of
the PS II S-states show that the structure
of the Mn4CaO5 cluster changes during the catalytic
cycle. In particular, the short Mn–Mn interactions undergo
distance changes in the range of 2.7–2.8 Å.[47,70,72−74] Such distance
changes can reflect several chemical parameters: Mn oxidation state
changes, protonation state changes of bridging n class="Chemical">oxygens, ligation modes
(e.g., bidentate/monodentate), and fundamental changes in geometry
(i.e., dimeric, trimeric, or cubane-like structure). In the first
case, Mn–ligand distances are shortened upon Mn oxidation from
Mn(III) to Mn(IV), while Mn–Mn distances within Mn(III)/Mn(IV)
and Mn(IV)/Mn(IV) multinuclear complexes strongly depend on the direction
of the Jahn–Teller axis.[75] When
the protonation states of the bridging oxygens are changed, di-μ-oxo
bridged Mn–Mn distance changes from 2.72 Å (bis-oxo),
2.84 Å (oxo/hydroxo), to 2.92 Å (bis-hydroxo).[76] The Mn–Mn distances are in general longer
in the cubane-like structure as compared with pure bis-μ-oxo
dimer complexes.[77] Therefore, such distance
changes could serve as an indicator of the chemical structural changes
that occur during the S-state transitions. Possible structural changes
of the Mn4CaO5 cluster during the S-state transitions
are derived from the Mn EXAFS and Sr EXAFS results shown in Figure 8.
Figure 8
(a) Fourier-transformed spectra of PS II solutions in
the S0 (green), S1 (black), S2 (red),
and
S3 (blue) states are shown. For comparison, the spectrum
of the S state is overlaid in
the S1, S2, and S3 spectra (gray).
Prominent changes between the S2 and the S3 state
and the S3 and the S0 state in peak II of the
FT spectra are indicated by a dashed line. All spectra are shown in
the same scale but with a vertical offset. (b) FTs from Mn EXAFS of
the S-states from Sr-PS II. (c) FTs from Sr EXAFS show the first FT
peak from Sr–O and the second FT peak from Sr–Mn. The
FT peak corresponding to Sr–Mn changes during the S-state cycle
and most significantly for the S2 to S3 transition.
Changes are indicated by dashed lines. (a) is adapted from ref (70). (b) and (c) are adapted
from ref (73).
(a) Fourier-transformed spectra of PS II solutions in
the S0 (green), S1 (black), S2 (red),
and
S3 (blue) states are shown. For comparison, the spectrum
of the S state is overlaid in
the S1, S2, and S3 spectra (gray).
Prominent changes between the S2 and the S3 state
and the S3 and the S0 state in peak II of the
FT spectra are indicated by a dashed line. All spectra are shown in
the same scale but with a vertical offset. (b) FTs from Mn EXAFS of
the S-states from Sr-PS II. (c) FTs from n class="Chemical">Sr EXAFS show the first FT
peak from Sr–O and the second FT peak from Sr–Mn. The
FT peak corresponding to Sr–Mn changes during the S-state cycle
and most significantly for the S2 to S3 transition.
Changes are indicated by dashed lines. (a) is adapted from ref (70). (b) and (c) are adapted
from ref (73).
S1 to S2 (g = 2, MLS State) Transition
Among
all the S-states,
the S2 state is spectroscopically the most studied state
as it is characterized by a rich EPR signal at g =
2. The OEC structure has been optimized by theoretical studies based
on the 1.9 Å crystal structure, EXAFS distances, and EPR parameters.[78] Therefore, conn class="Chemical">sidering the S-state structural
changes starting from the S2 state is the most logical
way to follow the EXAFS changes, and we will follow that method in
this review. A distance change in the dark stable S1 to
S2 state transition is observed, with a shortening of one
Mn–Mn interaction around 2.7 Å, which is likely due to
the oxidation state change of one Mn (formally Mn(III) to Mn(IV))
(Figure 9). The polarized EXAFS study of PS
II single crystals[41] supports the open-cubane-like
structure that was also suggested by the theoretical studies of Siegbahn
for S1 and S2[79] and
Neese’s group for the S2 state[80] characterized by the g = 2 multiline EPR
signal (MLS).
Figure 9
Possible structural changes described using the spectroscopic
model
for the Mn4CaO5 cluster for the S1 to S2 transition. The proposed oxidation states and the
main Mn–Mn distances are shown.
Possible structural changes described un class="Chemical">sing the spectroscopic
model
for the Mn4CaO5 cluster for the S1 to S2 transition. The proposed oxidation states and the
main Mn–Mn distances are shown.
S1 to S2 (g = 4 State) Transition
The S2 state
can also be prepared in a different spin state characterized by an
EPR signal at g = 4.1 in spinach preparations or
at g ≈ 6–10 for preparations from cyanobacteria.
The intricate relation between the S2-g4 and S2-g2 states and how they are related
to the S1 and S3 states and their interconvertibility
are shown in Figure 10a. The g2 and g4 EPR signals from the S2 state
are shown in Figure 10b. The S2-g4 state from spinach was shown to involve Mn oxidation
using Mn XANES, similar to the S2-g2 state.[81] Earlier EXAFS studies also showed that there
was a structural change between the S2-g2 and the S2-g4 states.[82] Magnetic resonance studies have indicated a closed cubane-like
structure for the S2-g4 state[78] similar to that proposed from XRD studies
in the S1 state.[21] EXAFS studies
of the Mn K-edge from the native spinach PS II show that the structure
of the Mn4CaO5 cluster in S2-g4 state is distinctly different from that in the S2-g2 state. The EXAFS studies seem to indicate
that the structure may not be a closed structure but another variation
of the open structure (unpublished results). Note that a similar g = 4 EPR signal (S2-g4* in
Figure 10a) can be produced by various chemical
treatments, such as with F– or amines. However,
these states do not advance to the S3 state. It is likely
that S2-g4 and the S2-g4* states are chemically different.
Figure 10
(a) Relation and interconvertibility
of the S2-g2 and -g4
states. The S2-g2 state characterized
by the multiline EPR signal can be
generated from the S1 state by flash illumination at room
temperature or 200 K (cw), while the S2-g4 state is generated by illumination of the S1 state at
140 K (cw)cor. The S2-g2 state can be
converted to the g4 state by IR illumination at 120–140
K, and the g2 state can be produced from the g4 state by annealing at 200 K. A g4* state
can be produced by treatment with F–, amines, and
other treatments but cannot advance to the S3 state. (b)
EPR signals from the g2 and g4 states
and the g2-MLS signal generated from the g4 state by annealing at 200 K. Adapted from ref (82).
(a) Relation and interconvertibility
of the S2-g2 and -g4
states. The S2-g2 state characterized
by the multiline EPR signal can be
generated from the S1 state by flash illumination at room
temperature or 200 K (cw), while the S2-g4 state is generated by illumination of the S1 state at
140 K (cw)cor. The S2-g2 state can be
converted to the g4 state by IR illumination at 120–140
K, and the g2 state can be produced from the g4 state by annealing at 200 K. A g4* state
can be produced by treatment with F–, n class="Chemical">amines, and
other treatments but cannot advance to the S3 state. (b)
EPR signals from the g2 and g4 states
and the g2-MLSsignal generated from the g4 state by annealing at 200 K. Adapted from ref (82).
S2 to S3 Transition
The S2 to S3 is clearly one of the most interesting
transitions that have been studied by EXAFS, as the S3 state
is the last stable S-state that can be cryogenically trapped and studied,
before the last step (S4), where the O–O bond formation
presumably occurs. Interestingly, elongations of the Mn–Mn
interactions are observed in the S2 to S3 trann class="Chemical">sition,
unlike the S0 to S1 or S1 to S2 transitions.[35,70,72−74] This suggests that the S2 to S3 step is not a simple one-oxidation state change of Mn, but is likely
accompanied by fundamental changes of the Mn4CaO5 geometry. Elongation of Mn–Mn due to the oxo-bridge protonation[83,84] is unlikely at the S2 to S3 transition, unless
protons from terminal water molecules are transferred to the neighboring
bridging oxygens. EXAFS studies suggest such structural change could
occur by the shift of oxygen (O-5) position as illustrated in Figure 11 from Mn1 side to Mn4side. Such O-5 shuffling
possibility has been suggested by Pantazis et al.,[78] but as a reason for the S2 low spin (S = 1/2)
(multiline species) to S2 high spin (S = 5/2) (g = 4 species for spinach and g = 6–10
for T. elongatus) state changes, and by Isobe et
al.[85] in the S2 to S3 state transition.
Figure 11
Possible structural changes described using the spectroscopic
model
for the Mn4CaO5 cluster for the S2 to S3 transition. The proposed oxidation states and the
main Mn–Mn distances are shown. The two proposed S3 state structures are shown, where one of them is a closed structure.
Possible structural changes described un class="Chemical">sing the spectroscopic
model
for the Mn4CaO5 cluster for the S2 to S3 transition. The proposed oxidation states and the
main Mn–Mn distances are shown. The two proposed S3 state structures are shown, where one of them is a closed structure.
If O-5 oxygen is moved toward
the n class="Chemical">Mn3Ca open cubane
site, a Mn3CaO4 closed cubane is formed in the
S3 state. Thus far, Mn/Ca heteronuclear complexes have
been synthesized by the Christou and Agapie groups.[86−88] The MnIV3Ca2O structure reported by Mukherjee
et al.[88] has Mn–Mn interactions
within the closed cubane structure with 2.73, 2.76, and 2.86 Å,
with an averaged Mn-bridging oxygen distance of 1.86 Å. The MnIV3CaO4 structure reported by Kanady
et al.[86] has three Mn–Mn interactions
at 2.83–2.84 Å with an average Mn-bridging oxygen distance
of 1.87 Å. These Mn–Mn distances are longer than what
is observed in the Mn dimer or trimer model complexes. The repositioning
of O-5 could be accompanied by the ligand symmetry changes of Mn4,
which becomes 6-coordinate from a 5-coordinate geometry.
Two
possible structural models for the S3 state can
be conn class="Chemical">sidered if an MnIV3CaO4 closed
cubane-like moiety is formed in this state: one with 6-coordinated
Mn1, and the other with 5-coordinated Mn1 upon O-5 shuffling. In the
former case, the N number for the ∼2.7 Å
Mn–Mn interactions becomes 2, and in the latter case it remains
as 1.5. While the EXAFS fitting result slightly prefers N = 1.5, the result is not conclusive based only on the EXAFS curve
fitting. In the latter case, Mn1 becomes 5-coordinated upon the S2 to S3 transition, or a new ligand, either water
or carboxylate, needs to be ligated to maintain 6-coordination at
Mn1. MnIV is in general 6-coordinate, but 5-coordinate
structures have been proposed.[89] In the
pre-edge region, which is generally sensitive to the ligand symmetry,
the intensity tends to increase upon S2 to S3 transition, which may suggest Mn1 to be 5-coordinated. This remains
an open question, and a detailed pre-edge analysis combined with theoretical
calculations will give us an insight into the ligand symmetry changes;
such an approach is underway.
S3 to S0 Transition
Upon S3 to
S0 transition via the S4 state, the Mn-ligand
and the ∼2.7 Å Mn–Mn distances
are shortened.[47] Tn class="Chemical">his is counterintuitive
if the Mn oxidation state changes from the most oxidized form (S3) to the most reduced state (S0). However, such
changes could be explained if the Mn4CaO5 geometry
in the S0 state is changed back to the one similar to the
S1 and S2 states, where the Mn3Ca
moiety takes the open-cubane-like structure (Figure 12).
Figure 12
Possible structural changes described using the spectroscopic
model
for the Mn4CaO5 cluster for the S3 to S0 transition. The proposed oxidation states and the
main Mn–Mn distances are shown. The two proposed S3 and S0 state structures are shown.
Possible structural changes described un class="Chemical">sing the spectroscopic
model
for the Mn4CaO5 cluster for the S3 to S0 transition. The proposed oxidation states and the
main Mn–Mn distances are shown. The two proposed S3 and S0 state structures are shown.
S0 to S1 Transition
The S0 to S1 transition is accompanied by
the shortening of Mn-ligand distances as well as a Mn–Mn distance
(∼2.8 to ∼2.7 Å) (Figure 13).[47] The recent EPR/ENDOR study supports
the formal oxidation state asn class="Chemical">signment of Mn4(III,III,III,IV)
in the S0 state and Mn4(III,III,IV,IV) state
in the S1 state.[90,91] Therefore, the shortening
of the Mn-ligand and Mn–Mn distances could be explained by
the elimination of the Jahn–Teller effect at one Mn.
Figure 13
Possible
structural changes described using the spectroscopic model
for the Mn4CaO5 cluster for the S0 to S1 transition.
Possible
structural changes described un class="Chemical">sing the spectroscopic model
for the Mn4CaO5 cluster for the S0 to S1 transition.
The entire S-state cycle and a summary of the proposed structural
changes based on X-ray spectroscopy data together with EPR and XRD
studies is shown in Figure 14.
Figure 14
Possible structural
changes during the S-state transitions are
illustrated. Note that the focus here is to accommodate the EXAFS
distance changes, and possible protonation states (at oxo-bridging
and terminal water molecules) or changes in the ligand environment
(type of ligands and ligation modes) are not included in the figure.
The Mn–Mn distances at ∼2.7 Å are indicated by
green arrows, ∼ 2.8 Å by blue arrows and ∼3.2 Å
by red arrows. The dashed line indicates that it may not be a bond.
For the S3 and the S0 states, two possible models
are presented. Mn atoms are shown in blue (MnIII), red
(MnIV), or magenta (MnIII or MnIV possible), Ca is shown in green, and the surrounding ligand environment
is shown in gray. Reproduced with permission from ref (70). Copyright 2013 American
Society for Biochemistry and Molecular Biology.
Possible structural
changes during the S-state trann class="Chemical">sitions are
illustrated. Note that the focus here is to accommodate the EXAFS
distance changes, and possible protonation states (at oxo-bridging
and terminal water molecules) or changes in the ligand environment
(type of ligands and ligation modes) are not included in the figure.
The Mn–Mn distances at ∼2.7 Å are indicated by
green arrows, ∼ 2.8 Å by blue arrows and ∼3.2 Å
by red arrows. The dashed line indicates that it may not be a bond.
For the S3 and the S0 states, two possible models
are presented. Mn atoms are shown in blue (MnIII), red
(MnIV), or magenta (MnIII or MnIV possible), Ca is shown in green, and the surrounding ligand environment
is shown in gray. Reproduced with permission from ref (70). Copyright 2013 American
Society for Biochemistry and Molecular Biology.
Structural Changes and Calcium
Calcium
is an essential element for the function of the OEC (reviewed in refs (46 and 92)). It has been speculated
that Ca controls substrate n class="Chemical">water binding to the catalytic Mn site,[93] and proposed mechanisms have suggested the involvement
of Ca.[46,94−98] Calcium can be depleted by several biochemical treatments
that produce preparations in which O2 evolution activity
is inhibited, but which can be reactivated by the addition of Ca2+. Reactivation of oxygen evolution in inhibited preparations
can also be achieved by addition of Sr2+, reactivating
the same number of centers as Ca2+, but with slower turnover
in the S-state cycle, producing a lower overall rate of oxygen evolution
at saturating light intensities.[99,100] One study
reports that vanadyl ion (VO2+) can also activate oxygen
evolution.[101] Na+, K+, Cd2+, and various lanthanides have been shown to compete
with Ca2+ for binding sites in PS II, but none of them
results in reactivation of oxygen evolution activity.[92]
Mn XAS and Ca or Sr
X-ray crystallography
has confirmed the presence of Ca as a part of the OEC cluster.[17−19,21,22,102−104] Prior to XRD, the presence
of Ca in the OEC was detected using Ca and n class="Chemical">Sr XAS.[38,69,105−107] A comparison of the
Mn EXAFS of Ca-containing, Sr-substituted, and Ca-depleted PS II samples
was the first study in 1995 that established the presence of a heteronuclear
Mn4Ca complex in the OEC of PS II; such a Ca–transition
metal mixed complex in biology had no precedence. Mn EXAFS on Sr-reactivated
PS II membranes was interpreted to indicate a 3.4–3.5 Å
distance between the Ca (Sr) and the Mn cluster.[69] This conclusion was based on the observation of increased
amplitude in Fourier peak III at 3.3 Å (Figure 15) upon replacement of Ca with Sr, a heavier atom and better
X-ray scatterer. Such a short Mn–Sr/Ca distance was interpreted
as indicating a direct Mn–O–Ca bridged structure in
the OEC. The numbers of Mn–Ca(Sr) and Mn/Ca(Sr)-ligand vectors
in the S0 to S3 states have been derived from
Sr XAS.[14,73,106,107] ESEEEM (electron spin echo envelope modulation) spectroscopy
has been important for studying PS II[108,109] and using 87Sr has shown the proximity of Sr to the Mn cluster,[110] and a 113Cd-NMR study showed that
Ca2+ is close to the Mn4-cluster.[111]
Figure 15
First Mn EXAFS study with Ca- and Sr-substituted
samples that showed
that there could be a Mn–Ca/Sr interaction at ∼3.3 Å.
The FT peak amplitude at ∼3.3 Å increased on Sr substitution
as expected from the higher scattering cross section of Sr compared
to Ca. Adapted from ref (69).
First Mn EXAFS study with Ca- and Sr-substituted
samples that showed
that there could be a Mn–Ca/n class="Chemical">Sr interaction at ∼3.3 Å.
The FT peak amplitude at ∼3.3 Å increased on Sr substitution
as expected from the higher scattering cross section of Sr compared
to Ca. Adapted from ref (69).
Mn
XAS and Ca Depletion
The structural
consequences of calcium depletion of PS II has been determined by
Mn XAS on PS II solutions[105] and also un class="Chemical">sing
polarized EXAFS of oriented samples (unpublished results). XANES of
Ca-depleted samples reveals that there is Mn oxidation for the Ca-depleted
S1 to S2 transition.[105] No evidence of Mn oxidation was found for the next illumination
step. This is in line with the results from EPR studies where it has
been found that the species oxidized to give the broad radical signal
found in Ca-depleted PS II is tyrosine Y (i.e., Ca-depleted S2YZ•).[115] EXAFS measurements of Ca-depleted
samples in the three modified S-states reveals that the Fourier peak
due to scatterers at ∼3.3 Å from Mn, although strongly
diminished, retains some amplitude.[105] More,
surprisingly, the orientation of the Mn–M vectors as determined
from the polarized EXAFS showed minimal changes in Ca-depleted samples.
The most pronounced differences are a lengthening of the Mn–Mn
interaction around 3.2 Å by >0.1 Å in the Ca-depleted
S1 state, accompanied by a tilt of ∼20° and
smaller
elongation of all Mn–Mn interactions upon advancement to higher
oxidation states (Figure 16). The fact that
Ca can be removed more easily in the S3 state (or that
Ca can be more easily exchanged in the higher S-state)[92,112,116−118] compared to the S1 and the S2 states, together
with the above observations, implies that the Mn–Ca binding
modes are changed upon S2 to S3 transition.
Figure 16
Possible
structural changes described using the spectroscopic model
for the Mn4CaO5 cluster for the S1 to Ca-depleted S1 state.
Possible
structural changes described un class="Chemical">sing the spectroscopic model
for the Mn4CaO5 cluster for the S1 to Ca-depleted S1 state.
Recent Mn ENDOR results have also shown that removal of Ca
does
not perturb the magnetic properties of the Mn cluster, suggesting
that the main bridging structure of Mn atoms is not changed by Ca
removal.[119] Therefore, we conclude that
the removal of the Ca2+ ion from the OEC does not lead
to fundamental distortion or rearrangement of the Mn cluster, indicating
that the n class="Chemical">Ca2+ in the OEC is not essential for structural
integrity of the cluster at least up to the S2 state. Presence
of Ca, however, is necessary for the formation of the S3 state. A recent study by Rappaport et al. showed that Ca and a tyrosine
residue of the D1 polypeptide (YZ) are involved in the
common hydrogen-bond network, with Ca2+ facilitating the
correct configuration of the hydrogen-bond network for proton transfer
and therefore being important for the S2 to S3 transition that is accompanied by the proton transfer. On the other
hand, there is no proton release upon S1 to S2 transition.
The fact that Ca-depletion does not affect the
n class="Chemical">Mn4 structure
together with the result that Ca can be removed or reconstituted during
the S-state transition[120−125] suggests that Ca is weakly bound to the Mn4 cluster in
PS II. Sensitivity of Ca-depletion to NH2OH and hydroquinone
treatment has suggested that the structural environment of the oxidizing
side of PS II may be flexible, rather than rigid.[117]
A detailed study of the requirement of Ca in the
S-state transitions
has been reported by Miqyass et al.[112] Despite
the inability to complete the reaction cycle, the n class="Chemical">Ca2+-depleted
S1 state can be advanced to higher oxidation states upon
illumination. A Ca-depleted S2 state can be generated that
is characterized by an EPR signal of at least 26 lines with an average
line spacing of 55 G, centered at g = 1.96, which
is different from that in the native state,[113,114] and therefore it is different from the S2 state in the
native system. A Ca-depleted S2YZ• can also be generated that exhibits a split EPR signal assigned
to the YZ• radical interacting magnetically
with the Mn complex in an S = 1/2 spin state.[115] Thus, the Ca2+-depleted S2YZ• state cannot advance to the S3 state,
as the Ca-depleted S2 state is not oxidized by YZ•.
Ca or Sr XAS
The first approach
that was tried as described above[69,126] was to substitute
other metals (such as n class="Chemical">Sr) for Ca and then use Mn XAS to detect changes
in the cluster. However, isolating the Mn–Ca or Sr component
from the Mn EXAFS is often difficult, as other Mn–ligand and
Mn–Mn interactions contribute in the same region. Alternatively,
the reverse experiment probing backscattering from Mn using Ca or
Sr EXAFS (Ca/Sr point-of-view for Mn) is more direct and definitive
than Mn EXAFS results. Such studies on both isotropic and oriented
PS II membranes have yielded unequivocal evidence for the proximity
and mode of binding of Ca to the Mn4 cluster, as well as
details about the orientation of the Ca/Sr in the PS II membrane and
the changes in the structure of the cluster as it advances through
the catalytic cycle.
Ca XAS
In a definitive experiment,
Ca EXAFS was used
to probe the structure of the Mn4CaO5 cluster
from the Ca point of view to detect the distances of Mn atoms in the
cluster. The use of Ca EXAFS spectroscopy has produced essentially
congruent results with those found by n class="Chemical">Sr EXAFS on Sr-reactivated PS
II[106] and Mn EXAFS on similar samples,[69] but it focused on native preparations, avoids
the treatments involving Ca depletion and Sr substitution, and is
a direct probe of the Ca binding site in PS II.
The FTs of the
Ca EXAFS in Figure 17 are remarkably similar
to the FTs of the n class="Chemical">Sr EXAFS study with Sr substituted for Ca. The first
FT peak corresponds to the coordinating oxygen atoms closest to Ca.
In contrast to the control (NH2OH-treated) sample where
the Mn cluster is disrupted, the PS II samples with only 1Ca/4Mn shows
a second FT peak, which corresponds to Mn at ∼3.4 Å. These
observations resulted in the proposal that there exists a motif where
the Ca/Sr is linked to at least two or three Mn.
Figure 17
Ca EXAFS (left) and
Sr EXAFS (right) both show scattering FT peaks
from Mn in active preparations that are not present when the Mn4CaO5 cluster is disrupted using NH2OH.
Both studies support a Mn–O–Ca bridging structure. Adapted
from ref (46).
Ca EXAFS (left) and
Sr EXAFS (right) both show scattering FT peaks
from Mn in active preparations that are not present when the n class="Chemical">Mn4CaO5 cluster is disrupted using NH2OH.
Both studies support a Mn–O–Ca bridging structure. Adapted
from ref (46).
Sr XAS
Sr is favored
for XAS study as the X-ray energies
involved (16 keV for the K-edge) are more penetrating and not attenuated
by air, and the higher X-ray absorption cross section and fluorescence
yield of n class="Chemical">Sr makes the experiment more practical than Ca XAS. The Sr
experiment requires PS II samples with Sr substituted for Ca while
maintaining activity and a stoichiometry of 1 Sr per PS II, which
were prepared biosynthentically from cyanobacteria grown in Sr medium.[127]
Sr EXAFS clearly showed the proximity
of n class="Chemical">Sr (and implicitly Ca) to within 3.5 Å of the Mn cluster.[106] The results are based on the presence of a
second FT peak (peak II, Figure 17) in the
Sr EXAFS from functional samples, a peak that is absent from inactive,
hydroxylamine-treated PS II. This FT peak was found to fit best to
two or three Mn at ∼3.5 Å rather than lighter atoms (C
or O).
Orientation of Ca/Sr–Mn
Interactions
in the PS II Membrane
In Mn XAS of PS II, the presence of
Fe in the sample places an inherent limit on the resolution of Mn
distances by limiting the data length. While EXAFS spectra of dilute
biological samples are normally collected with an energy-discriminating
solid-state detector by electronically windowing the Kα from
the n class="Chemical">metal atom, the low-energy resolution (∼ 200 eV) of the
solid-state detector cannot completely discriminate Mn Kα from
the Fe Kα signal, which limits the data length to k = 11.5 Å–1.[128−130] This limitation has
been overcome using a high-resolution crystal monochromator, to collect
EXAFS beyond the Fe K-edge to k = 16.5 Å–1, improving the distance resolution to ∼0.1
Å. The method confirmed two distances to the short Mn–Mn
interactions in the S1 and S2 states (two at
∼2.7 Å and one at ∼2.8 Å), whereas earlier
solution EXAFS studies could discern only one distance of ∼2.7
Å. This range-extended EXAFS method combined with polarized detection
using oriented PS II membranes for the first time allowed us to resolve
the FT peak at ∼3.3 Å into one Mn–Mn vector at
∼3.2 Å and Mn–Ca vectors at ∼3.4 Å
that are aligned at different angles to the membrane normal (Figure 18a–c).[42] The dichroic
behavior of peak IIIB is similar to that reported for the Mn–Sr
vector (Figure 18d).[38] The dichroism of these peaks has been used to determine the angle
the Mn–Mn and Mn–Ca vectors make to the membrane normal.
Figure 18
(a)
Membranes of PS II can be oriented, and polarized EXAFS can
be used to determine the orientation of Mn–Mn and Mn–Ca
vectors with respect to the membrane. (b) Relative orientations of
Mn–Mn and Mn–Ca vectors determined from the analysis
of the data shown in (c) and (d). (c) Range-extended EXAFS of oriented
membranes clearly shows the Mn–Mn vector at 3.2 Å and
the Mn–Ca vector at 3.3 Å oriented differently in the
PS II membrane. (d) Polarized Sr EXAFS shows the orientation of the
Mn–Sr vector with respect to the membrane. Adapted from ref (46).
(a)
Membranes of PS II can be oriented, and polarized EXAFS can
be used to determine the orientation of Mn–Mn and Mn–Ca
vectors with respect to the membrane. (b) Relative orientations of
Mn–Mn and Mn–Ca vectors determined from the analysis
of the data shown in (c) and (d). (c) Range-extended EXAFS of oriented
membranes clearly shon class="Chemical">ws the Mn–Mn vector at 3.2 Å and
the Mn–Ca vector at 3.3 Å oriented differently in the
PS II membrane. (d) Polarized Sr EXAFS shows the orientation of the
Mn–Sr vector with respect to the membrane. Adapted from ref (46).
Changes of the Ca–Mn Distances During
the S-State Transitions
Biosynthetically exchanged Ca/Sr-PS
II preparations have made it posn class="Chemical">sible to monitor Sr(Ca)–Mn
distance changes in the four intermediate S-states, S0–S3, of the catalytic cycle by Sr XAS.[131] Sr EXAFS data (Figure 8) in the S0–S3 states determined Sr–Mn distances at
∼3.5 Å (short) and ∼4.0 Å (long), and the
data shows that all four Sr/Ca–Mn distances are detectable
in all the S-state intermediates (Figure 19).[73] Previous results from Ca EXAFS of
plant PS II have shown that there are only 2–3 Mn–Ca
interactions[107] at <4 Å, compared
to the four we have observed in Sr-PS II; it is possible that the
longer interactions are at >4 Å or were not discernible at
the
S/N of the Ca EXAFS data.[107] It is, however,
difficult from Sr-XAS to determine if 2:2 or 3:1 short-to-long distance
ratios are best. There is evidence that Ca protects two of the four
Mn atoms from reductants, suggesting a closer interaction between
Ca and two of the four Mn atoms in the cluster.[132] The recent XRD data from Sr PS II indicates that Sr is
at a slightly different location compared to Ca due to the larger
ionic radius.[22] The Ca- and Sr-PS II are
slightly different in their kinetic and spectroscopic properties.[131] This is likely due to the small perturbations
of the hydrogen-bond network in the Sr-OEC, which reduces the proton-transfer
efficiency and possible water molecule(s) delivery to the OEC as shown
most recently by Rappaport et al.[133]
Figure 19
Summary of
the Mn–Ca(Sr) distances in the S-states determined
from Sr EXAFS. Adapted from ref (73).
Summary of
the Mn–Ca(Sr) distances in the S-states determined
from n class="Chemical">Sr EXAFS. Adapted from ref (73).
In the S1 state, there are three or two Sr–Mn
vectors at ∼3.5 Å and one or two n class="Chemical">Sr–Mn vectors
at ∼4.0 Å. There are no significant changes in the Sr–Mn
distances during the S1 to S2 transition, which
is in line with the Mn XAS result; no substantial changes were observed
in the Mn–Mn distances. Upon S2 to S3 transition, the FT peak II in Sr EXAFS splits in the S3 state (Figure 8c), indicating the distance
changes in the Sr(Ca)–Mn interactions. The fact that Mn–Sr
interactions change during the S2 to S3 transition
is also in agreement with the different efficiency of Ca depletion
in the S3-state from that in other S-states.[120] Ca/Sr-depleted PS II cannot advance to the
S3 state; instead, a state designated S2YZ• is formed, in which the Mn4-core structure is close to that of the S2 state and does
not resemble the structure of the native S3 state.[72,105] In the S3 to S0 transition, the Sr–Mn
distances at 3.4 and 3.9 Å increase to 3.5 and 4.0 Å, respectively.
These results implicate the involvement of at least one common
bridging oxygen atom between the Mn–Mn and Mn–n class="Chemical">Ca(Sr)
atoms in the S2 to S3 transition. Because PS
II cannot advance beyond the S2 state in preparations that
lack Ca(Sr), these results show that Ca(Sr) is one of the critical
components in the mechanism of the enzyme. The results also show that
Ca is not just a spectator atom involved in providing a structural
framework but is actively involved in the mechanism of water oxidation
and represents a rare example of a catalytically active Ca cofactor.
Ligands of Mn and Ca and Site-Specific Mutants
In addition to the Mn4CaO5 core structural
changes discussed previously, we expect that terminal ligands that
are derived from n class="Chemical">carboxylates, histidine, and water/hydroxo ligands
also likely change during the catalytic process. As shown in Figure 2, there are six carboxyl residues and one histidine
residue directly ligated to the Mn4CaO5 cluster.
Except for one glutamate residue (CP43-Glu354), other ligands to the
Mn4CaO5 cluster are provided from the D1 subunit.
There has been a series of mutagenesis studies to identify those amino
acid residues that control the assembly and functioning of the Mn4CaO5 cluster and to gain insight on which Mn are
involved in the different steps during the water oxidation reaction.[27,31,134]
Among the seven ligands,
it has been shown that some ligands seem to be more critical than
others in maintaining the OEC activity and the assembly process of
the metal cluster.[31] One of such ligands
is n class="Chemical">D1-His332. The replacement of His332 by a glutamate residue results
in a substantial geometric and electronic structural changes of the
Mn4CaO5 cluster, which was observed in the EXAFS
and XANES spectra (Figure 20, top).[135] Although the metal cluster can be assembled
in this altered ligand environment, the structure of the cluster is
perturbed with an elongation of Mn–ligand and Mn–Mn
distances. The Mn in the assembled cluster can be oxidized and advances
from the EPR silent dark stable state to the one with an altered multiline
EPR spectrum. It has also been shown by Sr EXAFS using Sr-substituted
mutant samples that the Sr(Ca) is a part of the assembled structure.
A possible reason for the altered structure of the cluster is a protonation
of the oxo-bridge to restore the charge balance because of the replacement
of a neutral histidine ligand by a negatively charged glutamate residue
(Figure 20, bottom). An alternative explanation
is a ligand environment change of the nearby carboxylate group, from
a bidentate to a monodentate ligand. Therefore, the inhibition of
the catalytic activity of the His332Glu mutant is because of the structural
changes of the OEC itself during the assembly process.
Figure 20
(Top) FT
of Mn EXAFS from the wild-type compared with the mutant
H332E. There is a clear difference in the FTs, indicating even more
asymmetry in the mutant with possible modification of a Mn–Mn
distance by replacement of a His ligand by a glutamate. (Bottom) Three
different structural modifications are shown as a consequence of His
replacement with Glu: (a) protonation of a bridge and (b) replacement
of the bidendate ligand in two different ways. One is where the Glu
binds only one Mn in a bidendate manner, while in the other it is
monodendate, with the possible addition of a hydroxide or water ligand.
Adapted from ref (135).
(Top) FT
of Mn EXAFS from the wild-type compared with the mutant
H332E. There is a clear difference in the FTs, indicating even more
asymmetry in the mutant with possible modification of a Mn–Mn
distance by replacement of a His ligand by a glutamate. (Bottom) Three
different structural modifications are shown as a consequence of His
replacement with Glu: (a) protonation of a bridge and (b) replacement
of the bidendate ligand in two different ways. One is where the Glu
binds only one Mn in a bidendate manner, while in the other it is
monodendate, with the possible addition of a hydroxide or water ligand.
Adapted from ref (135).Mutagenesis studies have also
examined the role of the n class="Chemical">CP43 protein.
CP43-Glu354 is a bridging ligand for two metal ions in the native
S1 state. First segment deletion studies were conducted,
followed by site-directed mutagenesis. The point mutant, Glu354Gln,
exhibits reduced photoautotrophic growth rate, although normal PS
II content (e.g., no photoinactivation) and an 80% reduction in the
O2 evolution rate were observed. Detailed spectroscopic
studies on this mutant showed that the Glu354Gln mutation leads only
to subtle changes in the structure and spin state of the Mn4CaO5 cluster in the S2 state. The water
exchange study showed that the mutant weakens the binding of substrate
water to the OEC.[136]
OEC in Monomeric and Dimeric PS II from Cyanobacteria
The existence of PS II in both a monomeric and dimeric form has
provided the basis for controvern class="Chemical">sial discussions, concerning their
contribution to the functionality of the photosynthetic apparatus.[137,138] This discussion in the past included the question whether PS II
always exists in a dimeric form or whether a monomeric PS II is an
equally active form. In general, the prevailing view is that the PS
II dimer is the fully assembled and functionally relevant form, whereas
the monomeric form is seen as an intermediate during the assembly
process of all known 20 subunits of PS II and the repair cycle of
photodamaged subunit D1.[139,140] The assembly of PS
II is a stepwise and highly regulated process,[141] which includes many auxiliary proteins that are absent
in the crystallized complexes. In cyanobacteria, an intermediate Psb27-PS
II complex, which has no functional Mn cluster,[142] regulates the assembly of the Mn4CaO5 cluster and the binding of the extrinsic subunits PsbO, PsbU, and
PsbV[143] prior to the dimerization of the
PS II core complex. Monomerization of photodamaged PS II was suggested
to be triggered by the detachment or structural reorganization of
PsbO on the lumenal side.[139,140]
The chromatographic
purification procedure of crude PS II extract yields essentially equal
amounts of reaction centers in the monomeric and dimeric forms.[144] The monomer/dimer ratio from 40 preparations
was estimated to be 1.05 ± 0.45 based on the quantity of Chla.[145] Moreover, it was shown
that the monomeric and dimeric forms of PS II are n class="Chemical">similar in their
oxygen-evolving capacity as well as in their subunit content. The
crystal structure of monomeric PS II, albeit available at medium resolution
so far, does not give indications of either a destabilization of subunit
D1 or a structural reorganization of subunit PsbO. The Mn EXAFS spectra
from dimeric and monomeric PS II in Figure 21 show that they are almost identical.
Figure 21
Mn EXAFS spectra from
monomeric and dimeric PS II. There are no
differences in the Mn4CaO5 structures that are
detectable from EXAFS between the dimeric and monomeric PS II.
Mn EXAFS spectra from
monomeric and dimeric PS II. There are no
differences in the n class="Chemical">Mn4CaO5 structures that are
detectable from EXAFS between the dimeric and monomeric PS II.
Electronic
Structure of the Mn4CaO5 Cluster
The Mn4CaO5 cluster goes through five intermediate
states during the n class="Chemical">water oxidation reaction triggered by the absorbed
light (Figure 1c). The cluster stores oxidizing
equivalents until the release of molecular oxygen in the final (S4–S0) step and then goes back to the most
reduced form in the S0 state by incorporating two water
molecules. The protein residues such as carboxylate and histidine
ligands provide a high degree of chemical flexibility to the cluster
and stabilize multiple oxidation states. To understand the mechanism
of water oxidation in detail, it is crucial to understand the changes
of the electronic structure in the OEC over the whole course of the
catalytic cycle.[146] The primary questions
include whether the electrons extracted from the OEC are directly
derived from bound water, or from the Mn atoms, or from any other
parts of the OEC accompanying each S-state transition,[23] and how the ligand environment modulates the d orbitals and the electronic structure of the Mn to tune
the redox potential during the catalytic process.[147−149]
Oxidation and spin states of the cryo-trapped intermediate
states
(S0–S3 states) have been studied intensively
by various spectroscopic methods such as EPR,[24,150] Fourier transform infrared (FTIR),[151−153] n class="Chemical">XANES,[146,154−159] and UV–vis spectroscopy.[160−162] EPR spectroscopy shows
that S0 and S2 have spin S = 1/2 ground states,
exhibiting MLS EPR (note that there are also high-spin species observed
in the S2 state; see section 3.2.2).[150,163−167] The S1 and S3 are
characterized by parallel-polarized EPR signals, indicating integral
spin ground states.[168−171] Through these studies, there is an overall consensus that Mn-centered
oxidation occurs through the S0, S1, to S2 transitions. Within the context of localized oxidation, the
formal oxidation state of the native S1 state has been
assigned to Mn2IIIMn2IV and S2 has been assigned to MnIIIMn3IV. In the S0 state, one of the questions is
whether MnII is present, in which case the oxidation states
are MnIIMnIIIMn2IV,[166,172,173] or whether the oxidation states
are Mn3IIIMnIV. Recent ENDOR studies
have provided the evidence of the latter case.[90] In the S2 to S3 transition, there
has been a discussion about whether a Mn-centered oxidation occurs[154,174] or a ligand-centered oxidation takes place.[159,175] There have also been proponents of a lower oxidation state process
for the entire cycle starting with a Mn4III for
the S1 state,[176−178] although the evidence from EPR[24] and X-ray spectroscopy,[14,154] and NH2OH and hydroquinone-induced reduction of the Mn,[179] supports the higher oxidation states, with
Mn2IIIMn2IV in the S1 state.
As described above, the nature of the electronic
structure has
been discussed only within the context of the formal oxidation state
of Mn. This is partly the reason for the controversy concerning the
assignment of the oxidation state in the S3 state.[74,159] Although there are distinct chemical characteristics in the formal
oxidation states, convenient, formal oxidation states do not necessarily
coincide with the effective number of electrons in the metal valence
shells. In this regard, for many transition-metal systems, the formal
oxidation state is an incomplete, and in many cases incorrect description
of the electronic structure of the system, because of other important
factors, especially metal–ligand covalency.[180−183] It is therefore necessary to understand the electronic structure
beyond the formal oxidation state and how PS II manages electron localization
and controls their paths using the flexible protein environment; this
may be an important aspect for learning the design concept of the
catalytic reaction and applying it to artificial photosynthetic devices.Since the 1.9 Å crystal structure has revealed the n class="Chemical">Mn4CaO4 cluster geometry, theoretical interpretations
of the EPR/ENDOR and X-ray spectra based on the geometry and detailed
comparison to the model compounds have become possible. Additionally,
advancement of the theoretical approaches, together with experimental
methods in the last few decades, has helped in advancing the understanding
of the nature of the Mn4CaO5 cluster as described
in the sections below.
Electronic Structure Probed
by X-ray Spectroscopy
Various X-ray spectroscopic approaches
have provided an element-specific
view of the electronic structural changes of the Mn4CaO5 cluster, and these techniques complement each other by probing
the electronic configurations with difn class="Chemical">ferent sensitivity and selection
rules. Figure 22 summarizes the energy level
diagram for the X-ray spectroscopy techniques we describe here, which
include X-ray absorption spectroscopy (XAS), X-ray emission spectroscopy
(XES), and resonant inelastic X-ray scattering spectroscopy (RIXS).
Figure 22
(Left)
Energy level diagram for K-emission. The dashed line (black)
is the excitation into the continuum. The solid lines are the emission
lines, black is 2p to 1s (Kα), red is 3p to 1s (Kβ1,3 and Kβ′), and green is valence orbitals to
metal 1s (Kβ2,5 and Kβ″). The exchange
interaction between the 3p and 3d makes the Kβ1,3 and the Kβ′ sensitive to the number of unpaired electrons
in the 3d level and therefore the charge density (oxidation states)
of Mn. The valence-to-core Kβ2,5 and Kβ″
involve the 2s and 2p levels from the ligand atoms and, hence, are
sensitive to the electronic environment of the ligand. (Right) 1s2p
RIXS energy level diagram. The excitation energy from 1s to 3d levels
is shown as a dashed red line and is scanned using a monochromator,
and the 2p-1s (Kα) emission is detected using a spherically
bent crystal analyzer spectrometer. The difference between the excitation
and the emission energies gives the 2p to 3d transitions, which is
equivalent to the L-edges. The RIXS spectra can be used to obtain
both the K-pre-edge and L-edge spectra.
(Left)
Energy level diagram for K-emission. The dashed line (black)
is the excitation into the continuum. The solid lines are the emisn class="Chemical">sion
lines, black is 2p to 1s (Kα), red is 3p to 1s (Kβ1,3 and Kβ′), and green is valence orbitals to
metal1s (Kβ2,5 and Kβ″). The exchange
interaction between the 3p and 3d makes the Kβ1,3 and the Kβ′ sensitive to the number of unpaired electrons
in the 3d level and therefore the charge density (oxidation states)
of Mn. The valence-to-core Kβ2,5 and Kβ″
involve the 2s and 2p levels from the ligand atoms and, hence, are
sensitive to the electronic environment of the ligand. (Right) 1s2p
RIXS energy level diagram. The excitation energy from 1s to 3d levels
is shown as a dashed red line and is scanned using a monochromator,
and the 2p-1s (Kα) emission is detected using a spherically
bent crystal analyzer spectrometer. The difference between the excitation
and the emission energies gives the 2p to 3d transitions, which is
equivalent to the L-edges. The RIXS spectra can be used to obtain
both the K-pre-edge and L-edge spectra.
XAS probes the unoccupied orbitals of the metals of interests.
The K-edge pre-edge region is due to the n class="Chemical">metal 1s to 3d transitions;
its peak position is sensitive to the d-orbital multiplicity and ligand
symmetry, and the intensity is related to the covalency. XES is complementary
to XAS and provides a direct probe of the occupied molecular orbitals.
In a Kβ XES spectrum, the Kβ1,3 and Kβ′
correspond to a transition from a metal 3p orbital to a metal1s hole.
Because of a strong contribution from 3p-3d exchange contribution,
this region of the spectrum is dominantly influenced by the spin state
of the metal. To higher energy are the valence-to-core transitions
(or also called Kβ2,5 and Kβ″). These
features correspond primarily to transitions from ligand 2p and 2s
orbitals to metal1s, respectively, and as such contain direct information
about the ligand identities.
The electronic structure of the
metal complex can be studied in
more detail un class="Chemical">sing RIXS, which is sensitive to both metal charge density
and spin state.[184,185] In 1s2p RIXS (Figure 22), a Mn 1s electron is excited into the lowest
unoccupied molecular orbitals (LUMOs). The orbitals have mainly Mn
3d character mixed with Mn 4p and ligand orbitals. The electronic
configuration can be approximated by 1s13d. After the absorption, the most probable decay
process is 2p to 1s to reach the final state configuration 2p53d. In the RIXS measurement,
both the incident X-ray energy (ν) and the emission energy (f) are scanned and the energy difference between the initial
state and the intermediate state (ν) is equivalent to the K-edge
pre-edge transition, and the difference between the initial state
and the final state (ν – f) is comparable
to L-edge spectroscopy. Because of the strong (2p, 3d) multiplet interaction,
L-edge spectra are more sensitive to the metal spin state; therefore,
RIXS is sensitive not only to the metal charge density but also to
the metal spin state. In general, L-edge spectroscopy is difficult
for biological samples because of severe radiation damage caused by
the higher X-ray absorption at lower X-ray energy region, and additionally
experiments need to be carried out under ultrahigh vacuum conditions.
In RIXS, on the other hand, L-edge-like spectra are obtained with
the excitation energy in the energy range of K-edge spectroscopy,
and therefore the degree of radiation damage is comparable to that
of the K-edge spectroscopy and can be avoided by collecting data at
cryogenic temperature. Hence, RIXS spectroscopy can circumvent some
difficulties in the L-edge spectroscopy, which makes this method suitable
for biological samples. In the following section, we summarize the
current understanding of the electronic structure of the OEC using
these three techniques.
Mn K-edge Absorption Spectra
of PS II
Figure 23a, b show the X-ray
absorption near
edge spectra (XANES) of PS II solutions from cyanobacteria and n class="Species">spinach
thylakoid membrane from S0 to S3 states. Pure
S-state spectra were obtained by deconvoluting the spectra of flash
samples (0F–3F) using the S-state distributions obtained from
EPR spectroscopy. A typical distribution of S-states is shown in Figure 7c.
Figure 23
(a) (Top) Mn XANES spectra from spinach PS II in all the
S-states
and (Bottom) the second derivatives of the Mn K-edge XANES. The inset
shows the pre-edges in all the S-states. (b) (Top) Mn XANES spectra
from cyanobacterial PS II in all the S-states and (Bottom) the second
derivatives of the Mn K-edge XANES. The similarities in the Mn K-edge
shifts in energy between spinach and cyanobacterial PS II are very
clear. There are only small changes in the shape as seen in the second
derivatives at around 6560 eV. The shifts in energy between the S0 to S1 and S1 to S2 are much
larger than that seen for the S2 to S3 transition.
This is particularly clear from the zero-crossing of the second derivatives.
(c) (Top) Mn XANES spectra from spinach PS II (BBYs) and cyanobacterial
PS II preparations in the dimeric form and (Bottom) the second derivatives
of the Mn K-edge XANES. There are small differences in shape between
the spinach and cyanobacterial PS II. These differences could arise
from differences in the second sphere interactions with the Mn4Ca cluster and H-bonding network. (d) Amino acid residues
within a radius of 20 Å around the Mn4Ca cluster according
to the structural model of PS II from T. elongatus at 2.9 Å resolution. The right panel shows in yellow (ribbon
mode) the amino acid residues within a radius of 20 Å around
the Mn4Ca cluster (Mn, purple spheres; Ca, green sphere).
Amino acids of subunit D1 different from spinach are highlighted in
pink and are labeled in the enlarged view on the left. For better
orientation, the amino acid residues Asp170, Glu189, and His332 (all
of subunit D1, yellow) are labeled and are shown in stick mode. The
extrinsic subunits PsbO (purple), PsbU (blue), and PsbV (light blue)
are shown in cartoon mode. The view is of one monomer looking onto
the monomer–monomer interface along the membrane plane (tilted
by 45° to the left), with the cytoplasm above and the lumen below.
(d) Adapted from ref (70).
(a) (Top) Mn XANES spectra from n class="Species">spinach PS II in all the
S-states
and (Bottom) the second derivatives of the Mn K-edge XANES. The inset
shows the pre-edges in all the S-states. (b) (Top) Mn XANES spectra
from cyanobacterial PS II in all the S-states and (Bottom) the second
derivatives of the Mn K-edge XANES. The similarities in the Mn K-edge
shifts in energy between spinach and cyanobacterial PS II are very
clear. There are only small changes in the shape as seen in the second
derivatives at around 6560 eV. The shifts in energy between the S0 to S1 and S1 to S2 are much
larger than that seen for the S2 to S3 transition.
This is particularly clear from the zero-crossing of the second derivatives.
(c) (Top) Mn XANES spectra from spinach PS II (BBYs) and cyanobacterial
PS II preparations in the dimeric form and (Bottom) the second derivatives
of the Mn K-edge XANES. There are small differences in shape between
the spinach and cyanobacterial PS II. These differences could arise
from differences in the second sphere interactions with the Mn4Ca cluster and H-bonding network. (d) Amino acid residues
within a radius of 20 Å around the Mn4Ca cluster according
to the structural model of PS II from T. elongatus at 2.9 Å resolution. The right panel shows in yellow (ribbon
mode) the amino acid residues within a radius of 20 Å around
the Mn4Ca cluster (Mn, purple spheres; Ca, green sphere).
Amino acids of subunit D1 different from spinach are highlighted in
pink and are labeled in the enlarged view on the left. For better
orientation, the amino acid residues Asp170, Glu189, and His332 (all
of subunit D1, yellow) are labeled and are shown in stick mode. The
extrinsic subunits PsbO (purple), PsbU (blue), and PsbV (light blue)
are shown in cartoon mode. The view is of one monomer looking onto
the monomer–monomer interface along the membrane plane (tilted
by 45° to the left), with the cytoplasm above and the lumen below.
(d) Adapted from ref (70).
The overall trend of the XANES
edge shift is n class="Chemical">similar between the
PS II from cyanobacteria and spinach thylakoid membrane preparations.
The edge position shifts to higher energy during the S0 to S3 transitions. The zero-crossing energies of the
rising edge spectrum, which are often used as an indicator of the
oxidation state, are 6550.9 eV for S0, 6553.5 eV for S1, 6554.1 eV for S2, and 6554.4 eV for S3 in cyanobacteria PS II,[70] and 6550.8,
6552.9, 6554.0, and 6554.3 eV, respectively, in spinach thylakoid
membrane. Note that these numbers differ depending on the methods
used,[174] but the zero-crossing of the second-derivative
method used by Messinger et al. avoids any problems with background
contributions.[159] On the basis of this
result, Messinger et al.[159] interpreted
that Mn oxidation occurs during the S0 to S1 and S1 to S2 state transitions based on the
1–2 eV XANES energy shifts. On the other hand, a much smaller
shift was observed in the S2 to S3 state transition,
which suggests that the chemical changes during the S2 to
S3 state transition are not the same as the ones during
the S0 to S1 and S1 to S2 state transitions. One possible explanation for this was that a
unit other than Mn (i.e., oxygen ligands) is oxidized during the S2 to S3 state transition. However, it is worth noting
that the XANES edge shape changes and edge shift could be more complicated
when the transition is accompanied by structural changes. Haumann
et al.[74] showed that the small edge shift
and the shape change could occur when one Mn coordination state changes
from five to six, based on the XANESfeature observed in the S2 and the S3 states.[186] Such coordination changes are also suggested by theoretical models
and EPR studies.[24,25,71] As shown in the previous section, the structural changes observed
in the S2 to S3 state transition are more substantial
compared with other S-state transitions. In the spectroscopic model
(Figure 6), Mn1 is suggested to be Mn(III)
in the S1 state from the EPR study, alternative to the
6-coordinated structure in the 1.9 Å crystal structure. If Mn1
is oxidized in the S2 to S3 transition and becomes
Mn(IV), it is likely that the sixth ligand, which could be O5 or,
as proposed by Siegbahn,[187] a water/hydroxo
ligand, is ligated to Mn1 in the S3 state.
In addition
to the main Mn K-edge, one can also study the 1s to
3d pre-edge spectra that arise from n class="Chemical">1s to 3d transitions, by probing
orbitals that are mainly localized around the metal ion. It shows
the immediate surrounding of the excited ion through the Coulomb interaction
between the core hole and the valence electrons. This pre-edge feature
is a quadrupole-allowed transition and without any dipole contribution
is usually very weak compared to the intensity of the dipole-allowed
main edge transition. The transition can gain intensity by the metal
4p mixing when the metal–ligand environment is distorted from
a centro-symmetric to a noncentro-symmetric coordination. The spectra
reflect coordination number, ligand environment, and oxidation state
of metals.
The pre-edge spectra of PS II in Figure 23a (inset, see also Figure 27 for fits to the
pre-edge) noticeably change during the S-state transition.[159] There are mainly three peaks, and the low-energy
component (∼6540 eV) decreases in intenn class="Chemical">sity during the S0 to S1 and S1 to S2 transitions
and is not present in the S3 spectrum. In the single-crystal
XANES of PS II S1 state, these three components show a
characteristic dichroism.[41] Such dichroism
has been investigated in Mn model complexes, like Mn(V) mononuclear
low spin complex,[188] which has shown the
potential of polarized experiments and also the complementary DFT
theory that can be used to understand the electronic structure. To
understand these pre-edge features and obtain information about the
electronic configuration of PS II, however, one needs to further investigate
the various model compounds and combine experimental data with theoretical
calculations based on ligand field theory and/or DFT.
Figure 27
(Top) K absorption
pre-edges and fits for PS II in S0–S3 states (red, experimental data; black, fit;
blue, pink, and green, peak components; dark gray, background). There
is a lower energy peak in the fits for the S0 and S1 states, which is decreased in the S2 and S3 states. The higher-energy component increases in intensity
in the S2 to S3 transition, which is consistent
with the increase in Mn(IV). (Bottom) RIXS contours for PS II in S0–S3 states. The spectral changes are more
subtle than those seen for the oxides and coordination complexes in
Figure 26 and also compared to the multinuclear
complexes in Figure 28. Adapted from ref (197).
Comparison of OEC from Plants and Cyanobacteria
While
overall trends are similar in the n class="Species">spinach PS II and cyanobacterial
PS II XANES, small changes are observed that are more visible in the
second derivative spectra. This is in agreement with the results of
Su et al.[189] who concluded that the electronic
structure of the Mn cluster is very similar but not identical between
both species, based on comparison of the S2 multiline and
the S2 Mn ENDOR signal from spinach and the cyanoacterial
PS II from T. elongatus.
The alignment of the
amino acid sequence of the D1 protein (subunit PsbA) from n class="Species">T. elongatus and spinach showed a sequence identity of 84.7%.
305 positions are identical and 36 are similar. Within a radius of
20 Å around the OEC, nine amino acid residues are not fully conserved,
but eight of them are conserved between groups with strongly similar
properties. This high conservation makes it unlikely that the slight
differences between spinach and T. elongatus PS II
XANES spectra (Figure 23c) are due to the differences
in the D1 protein. This is also true for subunit CP43 (providing one
of the ligands to the Mn4CaO5 cluster) with
all residues in the vicinity of the OEC being highly conserved between T. elongatus and spinach.
The subunit compositions
of n class="Species">spinach PS II and cyanobacterial PS
II are slightly different. While PS II from cyanobacteria have the
extrinsic subunits PsbO, PsbU, and PsbV, spinach PS II has neither
PsbU nor PsbV but the proteins PsbP and PsbQ instead. The exact localization
of PsbP and PsbQ is not yet resolved, but various studies found these
two subunits to be important for the stability and activity of the
OEC (for review, see refs (190−192) and references therein). PsbV and PsbU are found to bind in the
vicinity of the OEC with closest distances to Mn of 11 Å (PsbV-K160)
and 14 Å (PsbU-Y133), respectively, and thereby they directly
interact with the C-terminus of the D1 protein. Replacing these subunits
with PsbP and PsbQ might induce structural changes in the vicinity
of the OEC, which could cause the slight variations visible in the
XAS spectra, demonstrating the sensitivity of XAS for subtle changes
in the electronic structure.
Mn X-ray
Emission Spectra of PS II
Kβ1,3 Transitions
Spectral changes in the Kβ main lines
reflect the effective
number of unpaired n class="Chemical">metal 3d electrons through the exchange interaction
between the core hole (1s or 2p) and the net electron spin in the
metal valence shell by the (3p, 3d) exchange interaction. In general,
spectral changes for Kβ lines are more pronounced than for Kα,
because the 3p and 3d orbitals interact more with each other than
the 2p and 3d orbitals. In a very simplified model of the two final
spin states, Kβ1,3 is a constructive and Kβ′
is a destructive spin-exchange interaction between the unpaired 3p
and 3d electrons (Figure 24). The magnitude
of the exchange interaction depends on the number of unpaired electrons
in the 3p and 3d orbitals. Thus, the Kβ spectrum serves as an
indicator of oxidation state that is different from XANES, which monitors
oxidation state through 1s core hole shielding effect. Figure 24a shows the Kβ emission spectra of a series
of Mn oxides, Mn(IV)O2, Mn2(III)O3, and Mn(II)O, which illustrate the sensitivity of Kβ spectra
to the oxidation state of Mn.[193] As the
oxidation state of Mn increases from Mn(II) to Mn(III) to Mn(IV),
fewer unpaired 3d valence electrons are available to interact with
the 3p hole; concomitantly, the magnitude of the 3p-3d spin exchange
interaction becomes smaller. Accordingly, the Kβ1,3 transition shifts to a lower energy, the Kβ′ transition
shifts to a higher energy, and the Kβ′–Kβ1,3 splitting becomes smaller (Figure 24b).
Figure 24
(a) Kβ1,3 and Kβ′ spectra from MnII, MnIII, MnIV oxides. (b) 3p-3d exchange
coupling that shows how the Kβ1,3 and Kβ′
spectra are sensitive to the oxidation and spin state of Mn. (c) Kβ1,3 spectra of the S-states. (d) Difference spectra which show
that the shifts are larger for the S0 to S1,
S1 to S2, and S3 to S0 transitions. The S2 to S3 transition is the
smallest. Adapted from ref (146).
(a) Kβ1,3 and Kβ′ spectra from MnII, MnIII, MnIV oxides. (b) 3p-3d exchange
coupling that shon class="Chemical">ws how the Kβ1,3 and Kβ′
spectra are sensitive to the oxidation and spin state of Mn. (c) Kβ1,3 spectra of the S-states. (d) Difference spectra which show
that the shifts are larger for the S0 to S1,
S1 to S2, and S3 to S0 transitions. The S2 to S3 transition is the
smallest. Adapted from ref (146).
Kβ XES is less
sensitive to the ligand environment compared
to n class="Chemical">XANES, as the 3p orbitals have less overlap with the ligand orbitals
compared to the 4p orbitals. This is demonstrated in the XANES and
XES spectra of two types of Mn compounds, with very different structures
such as the “trimers” (trinuclear complexes) and “butterflies”
(tetranuclear), in different oxidation states.[194] In the XANES spectra, the Mn oxidation state changes have
clear effects on the inflection point energy (IPE) shifts: the shift
is 1.64 eV for Mn3O and 2.20 eV for Mn4O2, with the magnitude of the shift depending on the type of
compounds. On the other hand, the Kβ energy shifts from one
oxidation state to another are more or less the same between these
two series; the shifts scale with the fractional change in oxidation
state: 0.12 eV for Mn3O (1 of 3) and 0.09 eV for Mn4O2 (1 of 4). This shows that, compared to XANES,
Kβ XES primarily reflects the changes in oxidation state rather
than differences in the overall ligand environment.
The Kβ
emission spectra of PS II in S0–S3 and
their difference spectra are shown in Figure 24c, d. The derivative shape of the S0 to S1 and
the S1 to S2 difference
spectra show that the Kβ1,3 peak shifts to lower
energy during these transitions. By contrast, the change is not apparent
upon the S2 to S3 transition, suggesting that
the change in the metal charge density is much less than for the other
transitions. Mn reduction occurs during the S3 to S0 state transition, and the Kβ1,3 peak shifts
to a higher energy; the difference spectrum reflects a return to the
starting oxidation states. The first moment energy was calculated
for each spectrum using the following equation (eq 2),and used as an indicator
of the effective
charge density,[159,193] where E and I are the energy and fluorescence intensities of the jth data point. The method is suited for very small shifts
because the statistics from the entire Kβ1,3 (6485–6495
eV) peak is considered rather than just the peak energy.Figure 25 summarizes the IPE (inflection
point energy) from the n class="Chemical">XANES and the 1st moments of the Kβ1,3 spectra of flash-induced spinach PS II samples. The first
moment shifts observed in PS II are much smaller than those for Mn
oxides. As Mn is oxidized from Mn(II), Mn(III), to Mn(IV) in the oxide
series, the first moments shift to lower energy by ∼0.3 eV
step. For the S1 to S2 transitions of PS II,
the shift is 0.06 eV, which is ∼3 times the value observed
for the S2 to S3 transition (0.02 eV). The first
moment shift of 0.06 eV for the S1 to S2 transition
is one-fourth of that seen for the Mn(III) to Mn(IV) oxides.
Figure 25
Mn K-edge
inflection points from the XANES spectra (Figure 23) and the first moments from the Kβ1,3 spectra
(Figure 24) shown on top and bottom,
respectively. The inflection points and the first moments show that
the oxidation of the OEC from the S0 to S1 and
S1 to S2 is different from the S2 to S3 advance. Adapted from ref (146).
Mn K-edge
inflection points from the XANES spectra (Figure 23) and the first moments from the Kβ1,3 spectra
(Figure 24) shown on top and bottom,
respectively. The inflection points and the first moments show that
the oxidation of the OEC from the S0 to S1 and
S1 to S2 is difn class="Chemical">ferent from the S2 to S3 advance. Adapted from ref (146).
One point to consider is whether major structural changes
can cause
the lack of an energy shift in the XES data, even when there is a
Mn-centered oxidation state change. A detailed understanding of Kβ1,3 XES requires a ligand-field multiplet theory that conn class="Chemical">siders
symmetry-dependent perturbations such as (1) spin–orbit coupling,
(2) ligand-field splitting, (3) Jahn–Teller distortion, and
(4) spin–spin interaction between different metal atoms.[195] Each of these perturbations will split the
spin states into a multiplet of states, causing an asymmetric broadening
of the observed emission peaks, indicating that there is some dependence
of the Kβ1,3 XES spectra on the ligand environment.
Although it is expected to be subtle, the details of such effects
need to be further investigated.
Resonant
Inelastic X-ray Scattering
The electronic structure of the
Mn complex in PS II has been studied
in detail using RIXS, owing to the developments in the instrumentation
for conducting such two-dimenn class="Chemical">sional X-ray spectroscopy experiments.
The potential of the RIXS methodology for understanding the details
of electronic structure of the Mn4CaO5 cluster
has been demonstrated using comparisons to Mn complexes that are very
basic such as oxides and mononuclear Mn coordination complexes in
different oxidation states, and more recently using other Mn complexes
with oxo-bridged multinuclear structures in various oxidation states.
The RIXS contour plots of the simpler types of Mn compounds, the
n class="Chemical">oxides of Mn and mononuclear coordination compounds of Mn, which are
all 6-coordinate with formal oxidation states, Mn(II), Mn(III) and
Mn(IV) are shown in Figure 26A, B. Despite
the differences in the ligand environment, similar trends are observed
in the Mn oxides and the mononuclear coordination compounds with the
same oxidation states. The spectrum from Mn(II)O shows one broad peak
centered at ∼6540 eV that is well-explained by the contribution
of the crystal field splitting between the t2g and eg orbitals.[196] For Mn in oxidation
state (III) and Mn(IV), the electron (3d-3d) interaction becomes dominant.
In Mn(III) compounds, for example, the two strong resonances observed
in the contour plots (ν = 6540 and 6543 eV) are due to the (3d,
3d) multiplet interactions (∼3 eV separation). The separation
of two strong features is smaller in Mn coordination compounds (∼2
eV) due to the reduced magnitude of the (3d-3d) interaction; i.e.,
a more covalent electron configuration decreases the electron–electron
interaction.
Figure 26
Contour plots of the 1s2p3/2 RIXS planes for
four Mn
oxides (A) in oxidation states II, III, and IV and the four molecular
complexes (B) MnII(acac)2(H2O)2, MnIII(acac)3, [MnIII(5-Cl-Salpn)(CH3OH)2]+, and MnIV(sal)2(bipy).[233,234] The abscissa is the excitation
energy, and the ordinate is the energy transfer axis. (C) Line plots
extracted from the RIXS planes for the coordination complexes in oxidation
states III or IV and the S1 state of PS II. The PS II plots
are between oxidation states III and IV. An integration of the 2D
plot parallel to the ordinate yields L-edge like spectra, the feature
at ∼640 eV corresponds to transitions to J = 3/2 like states
(L3 edges). Integrations parallel to the energy transfer
axis sort the spectrum according to the final state. Adapted from
ref (196).
Contour plots of the 1s2p3/2 RIXS planes for
four Mnn class="Chemical">oxides (A) in oxidation states II, III, and IV and the four molecular
complexes (B) MnII(acac)2(H2O)2, MnIII(acac)3, [MnIII(5-Cl-Salpn)(CH3OH)2]+, and MnIV(sal)2(bipy).[233,234] The abscissa is the excitation
energy, and the ordinate is the energy transfer axis. (C) Line plots
extracted from the RIXS planes for the coordination complexes in oxidation
states III or IV and the S1 state of PS II. The PS II plots
are between oxidation states III and IV. An integration of the 2D
plot parallel to the ordinate yields L-edge like spectra, the feature
at ∼640 eV corresponds to transitions to J = 3/2 like states
(L3 edges). Integrations parallel to the energy transfer
axis sort the spectrum according to the final state. Adapted from
ref (196).
For Mn coordination compounds, the energy position
shifts more
toward lower energy compared to the n class="Chemical">Mn oxides (Figure 26B), which indicates that Mn coordination compounds have much
stronger covalency compared to the Mn oxides. The changes per oxidation
state in the positions are more pronounced between the Mn oxides than
are those between the Mn coordination compounds.
In Figure 26C, the two types of line plots
(constant incident energy (CIE, left) and constant energy transfer
(CET, right)) of the PS II S1 state are shown together
with those of Mn coordination compounds. The S1 CET spectrum
shon class="Chemical">ws mixed features characteristic of Mn(III) and Mn(IV) compounds,
having two main peaks, which supports the mixed oxidation states of
Mn(III) and Mn(IV) in the S1 state assigned earlier by
the EPR and XANES studies.
More recently, RIXS has been used
to study the entire S-state cycle
to specifically address the issue of the electronic structure of the
S3 state.[197] Figure 27 shows Mn XAS pre-edges
and RIXS spectra of the S0 to S3 states of PS
II. RIXS spectroscopy considerably improves the pre-edge separation
from the main edge feature as compared to XANES in which it is usually
difficult to distinguish the contribution of strong main edge transition.
The main spectral features in the RIXS spectra extend along a diagonal
streak in the 1s2p RIXS plane. Spectral features off this diagonal
line result from different interactions of the 1s and the 2p core
hole with the valence electrons. These direct Coulomb and exchange
interactions are considerably stronger for a 2p core hole, making
the technique also sensitive to the spin density of Mn.(Top) K absorption
pre-edges and fits for PS II in S0–S3 states (red, experimental data; black, fit;
blue, pink, and green, peak components; dark gray, background). There
is a lower energy peak in the fits for the S0 and S1 states, which is decreased in the S2 and S3 states. The higher-energy component increases in intensity
in the S2 to S3 trann class="Chemical">sition, which is consistent
with the increase in Mn(IV). (Bottom) RIXS contours for PS II in S0–S3 states. The spectral changes are more
subtle than those seen for the oxides and coordination complexes in
Figure 26 and also compared to the multinuclear
complexes in Figure 28. Adapted from ref (197).
Figure 28
Contour plots of Mn 1s2p RIXS planes
of model compounds (a) salpn2MnIV2(OH)2, (b) salpn2MnIV2(O)(OH), (c) salpn2MnIV2(O)2, (d) phen4MnIV2(O)2, (e) MnIV3Ca2, and (f) MnIV3(O)4Acbpy.[76,88,235,236] The spectral features
in (a), (b), and (e) with protonated bridge
or with Ca are more similar to PS II S3 state spectra shown
in Figure 27. Detailed theoretical analysis
of spectra from model compounds such as these has the potential for
understanding the electronic structure and the changes in PS II. Adapted
from ref (197).
RIXS spectra of a series of multinuclear Mn model complexes
are
shown in Figure 28 to compare with the S3 state spectrum.[196−199] The Mn(IV) complexes show a characteristic off-diagonal intensity,
suggesting a stronger (2p, 3d) final-state interaction. Strong variations
are seen in the electronic structure within the series of MnIV model systems. The spectrum of the S3 state best resembles
the MnIV complexes, n class="Chemical">Mn3IVCa2 and saplnMn2IV (OH)2, i.e., the
oxo-bridge protonation of Mn dimer complexes and the presence of Ca
in one corner of a Mn cubane structure show a similar spectroscopic
response, suggesting that Ca in PS II and protonation of the oxo-bridge
may give rise to analogous modifications of the electronic structure
at the Mn sites, e.g., ligand symmetry changes. These results emphasize
that the assignment of formal oxidation states alone is not sufficient
for understanding the detailed electronic structural changes and describing
the complex nature of the electronic structure in multinuclear clusters
like the Mn4CaO5 cluster in PS II. The Mn RIXS
spectral changes are subtle during the S-state transitions (Figure 27), which suggests that electrons are strongly delocalized
in the Mn4CaO5 cluster, and ligands may be intimately
involved in the redox chemistry.
Contour plots of Mn 1s2p RIXS planes
of model compounds (a) salpn2MnIV2(OH)2, (b) salpn2MnIV2(O)(OH), (c) salpn2MnIV2(O)2, (d) phen4MnIV2(O)2, (e) n class="CellLine">MnIV3Ca2, and (f) MnIV3(O)4Acbpy.[76,88,235,236] The spectral features
in (a), (b), and (e) with protonated bridge
or with Ca are more similar to PS II S3 state spectra shown
in Figure 27. Detailed theoretical analysis
of spectra from model compounds such as these has the potential for
understanding the electronic structure and the changes in PS II. Adapted
from ref (197).
S-States
and Summary of the Oxidation States
Current understanding
of the oxidation state changes of the Mn4CaO5 cluster during the reaction cycle is a stepwise
oxidation of Mn in each S-state trann class="Chemical">sition; within the context of
the formal oxidation state, the Mn oxidation state could be assigned
as Mn(III3, IV) for S0, Mn(III2,
IV2) for S1, Mn(III, IV3) for S2, and Mn(IV4) for S3. The RIXS study,
however, suggests that formal oxidation states may be insufficient
for describing the complex nature of the electronic structure in multinuclear
clusters like the Mn4CaO5 cluster in PS II,
and that electrons are strongly delocalized in the Mn4CaO5 cluster. It is likely that ligands are intimately involved
in modulating the redox chemistry and are also involved in the delocalization
of the electron density as the cluster is oxidized.
The RIXS
data show that, among the compounds with the same formal oxidation
states, the spectroscopic response is modulated by the delocalization
of the charge density. Tn class="Chemical">his is illustrated by the differences in the
RIXS spectra of the oxides and the coordination complexes. The trend
is that, for similar oxidation states, the charge distribution as
determined by the RIXS spectra seems to be higher in the Mn4Ca cluster than in coordination complexes, and higher in coordination
complexes than in oxides. Therefore, the data from PS II suggest that
the distribution of the charge between the metal and ligands could
be critical for the mechanism of water oxidation.
Of the two
main mechanisms that are being considered for the O–O
bond formation catalyzed by the OEC, one involves the generation of
a n class="Chemical">MnV-oxo group in the final step when the O–O bond
is formed, and the alternate mechanism involves the oxo-oxyl bond
formation.[71,200] The current RIXS results show
that the change of the electronic charge includes the ligands in all
S-state transitions, and such delocalization of charge on the ligands
may play an important role in the reaction. However, interpretation
of the electronic structure of the Mn4CaO5 cluster
beyond the formal oxidation state requires chemical structural information
of the cluster in each S-state, as well as further development of
theoretical tools to interpret the spectroscopic data, and such efforts
are underway. Additionally, whether or not Ca plays a role in modulating
the redox-active cluster is still an open question, but recent studies
with heteronuclear Mn and Ca/Sr complexes have implicated the role
of Ca in this process.[87]
Lessons from XES of Model Complexes about
the Intermediate States
High-Spin and Low-Spin
MnV with
Kβ1,3/Kβ″ Spectra
Mn(V) has
been proposed as a posn class="Chemical">sible transient intermediate in the water oxidation
in PS II as well as some of the Mn-based catalytic reactions like
epoxidation reactions and C–H bond functionalization. Mn(V)-oxo
complexes have been synthesized, and most of them are stable at room
temperature, with low-spin S = 0 ground states.[201−203] On the other hand, there are only a few reports on high-spin Mn(V)-oxo
species with S = 1 spin ground states, one with a porphyrin ligand[204] and another that has trigonal symmetry.[89] The metal–oxo interactions in trigonal
symmetry are weaker compared to those with tetragonal symmetry, rendering
the Mn(V)-oxo species with high-spin configuration. This species is
unstable at room temperature and more reactive.[89] We would therefore expect a high-spin, reactive Mn(V) to
be involved in the catalytic reaction if indeed a Mn(V) is involved
in the O–O bond formation in the OEC. It is known that up to
the S3 state all four Mn are in a high-spin configuration;
hence, it is likely that if Mn(V) is involved it would also be high
spin.
The high-spin mononuclear Mn(V) complex recently prepared
by the Borovik group is 5-coordinate.[89] It is very reactive and short-lived at room temperature, and therefore
can be studied only at low temperature. It is likely that having a
5-coordinate geometry is required to generate a high-spin n class="Chemical">Mn(V) species.
The Mn XAS and Kβ1,3/Kβ′ XES of this
complex can be a good indicator of identifying low/high-spin Mn species
as well as the presence or absence of the Mn(V) species (Figure 29).[89]
Figure 29
Mn Kβ1,3 and Kβ′ spectra from the
high-spin and low-spin Mn(V) complexes.[89,202] The exchange
coupling scheme describing the two cases is shown below the spectra,
which explains the lack of a Kβ′ peak in the low-spin
case and the energy difference in the Kβ1,3 peak
between the low- and high -pin complexes. Adapted from ref (89).
Mn Kβ1,3 and Kβ′ spectra from the
high-spin and low-spin Mn(V) complexes.[89,202] The exchange
coupling scn class="Chemical">heme describing the two cases is shown below the spectra,
which explains the lack of a Kβ′ peak in the low-spin
case and the energy difference in the Kβ1,3 peak
between the low- and high -pin complexes. Adapted from ref (89).
XES for Direct Detection of Oxo-Bridges
Using Kβ2,5/Kβ″ Spectra
Direct
detection of O-ligation to the Mn4CaO5 cluster
provides a method for studying the role of the O ligands in the mechanism
of n class="Chemical">water oxidation and dioxygen formation. However, there are few,
if any, methods that can specifically monitor the electronic spectra
of the O ligand atoms of Mn from the Mn4CaO5 cluster in the PS II complex. Kβ2,5 emission is
predominantly from ligand 2p (metal 4p) to metal1s, and the Kβ″
emission is assigned to a ligand 2s to metal1s, known as crossover
transitions.[205−207] Therefore, only direct ligands to the metal
of interest are probed with Kβ,2,5/Kβ″
emission. In particular, the Kβ″ energy is affected by
the charge density on the metal, the ligand protonation state, and
changes in the coordination environment. The intensity is influenced
by the overlap between the Mn 3d and ligand molecular orbital wave
functions and is thus affected by the metal-to-ligand distance, as
well as the number of ligands per metal ion. Shorter distances (e.g.,
from higher bond order or deprotonation) result in increased Kβ″
intensity with an approximate exponential dependence.[205] On the other hand, a spread of the molecular
wave function over next-nearest neighbor atoms will decrease the Kβ″
spectral intensity. Therefore, contribution from single atom ligands
such as oxo-bridges, or terminal oxo ligands bonded to Mn, is predominant.
These combination of factors makes the Kβ″ spectrum a
powerful tool for detection and characterization of oxo-bridges in
the Mn4CaO5 cluster of PS II.[208]
Figure 30 shows a comparison
of the PS II S1 state spectrum with those of a series of
Mn coordination compounds, n class="Chemical">MnV-oxo (a), di-μ-oxo
bridged Mn2III,IV and Mn2IV (b, c), cubane-type Mn2IIIMn2IV and MnIIIMn3IV (d, e),
and a μ-alkoxide bridged Mn2II (f). These
compounds have oxo-bridged Mn (except for MnV-oxo) with
O or N/O terminal ligands. The strongly delocalized molecular orbitals
from O and N terminal ligands, and the μ-alkoxide/carboxylate
bridges, do not contribute significantly to the spectra. Moreover,
terminal ligands are generally at longer distances to Mn than the
bridging ligands, and their contributions are smaller.[205] There is a strong peak in the MnV-oxo compound, indicating that the Kβ″ peak intensity
is predominantly sensitive to single atom ligands and short metal–ligand
atom distances, namely, bridging O ligands and double/triple bonds,
which all have localized 2s orbitals. The sensitivity of the spectra
to even one-electron changes is illustrated in Figure 30. The spectra are different for the two binuclear and the
two cubane molecules in which one of two or four Mn is oxidized from
(III) to (IV). The Kβ″ peak of the PS II S1 state is relatively intense compared to other Mn2III,IV di-μ-oxo bridged compounds. This suggests that
there are several μ-oxo bridged Mn–O bonds in the S1 state.[14]
Figure 30
(a) Kβ′′
of the Mn in oxidation states II,
III, and IV is compared to PS II in the S1 state. The position
of the peak in the S1 state shows that the oxidation state
is III2,IV2, and the intensity as exemplified
in (b) by comparison to Mn complexes makes it clear that it is from
an oxo-bridged Mn. The Mn model compounds were the following: a bridging
μ-alkoxide Mn2 compound[237] ([Mn2(II)μ-ClCH2CO2](CH3CO2)2(ClO4)2),
two di-μ-oxo bridged Mn2 compounds[238,239] ([Mn2(III,IV)O2bipy4](ClO4)3 and [Mn2(IV,IV)O2terpy2(SO4)2]6(H2O)), two Mn4 cubane compounds[240] (hexakis(μ2-diphenylphosphinato)tetrakis(μ3-oxo)Mn4(III,III,IV,IV) and [hexakis(μ2-diphenylphosphinato)tetrakis(μ3-oxo)Mn4(III,IV,IV,IV)]CF3SO3), and a macrocyclic Mn(V)-oxo complex.[202] Adapted from ref (208).
(a) Kβ′′
of the Mn in oxidation states II,
III, and IV is compared to PS II in the S1 state. The position
of the peak in the S1 state shon class="Chemical">ws that the oxidation state
is III2,IV2, and the intensity as exemplified
in (b) by comparison to Mn complexes makes it clear that it is from
an oxo-bridged Mn. The Mn model compounds were the following: a bridging
μ-alkoxideMn2 compound[237] ([Mn2(II)μ-ClCH2CO2](CH3CO2)2(ClO4)2),
two di-μ-oxo bridged Mn2 compounds[238,239] ([Mn2(III,IV)O2bipy4](ClO4)3 and [Mn2(IV,IV)O2terpy2(SO4)2]6(H2O)), two Mn4 cubane compounds[240] (hexakis(μ2-diphenylphosphinato)tetrakis(μ3-oxo)Mn4(III,III,IV,IV) and [hexakis(μ2-diphenylphosphinato)tetrakis(μ3-oxo)Mn4(III,IV,IV,IV)]CF3SO3), and a macrocyclic Mn(V)-oxo complex.[202] Adapted from ref (208).
The involvement of bridging oxo
groups[13] or the high-valent MnIV=O• or MnV≡O species[23,209] has been implicated in the mechanism
for the formation of the critical O–O bond in the n class="Chemical">water-oxidation
reaction of PS II. It is feasible to obtain Kβ2,5/Kβ″ spectra for the Mn4CaO5 cluster
in PS II, and the outlook for Kβ2,5/Kβ″
spectroscopy as a tool for studying the nature of the O ligand binding
modes and, therefore, the mechanism of the water-splitting reaction
seems promising.
Protonation State of
the Oxo-Bridges with
Kβ2,5/Kβ″ Spectra
Protonation
of the oxo bridges is another issue where the study of model complexes
un class="Chemical">sing Mn Kβ2,5/Kβ″ XES has the potential
for being useful for understanding the OEC. In PS II, protonation
state changes in the bridging oxygen (μ-oxo or μ-hydroxo)
and/or a terminal substrate water as aqua or hydroxo have been proposed
during the four-electron redox chemistry.[71,210,211] The generally accepted proton-release
pattern during the S0 to S1, S1 to
S2, S2 to S3, and S3 to
S0 transitions is 1, 0, 1, 2, respectively,[212] although it has been reported that the proton-release
pattern is pH-dependent.[162]
Several
techniques have the potential to detect a single protonation event,
while n class="Chemical">few of them can directly and selectively probe the protons in
the first coordination sphere of a transition metal ion. Potential
methods include vibrational spectroscopy and ligand-sensitive EPR
techniques such as ENDOR (electron nuclear double resonance), ESEEM
(electron spin echo envelope modulation), and HYSCORE (hyperfine sublevel
correlation). EXAFS can also provide indirect information about protons,
by the effect that protonations or deprotonations might have on the
Mn–Mn/Sr(Ca) and Mn–O,N ligand atom distances.
The shortening of one Mn–Mn distance observed in the EXAFS
during the S0 to S1 transition could be a consequence
of the deprotonation of an hydrn class="Chemical">oxo (OH–) bridge
between Mn atoms to an oxo (O2–) bridge.[47,76] The S0 to S1 transition is accompanied by
the release of one proton from the OEC. During the S1 to
S2 transition, there is no net release of protons and there
are no significant changes in Mn–Mn/Ca distances. The following
S2 to S3 state transition is accompanied by
the release of a proton, and a structural change in the cluster is
observed. There are no EXAFS studies on intermediate states between
S3 and S0, when two protons are released. Ca
can accommodate 7 or 8 ligands, and it is therefore ideally suited
for ferrying in H2O/OH– groups to the
catalytic site (Hillier and Messinger in ref (9)).
Unlike EXAFS, the
Kβ2,5/Kβ″ XES can
be a direct method to probe single protonation events or other changes
in ligand environment. The advantage of tn class="Chemical">his method over EPR techniques
is its element specificity, and they are sensitive only to the protonation
states of the ligands of metals. Moreover, they are unrestricted by
the spin states of the compounds.
A set of homologous dinuclear
MnIV complexes (Figure 31a) that
were initially synthesized by Baldwin et
al.,[76] in which the protonation state of
the bridging n class="Chemical">oxygen atoms was changed systematically, has been explored.
Although these complexes are not mimics of the Mn4Ca cluster
in the OEC, they contain motifs such as oxo and protonated oxo-bridges
that are implicated in the mechanism of water oxidation. A combination
of XES data (Figure 31b) and DFT calculations
(Figure 31c)[213] provides
a detailed understanding of the origin of valence-to-core emission
peaks, making XES an important tool to resolve protonation states
of bridging oxygen atoms in biological catalysts. The technique also
allows one to differentiate between two possible structures for the
doubly protonated species, namely, a bis-μ-hydroxo versus a
μ-oxo-μ-aqua bridged complex. Although a symmetric doubly
protonated bridging motif is chemically more reasonable, the ability
of valence-to-core XES and the accompanying DFT calculations to distinguish
between these isomers is of interest, as such aquo units have been
formulated recently as bridging motifs for the Mn4CaO5 core of the OEC.[214]
Figure 31
Sensitivity
of the Kβ2,5 and Kβ′′
spectra to the protonation state. This is demonstrated in the spectra
of three Mn2IV oxygen-bridged complexes in which
the protonation states of oxo-bridges are changed. (a) 1 and 2 show
the structures of the di-μ-oxo and oxo-hydroxo bridged complexes,
and 3a and 3b show the two possibilities, dihydroxo or oxo-aquo bridges.
(b) Spectra of 1, 2, and 3 shown in solid black, dashed red, and dashed
blue, respectively. (c) Assignments of the calculated XES valence-to-core
region based on the orbital character corresponding to the individual
transitions for compounds 1, 2, 3a and 3b shown in (a). The left side shows the Kβ″
region, and the right side shows the Kβ2,5 region.
Adapted from ref (241).
Sensitivity
of the Kβ2,5 and Kβ′′
spectra to the protonation state. Tn class="Chemical">his is demonstrated in the spectra
of three Mn2IVoxygen-bridged complexes in which
the protonation states of oxo-bridges are changed. (a) 1 and 2 show
the structures of the di-μ-oxo and oxo-hydroxo bridged complexes,
and 3a and 3b show the two possibilities, dihydroxo or oxo-aquo bridges.
(b) Spectra of 1, 2, and 3 shown in solid black, dashed red, and dashed
blue, respectively. (c) Assignments of the calculated XES valence-to-core
region based on the orbital character corresponding to the individual
transitions for compounds 1, 2, 3a and 3b shown in (a). The left side shows the Kβ″
region, and the right side shows the Kβ2,5 region.
Adapted from ref (241).
While detecting very weak valence
to core Kβ2,5/Kβ″ XES signals is challenging
in the dilute biological
samples, recent progress in the design of the spectrometer and the
high brilliance available at third-generation synchrotron facilities
make it possible to detect such weak signals, and the effort is underway.
Current Understanding of the Water-Oxidation
Mechanism
There is limited direct experimental information
about the O–O
bond-formation step, and the mechanisms that have been proposed are
largely based on what is known about the structures in the stable
S-states and theoretical calculations.[71,85,215−217] There is consensus that the
O–O bond is not formed before the S3 state. It is
thought that, after the third flash, one electron and one proton is
released from the S3 state, forming the putative S4 state where the O–O bond is formed prior to the release
of molecular oxygen.[162,218] The presence of a kinetic intermediate,
S3Yz•, before the final oxidation of
the OEC (S4) has been reported from several experiments
based on the detection of a lag phase (150–200 μs): UV–vis[133,219] and EPR spectroscopy[220] by following
Yz• kinetics, time-resolved XAS by following the
Mn oxidation state,[157] and FTIR spectroscopy.[153] It is proposed that, at tn class="Chemical">his stage, the OEC
is not oxidized, but an electron is released from Yz, and the OEC
is oxidized only after a release of proton in the S4 state.
The chemical nature of the transient S4 state has not yet
been experimentally determined.
Since the 1.9 Å crystal
structure was reported in 2011, it
has become posn class="Chemical">sible to interpret the various experimental data within
the context of the reliable geometric structure of the Mn4CaO5 cluster.[221] Although there
are questions about the chemical nature of the cluster such as the
protonation state of the bridging oxygens and the identity of the
terminal ligands such as aquo or hydroxo, it is now possible to narrow
down the many proposed water oxidation mechanisms and the probable
site on the Mn4CaO5 cluster. The O–O
bond formation mechanisms proposed thus far are shown in Figure 32: (a) nucleophilic attack (high-valent Mn(V)-oxo
or Mn(IV)-oxo);[85,209,210,222−224] (b) between two oxo-bridges;[13] (c) terminal
oxo-radical and bridging oxygen;[23,24,71] and (d) two terminal oxygen ligands.[217] A radical coupling mechanism (e) has been suggested
for a Ru catalyst, although not for the OEC in PS II.[225]
Figure 32
Main mechanistic schemes that are being considered
for the O–O
bond formation by the Mn4CaO5 cluster.
Main mechanistic schemes that are being conn class="Chemical">sidered
for the O–O
bond formation by the Mn4CaO5 cluster.
Among these mechanisms, the most
likely one suggested from the
substrate water exchange studied by TR-MIMS (time-resolved membrane
inlet mass spectroscopy) un class="Chemical">sing isotope-labeled water is (c) the oxo/oxyl
radical coupling mechanism. The isotope-labeled water-exchange studies
have provided important information that informs about the possible
O–O bond-forming mechanisms.[226,227] From the
distinct exchange rate of the two substrate water molecules, Wf (fast-exchanging water) and Ws (slow-exchanging
water), at each S-state, as well as currently available structural
information and the EPR results,[228] Messinger
et al. suggested that Wf is the bridging oxygen, O5 (Figure 6), which reacts with a terminal oxyl radical formed
in the S4 state that is bound to either Mn1 or Mn4. A detailed
review by Cox and Messinger is found in the literature.[25] This mechanism is basically in line with what
Siegbahn has suggested based on theoretical calculations.[71]
RIXS results described in section 4.3.2 have
shown that the electrons are strongly delocalized in the Mn4CaO5 cluster, and if so, ligands may be intimately involved
in the redox chemistry. Tn class="Chemical">his suggests that such delocalization may
play a role during the O–O bond-formation step in the S4 state. The structural changes that occur in the S2 to S3 transition described in section 3.2.3 show that, accompanying both Mn–Mn and Mn–Ca(Sr)
distance changes, the closed cubane structure may be formed in the
S3 state. If such a structural change occurs, it will affect
the charge distribution in the OEC.
In the oxo/oxyl mechanism
proposed on the ban class="Chemical">sis of the substrate
water-exchange results, redox tautomerism in the S1, S2, and S3 states is suggested to play an important
role for the fast water-exchange rate.[24,229] Although
such interconvertible multistates are observed in the S2 state (g = 2 and g = 4 spin states),
there is no experimental evidence so far in the S1 and
the S3 states, and further structural study is necessary.
The presence of such multistates also implies the importance of the
dynamic nature of the metal cluster to the catalytic function. Therefore,
it becomes important to study the structural and chemical dynamics
of the Mn4CaO5 cluster and PS II under functional
conditions, which is the future direction using XFELs; the approach
is described in the next section.
Toward
an Understanding of Protein and Chemical
Dynamics of PS II
Synchrotron radiation (SR)-based X-ray
techniques (crystallography
and spectroscopy) have been the major tools for studying the architecture
of PS II and its n class="Chemical">water-oxidation reaction in the OEC. As described
above, X-ray spectroscopy has provided the element-specific information
of structural and electronic structure of the cryo-trapped intermediate
S-states, from the S0, S1, S2 to
S3 state. However, accessing the kinetically unstable S4 state (Figure 1) that appears in the
last step of the catalytic cycle is difficult using the traditional
cryo-trapping methodology, and characterizing the nature of such a
transient state requires time-resolved detection at room temperature.
Within the total time scale of ∼1.3 ms of the S3–S4–S0 transition after initiation
by the third flash, sequential events occur that include release of
one electron, release of two protons, release of molecular oxygen,
and water substrate(s) binding, along with four-electron reduction
of Mn. Understanding the nature of the S4 state and following
the sequential chemistry during the S3–S4–S0 transitions is therefore at the heart of understanding
the water-splitting mechanism. Additionally, how the protein environment
cooperates with this catalytic reaction by providing the environment
for stabilizing the wide range of Mn redox states, reassembling the
cluster with two water molecules, and providing a pathway for protons,
water, and oxygen are the key questions for understanding the mechanism
of thismetalloenzyme. Recent development of femtosecond XRD[58] with its “collect before destroy”
approach opens a new way of studying the structural dynamics of enzyme
systems under functional conditions at ambient conditions. The very
short, intense fs pulses make it possible to outrun damage and collect
data. This method also makes simultaneous data collection of XRD and
X-ray spectroscopy possible as shown in Figure 33.
Figure 33
Schematic of the simultaneous detection
of X-ray diffraction and
X-ray emission spectra of photosystem II crystals using the femtosecond
pulses from a X-ray free electron laser (XFEL) at room temperature.
The ultrashort, intense pulses from the XFEL allow one to collect
data at room temperature without radiation damage, thus opening up
possibilities for conducting time-resolved studies. The crystal suspension
is injected using a microjet that intersects the X-ray pulses. XRD
data from a single crystal are collected downstream, and XES data
from the same crystal are collected at ∼90° to the beam
using an XES spectrometer and a position-sensitive detector. A visible
laser (527 nm) is used to illuminate the crystals to advance the PS
II crystals through the S-states.
Simultaneous data collection of XRD and X-ray spectroscopy
has
several important advantages; the method probes the overall protein
structure from XRD and the electronic structure of the n class="Chemical">Mn4CaO5 cluster in the oxygen-evolving complex from the spectroscopic
data using the same samples under the same conditions. This eliminates
the controversy that often exists between the spectroscopic and XRD
results due to the different experimental conditions and the different
sensitivity and error of each method. The use of the X-ray free electron
laser (XFEL) pulses is critical for this approach.
Among the
various spectroscopic methods, nonresonant XES probes
occupied electronic levels. In particular, the Kβ1,3 and Kβ′ lines are a probe of the number of unpaired
3d electrons, hence providing information about the oxidation and/or
spin state. Experimentally, XES using an energy-dispern class="Chemical">sive X-ray spectrometer[230] is well-suited for such combined shot-by-shot
studies, as excitation energies above the 1s core hole of first-row
transition metals are also ideal for XRD, and therefore neither incident
nor emitted photon energy have to be scanned.[231]
Simultaneous XRD/XES data were collected from microcrystals
of
PS II. Figure 34 shon class="Chemical">ws
the electron density map of PS II isolated from the thermophilic cyanobacterium T. elongatus, collected at Linac Coherent Light Source (LCLS)
using the single-shot approach. The data is comparable to that obtained
using synchrotron radiation studies as seen by the similarities in
the overall structure of the helices, the protein subunits, and the
location of the various cofactors.
Figure 34
PS II structure from diffraction of micrometer-sized crystals using
∼50 fs X-ray pulses at room temperature at the XFEL. (a) Electron
density map, 2mFo-DFc, for the PS II in the S1 state shown in yellow,
and the electron density contoured at 1.2 σ (blue mesh) shown
for a radius of 5 Å around the protein. (b) Detail of the same
map in the area of the Mn4CaO5 cluster in the
dark S1 state, with mesh contoured at 1.0 σ (gray)
and 4.0 σ (blue). Selected residues from subunit D1 are labeled
for orientation; Mn is shown as violet spheres, and Ca is shown as
as an orange sphere (metal positions taken from pdb file 3bz1). (c) (Left) Kβ1,3 X-ray emission spectra from crystals (red dashed) and solutions
(green) of PS II at room temperature using the XFEL. (Right) Kβ1,3 X-ray emission spectra of PS II solutions in the S1 state collected at the XFEL at room temperature (green),
spectra collected using synchrotron radiation under cryogenic conditions
with low dose (“8K intact”, light blue) and using synchrotron
radiation at room temperature under high-dose conditions (“RT
damaged”, pink). The spectrum from MnIICl2 in aqueous solution collected at room temperature is shown (gray)
for comparison. The XES spectra show that the Mn4CaO5 cluster is intact in the crystals under the conditions of
the XFEL experiment. Adapted from ref (62).
Schematic of the simultaneous detection
of X-ray diffraction and
X-ray emisn class="Chemical">sion spectra of photosystem II crystals using the femtosecond
pulses from a X-ray free electron laser (XFEL) at room temperature.
The ultrashort, intense pulses from the XFEL allow one to collect
data at room temperature without radiation damage, thus opening up
possibilities for conducting time-resolved studies. The crystal suspension
is injected using a microjet that intersects the X-ray pulses. XRD
data from a single crystal are collected downstream, and XES data
from the same crystal are collected at ∼90° to the beam
using an XES spectrometer and a position-sensitive detector. A visible
laser (527 nm) is used to illuminate the crystals to advance the PS
II crystals through the S-states.
PS II structure from diffraction of micrometer-sized crystals un class="Chemical">sing
∼50 fs X-ray pulses at room temperature at the XFEL. (a) Electron
density map, 2mFo-DFc, for the PS II in the S1 state shown in yellow,
and the electron density contoured at 1.2 σ (blue mesh) shown
for a radius of 5 Å around the protein. (b) Detail of the same
map in the area of the Mn4CaO5 cluster in the
dark S1 state, with mesh contoured at 1.0 σ (gray)
and 4.0 σ (blue). Selected residues from subunit D1 are labeled
for orientation; Mn is shown as violet spheres, and Ca is shown as
as an orange sphere (metal positions taken from pdb file 3bz1). (c) (Left) Kβ1,3 X-ray emission spectra from crystals (red dashed) and solutions
(green) of PS II at room temperature using the XFEL. (Right) Kβ1,3 X-ray emission spectra of PS II solutions in the S1 state collected at the XFEL at room temperature (green),
spectra collected using synchrotron radiation under cryogenic conditions
with low dose (“8K intact”, light blue) and using synchrotron
radiation at room temperature under high-dose conditions (“RT
damaged”, pink). The spectrum from MnIICl2 in aqueous solution collected at room temperature is shown (gray)
for comparison. The XES spectra show that the Mn4CaO5 cluster is intact in the crystals under the conditions of
the XFEL experiment. Adapted from ref (62).
At the published resolution of 5.7 Å, there are no large
protein
structural changes taking place between the S1 and S2 states.[61,62] The isomorphous difference map
computed between the dark and illuminated data set, contoured at ±3.0
σ, showed no n class="Chemical">significant peaks above the noise level in the
region of the Mn4CaO5 cluster and the stromal
electron-acceptor side of the complex. Although the S1–S2 transition is accompanied by a number of changes in carboxylate
and backbone vibration frequencies as detected by infrared spectroscopy,[31,152,232] the associated structural changes
are most likely too small to be detected by the resolution achieved
in the present study of ∼5 Å (Figure 35).
Figure 35
First data from the S1 and illuminated S2 state using the femtosecond pulses from the XFEL. This experiment
shows that time-resolved crystallography of PS II at room temperature
is possible. Isomorphous difference map between the XFEL-illuminated
(S2 state) and the XFEL-dark (S1 state) XRD
data set in the region of the Mn4CaO5 cluster,
with F–F difference contours shown
at +3 s (green) and −3 s (red); analysis indicates that this
map is statistically featureless showing that at this resolution there
are no major changes in the protein or the metal ion positions. Metal
ions of the Mn4CaO5 cluster are shown for orientation
as violet (Mn) and orange (Ca) spheres; subunits are indicated in
yellow (D1), orange (D2), pink (CP43), and green (PsbO). Adapted from
ref (62).
First data from the S1 and illuminated S2 state using the n class="Chemical">femtosecond pulses from the XFEL. This experiment
shows that time-resolved crystallography of PS II at room temperature
is possible. Isomorphous difference map between the XFEL-illuminated
(S2 state) and the XFEL-dark (S1 state) XRD
data set in the region of the Mn4CaO5 cluster,
with F–F difference contours shown
at +3 s (green) and −3 s (red); analysis indicates that this
map is statistically featureless showing that at this resolution there
are no major changes in the protein or the metal ion positions. Metal
ions of the Mn4CaO5 cluster are shown for orientation
as violet (Mn) and orange (Ca) spheres; subunits are indicated in
yellow (D1), orange (D2), pink (CP43), and green (PsbO). Adapted from
ref (62).
This technique can be used for future time-resolved
studies of
light-driven structural changes within proteins and cofactors, and
of chemical dynamics at the catalytic n class="Chemical">metal center under functional
conditions.
Conclusions
Since the inn class="Chemical">sightful hypothesis
of the S-state intermediates in
the process of the water oxidation reaction by Bessel Kok in the 1970s,
which were based on the kinetics of dioxygen release as a function
of flash number, we are now in a position to look into the mechanism
of the catalytic machine. One of the major advances has been the developments
in the field of X-ray crystallography that have now revealed the structure
of PS II and the Mn4CaO5 cluster at very high
resolution. In conjunction, the developments in X-ray spectroscopy
techniques have contributed to revealing both the geometric and electronic
structure of the Mn4CaO5 cluster, not only in
the native dark state but also in the other intermediate states. EPR
and FTIR spectroscopy have also been instrumental in advances that
have led to mapping the geometric structures obtained from X-ray spectroscopy
and crystallography on to the electronic structures of the native
and intermediate states. All of above techniques are taking advantage
of the biochemical advancements that have been able to isolate and
characterize ever purer PS II solution and crystal samples. We also
note the importance of the availability of appropriate inorganic model
complexes that are critical for the interpretation of the spectroscopic
data as well as theoretical developments.
In concluding our
last review in Bioinorganic Enzymology
I, we noted some of the questions about the OEC that needed
to be answered. Some of these, especially the question about what
is the structure of the Mn4CaO5 cluster in the
dark native state, may be approaching final resolution. However, there
are some critical questions that are still open. What is the structural
or mechanistic role of n class="Chemical">Ca2+ and how is Cl– involved in maintaining a high level of activity? What are the oxidation
states and the structure of the Mn cluster in the transient S4 state? How are the oxygen atoms coupled to form an O–O
bond? In spite of many interesting leads and studies, the S3 → (S4) → S0 process has not
yet been understood in detail. These questions, along with the need
to characterize the proposed transient S4 state and other
kinetic intermediates that may be present, offer challenges for the
future; they need to be addressed before we can fully understand where
and how plants oxidize water to dioxygen.
Finally, the studies
in the natural photosynthetic water oxidation
reaction have had a profound influence and inspiration on the development
of artificial n class="Chemical">water-oxidizing catalysts. Given the vision of a renewable
energy economy, understanding the design principles of the natural
water oxidation reaction could be of great practical use to create
an artificial photosynthetic device and for developing cleaner, renewable
carbon-neutral energy sources.
Authors: Jena E Johnson; Samuel M Webb; Katherine Thomas; Shuhei Ono; Joseph L Kirschvink; Woodward W Fischer Journal: Proc Natl Acad Sci U S A Date: 2013-06-24 Impact factor: 11.205
Authors: Heui Beom Lee; Angela A Shiau; Paul H Oyala; David A Marchiori; Sheraz Gul; Ruchira Chatterjee; Junko Yano; R David Britt; Theodor Agapie Journal: J Am Chem Soc Date: 2018-11-30 Impact factor: 15.419
Authors: Thomas Fransson; Ruchira Chatterjee; Franklin D Fuller; Sheraz Gul; Clemens Weninger; Dimosthenis Sokaras; Thomas Kroll; Roberto Alonso-Mori; Uwe Bergmann; Jan Kern; Vittal K Yachandra; Junko Yano Journal: Biochemistry Date: 2018-06-28 Impact factor: 3.162
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
Figure 15
Figure 16
Figure 17
Figure 18
Figure 19
Figure 20
Figure 21
Figure 22
Figure 23
Figure 27
Figure 24
Figure 25
Figure 26
Figure 28
Figure 29
Figure 30
Figure 31
Figure 32
Figure 33
Figure 34
Figure 35