Broken Symmetry DFT Calculations/Analysis for Oxidized and Reduced Dinuclear Center in Cytochrome c Oxidase: Relating Structures, Protonation States, Energies, and Mössbauer Properties in ba3 Thermus thermophilus.

The Fea3(3+)···CuB(2+) dinuclear center (DNC) structure of the as-isolated oxidized ba3 cytochrome c oxidase (CcO) from Thermus thermophilus (Tt) is still not fully understood. When the proteins are initially crystallized in the oxidized state, they typically become radiolyticly reduced through X-ray irradiation. Several X-ray crystal structures of reduced ba3 CcO from Tt are available. However, depending on whether the crystals were prepared in a lipidic cubic phase environment or in detergent micelles, and whether the CcO's were chemically or radiolyticly reduced, the X-ray diffraction analysis of the crystals showed different Fea3(2+)···CuB(+) DNC structures. On the other hand, Mössbauer spectroscopic experiments on reduced and oxidized ba3 CcOs from Tt (Zimmermann et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5779-5783) revealed multiple (57)Fea3(2+) and (57)Fea3(3+) components. Moreover, one of the (57)Fea3(3+) components observed at 4.2 K transformed from a proposed "low-spin" state to a different high-spin species when the temperature was increased above 190 K, whereas the other high-spin (57)Fea3(3+) component remained unchanged. In the current Article, in order to understand the heterogeneities of the DNC in both Mössbauer spectra and X-ray crystal structures, the spin crossover of one of the (57)Fea3(3+) components, and how the coordination and spin states of the Fea3(3+/2+) and Cu(2+/1+) sites relate to the heterogeneity of the DNC structures, we have applied density functional OLYP calculations to the DNC clusters established based on the different X-ray crystal structures of ba3 CcO from Tt. As a result, specific oxidized and reduced DNC structures related to the observed Mössbauer spectra and to spectral changes with temperature have been proposed. Our calculations also show that, in certain intermediate states, the His233 and His283 ligand side chains may dissociate from the CuB(+) site, and they may become potential proton loading sites during the catalytic cycle.

Introduction

Located in the inner mitochondrial membrane in eukaryotes or alternatively in the periplasmic membrane in aerobic bacteria, cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory chain that reduces O2 to H2O and pumps protons across the membrane.[1−4] The catalytic site of CcO, which binds and reduces O2, contains a heme A (Fea3) and a Cu ion (CuB), forming the dinuclear (or binuclear) center (DNC or BNC). CuB is in the proximity (∼5 Å) of Fea3.[5−15] In addition, CcO protein contains another two redox centers: a homodinuclear Cu dimer (CuA), which serves as the initial site of electron entry to CcO,[16,17] and another heme, which is heme A (Fea) in the case of the aa3 type of CcO or heme B (Feb) in the ba3 type.

Zimmermann et al. reported Mössbauer spectra of the as-isolated oxidized and reduced ba3 CcO from Thermus thermophilus (Tt).[18] In the reduced state, two high-spin ferrous components (at 4.2 K) were observed for the Fea3 site with the majority component (86%) having an isomer shift δ = 0.95 mm s–1 and quadrupole splitting ΔEQ = 1.43 mm s–1 and a minority species (14%) with δ = 0.92 mm s–1 and ΔEQ = 2.34 mm s–1.[18] The oxidized state was even more complicated. When the temperature was between 4.2 and 190 K, the Mössbauer spectra revealed two components attributable to 57Fea33+: a quadrupole doublet with splitting ΔEQ = 0.71 mm s–1 and δ = 0.41 mm s–1, which was proposed to be a high-spin (HS) ferric heme and accounts for ∼46% of 57Fea33+, and a second doublet (54%) with ΔEQ = 2.24 mm s–1 and δ = 0.29 mm s–1, which was proposed to be a low-spin (LS) ferric heme.[18] When the temperature was >190 K, the “low-spin” 57Fea33+ component was reported to undergo a transition to “high-spin” that was essentially complete at 245 K. However, the new “high-spin” compound yielded ΔEQ ≈ 1 mm s–1, and the two distinct “high-spin” species (ΔEQ = 0.7 and ∼1 mm s–1) coexisted at 245 K.[18] For easier comparison, we assembled the 57Fea33+/2+ Mössbauer experimental data of ba3 CcO from Tt in Figure .

Figure 1

Data summarized from ref (18). The observed 57Fea32+ and 57Fea33+ Mössbauer isomer shifts (δ, mm s–1) and quadrupole splittings (ΔEQ, mm s–1) for ba3 CcO from Tt and the spectra variation with temperature (T).[18] Note that only ΔEQ ≈ 1 mm s–1 was given in ref (18). for the third 57Fea33+ species (HS state), which was transformed from the second 57Fea33+ species (supposed to be in LS state) as the temperature increased to >190 K, and the transition was complete at 245 K. “nd” stands for “not determined from experiment”.

Currently, detailed DNC structures and comparative energies in the as-isolated, fully oxidized, and fully reduced states of CcO are still not clear.[1−3,6,7,10−15,19−22] Although crystals of the fully oxidized as-isolated CcOs were well prepared, when exposed to X-ray irradiation, the metal sites are easily reduced.[12,13,15] Several reduced X-ray crystal structures of ba3 CcO from Tt have been obtained.[10−12,15] However, depending on whether the crystals were prepared in a lipidic cubic phase environment or in detergent micelles, and whether the CcOs were chemically or radiolyticly reduced, X-ray diffraction analyses of the crystals obtained from different protocols show different DNC structures.[12,15] The PDB entries of these Tt X-ray structures are 1EHK,[10]1XME,[11]3EH3,[12]3EH4,[12]3EH5,[12]3S8F,[15] and 3S8G,[15] in which both 3S8F (wild-type) and 3S8G (A120F mutant) were obtained in a lipidic cubic phase environment and others in detergent micelles. 1EHK, 1XME, 3EH4, 3S8F, and 3S8G are radiation-reduced crystal structures. 3EH3 and 3EH5 represent chemically reduced X-ray crystal structures. However, 3EH3 was obtained by chemically reducing the crystals of the oxidized, as-isolated protein, and 3EH5 was obtained by a path in which the oxidized as-isolated proteins were reduced with dithionite in an anaerobic environment prior to being crystallized and frozen in liquid nitrogen.[12]

The resolutions and the main geometric parameters in the DNCs from these X-ray structures are compared in Table . As an example, the DNC of 1EHK is shown in Figure . In the observed structures, the Fea3 site has one axial histidine ligand (His384), and the CuB site has three histidine ligands: His233, His282, and His283. His233 covalently links with Tyr237, a linkage that is common to all CcOs but is otherwise unknown in metalloenzymes. These structures obviously vary in the Fea3···CuB distances, in what resides in the space between the Fea3 and CuB sites, and in the Fea3–ligand distances. Because only the DNC of CcO is studied in the current Article, for simplicity, the Fea3 and CuB will be noted as Fe and Cu hereafter. An open Fe2+···Cu+ (with no atoms between Fe2+ and Cu+) DNC structure was obtained in 3EH3 and 3EH5.[12] A bridging oxygen species was observed between the Fe2+ and Cu+ sites in 1EHK (Figure ),[10] where the His384 side chain was found to be far from the Fe atom with an Fe···N(His384) distance of 3.31 Å. In 1XME(11) and 3EH4,[12] an oxygen species is also found in the space between Fe2+ and Cu+, but it appears bound only to the Cu+ site, and the axial His384 side chain ligand is much closer to the Fe2+ site. Although the bound solvent was called “the water molecule residing between Fe and Cu” in later publications,[12] it is not evident whether this oxygen species in the DNC’s of 1EHK, 1XME, and 3EH4 is a water molecule (H2O) or a hydroxide anion (OH–).[10]

Table 1

Resolutions (Å) and the Main DNC Geometrical Parameters (Å) of the Reduced X-ray Crystal Structures of ba3 CcO from Tt and the Experimental 57Fea32+ Mössbauer Isomer Shifts (δ, mm s–1) and Quadrupole Splittings (ΔEQ, mm s–1) for the Reduced DNC in Different Cytochrome Oxidase Proteins

crystal structure	resolution	Fe–N (H384)	Fe–O/Fe–O1	O1–O2	Cu–O/Cu–O2	Fe···Cu	Cu–N (H233)	Cu–N (H282)	Cu–N (H283)	O···O (Y237)	ref
1EHK	2.4	3.31	2.26		2.31	4.40	2.10	2.22	2.13	2.52	(10)
1XME	2.3	2.48	2.44		2.07	4.39	1.96	1.96	1.97	2.67	(11)
3EH4	2.9	2.39	3.08		2.20	4.73	1.96	2.03	1.97	2.47	(12)
3EH3	3.1	2.39				5.06	1.92	2.05	2.06	2.91	(12)
3EH5	2.8	2.10				5.04	2.16	1.94	2.18	2.61	(12)
3S8F	1.8	2.09	2.25	1.54	2.29	4.87	1.94	1.99	1.91	2.56	(15)
3S8G	1.8	2.22	2.39	1.52	2.25	4.92	1.95	2.03	1.98	2.66	(15)

Figure 2

Fe–Cu DNC of the X-ray radiolyticly reduced crystal structure 1EHK (2.4 Å resolution),[10] which was obtained for the native Tt ba3 CcO solubilized in detergent micelles. The protonation state of the Tyr237 side chain and the oxygen species between the Fe and Cu sites in the DNC are not known from the X-ray crystal structure alone. A water molecule (“HOH” in red) was added based on other X-ray structures.

Further, a peroxide-type dioxygen species bridging the Fe2+···Cu+ DNC was observed in both 3S8F and 3S8G.[15] To evaluate whether this bridging dioxygen species is O22–, O2·–, HO2–, or H2O2, in ref (23) we performed a series of density functional theory (DFT) calculations (using the OLYP[24,25] functional) on the quantum cluster models based on 3S8G DNC and have proposed that the X-ray crystal structure 3S8G is likely to represent the superposition of the Fe2+-(HO2–)-Cu+ DNC’s in different states (Fe2+ in low-spin, intermediate-spin, or high-spin) with the majority species having the proton of the HO2– residing on the oxygen atom that is closer to the Fe2+ site in the Fe2+-(HO-O)−-Cu+ conformation.[23]

Clearly, the X-ray crystal structures and the Mössbauer experiments show structural heterogeneity within the DNC of ba3 CcO from Tt. Relating the detailed structures of the oxidized and reduced DNC’s is an essential step in analyzing the path(s) by which CcO reduces O2 to H2O and in finding what paths effectively couple O2 reduction chemistry to proton pumping across the membrane. In the current Article, we continue our DFT calculations on DNC models starting from different X-ray crystal structures to address the following questions: (1) Is the bridging/Cu-binding oxygen species observed in the DNC’s of 1EHK, 1XME, and 3EH4 an H2O molecule or an OH– anion? (2) How do the axial His384 and nearby solvent ligands influence the 57Fe2+ Mössbauer properties? (3) What are the DNC structures representing the two 57Fea32+ components observed in Mössbauer experiments, and what do the DFT calculations predict for Fe2+ spin states and their relative energies in the DNC? (4) Will the geometry optimized 3S8G-Fe2+-(HO2–)-Cu+ DNC structures obtained in ref (23) reproduce the Mössbauer properties of any of the two 57Fea32+ components? (5) What are the feasible DNC structures of the three 57Fea33+ components observed in the Mössbauer experiments? (6) How does spin-crossover link different states and structures in the transition of Fe3+(state 2) → Fe3+(state 3) when the temperature is increased from 190 to 245 K (Figure )? (7) What do the comparative structures and energies tell us about potential reaction pathways and protonation states (especially for Tyr237) in this early part of the catalytic cycle?

Models and Calculational Methods

Several water molecules are normally found above the DNC that have H-bonding interactions with the two propionate carboxylate groups (prop-A and prop-D) and the side chains of Arg449 and Asp372. In many CcO X-ray crystal structures,[6,11−15] a water molecule (“HOH” in red) was observed in a position to have hydrogen bonding interactions with both prop-A and prop-D and also with the His283 side chain (Figure ). However, this water molecule was not present in the 1EHK.pdb file. We are not sure if this water molecule missed detection in 1EHK or if it is displaced (a water molecule HOH941 above this position was observed in 1EHK). To be consistent with the calculations for the DNC clusters of other X-ray crystal structures, we also placed a water molecule in this position (HOH) for the 1EHK DNC calculations.

The O···O distance between the carboxylate groups of Prop-A and Asp372 side chain in 1EHK is only 2.49 Å. Therefore, the Asp372 side chain is likely in the neutral protonated state and has a H-bonding interaction with the anionic carboxylate group of prop-A.[17] Prop-A also H-bonds to HNδ1 of His376 side chain with an O···Nδ1 distance of 2.90 Å. Nε2 (near the top of Figure ) of the His376 side chain is within a strong H-bonding distance (2.68 Å) with one of the oxygen atoms of the carboxylate group of Glu126B side chain (not shown in Figure ). Therefore, the His376 side chain is likely in the neutral state, and its Nε2 site H-bonds to the neutral protonated Glu126B side chain.

The initial geometries of our DNC models are taken from the different X-ray crystal structures. The size of the model clusters taken from 1EHK, 3EH3, 3EH4, and 3EH5 is the same as shown in Figure (but with different species between the Fe and Cu sites). No water molecules were reported in 3EH3.pdb, we therefore manually added the three water molecules above the DNC in that structure according to their relative positions in 3EH5.pdb. The ending Cα/Cγ/Cgeranyl-side chain atoms shown in Figure were each replaced by a link hydrogen (Hlink) atom along the original C–C direction with the C–Hlink distance of 1.09 Å. The positions of the Hlink atoms that replace the Cα/Cγ atoms were fixed during all geometry optimization calculations.

Additional crystallographic water molecules near the DNC were observed in 3S8G and 3S8F compared to the other structures. The size of the model cluster taken from 3S8G, which was used in both ref (23) and in the current study is a little larger than that in Figure by including the Gly232 residue and four more water molecules that are in the positions of HOH604, HOH608, HOH607, and HOH610 in 3S8G (see Figures 1 and 2 of ref (23)).

On the basis of the calculations for relative spin-state energetics of Fe2+ and Fe3+ heme models performed by Vancoillie et al.,[26] none of the tested density functionals (B3LYP, B3LYP*, OLYP, BP86, TPSS, TPSSh, M06, or M06-L) consistently provide better accuracy than CASPT2 (multiconfigurational perturbation theory) for all their model complexes against available high-level coupled cluster singles and doubles (CCSD) results. However, the pure functional OLYP yields similar results to the hybrid functionals B3LYP* and B3LYP. And for their large heme models, the results of OLYP, B3LYP, and B3LYP* are reasonably close to the best estimate of the spin-splittings with errors typically ≤6 kcal mol–1.[26] Radoń and Pierloot also investigated the performance of the CASSCF/CASPT2 approach and several DFT functionals (PBE0, B3LYP, BP86, and OLYP) in calculating the bonding of CO, NO, and O2 molecules to two model heme systems.[27] They found that the experimentally available binding energies are best reproduced by the CASPT2 method and with the OLYP functional. The CASSCF spin populations most closely correspond to the results obtained with the pure OLYP or BP86 rather than with the hybrid functionals.[27] Therefore, we used the OLYP functional in studying the 3S8G-DNC[23] geometric and energetic properties and will continue using this functional in the current study.

The DNC model geometries were optimized using the Amsterdam Density Functional Package (ADF2012.01)[28−30] with integration grid accuracy parameter 4.0 within the conductor-like screening (COSMO) solvation model.[31−34] Because both the cluster and the surrounding protein environment are quite polar and contain many water molecules, to be consistent with refs (17) and (23), a large dielectric constant of a simple ketone (ε = 18.5) was applied to the environment in all COSMO calculations. The van der Waals radii of 1.5, 1.4, 1.7, 1.52, 1.55, and 1.2 Å were used for atoms Fe, Cu, C, O, N, and H, respectively.[17,23] During geometry optimizations, the all-electron triple-ζ plus polarization (TZP) Slater-type basis set was applied for the major atoms at the DNC, including Fe3+/2+, Cu2+/1+, the oxygen species (H2O or OH–) between Fe3+/2+ and Cu2+/1+, and all the N atoms binding with Fe3+/2+/Cu2+/1+, and the double-ζ plus polarization (DZP) basis set with frozen cores of C(1s), N(1s), and O(1s) was applied to the rest of the atoms.

The Fe2+ and Fe3+ sites may exist in one of the three spin states: low-spin (LS) with SFe2+ = 0 and SFe3+ = 1/2, intermediate-spin (IS) with SFe2+ = 1 and SFe3+ = 3/2, or high-spin (HS) with SFe2+ = 2 and SFe = 5/2. The Cu+ has spin SCu+ = 0, and Cu2+ has SCu2+ = 1/2. Further, the Fe3+ site may couple with the Cu2+ site ferromagnetically (F) with a total spin Stotal = SFe3+ + SCu2+ or antiferromagnetically (AF) with Stotal = SFe3+ – SCu2+. The AF-coupling spin state cannot be obtained directly from the normal DFT calculations. As in previous work,[23,35−42] we represent the AF spin-coupled state in DFT by a “broken-symmetry” (BS) state,[43−45] where a spin-unrestricted determinant is constructed in which the Fe3+ site has spin-up electrons as majority spin and the Cu2+ site has majority spin-down electrons.

After geometry optimizations, to obtain more accurate energies (which will be reported in the Results section) and the Mössbauer isomer shift and quadrupole splitting properties, we applied a single-point energy calculation at each optimized geometry using an all-electron/all-TZP basis set to all atoms within the COSMO solvation model. Our own program is then used to calculate the electron density ρ(0) at the Fe nuclei.[40,46,47] The Mössbauer isomer shift δ is calculated based on ρ(0)

A is a constant and taken as 11877. In our previous study,[40] α = −0.328, C = 0.622 mm s–1 for 57Fe2+ and α = −0.307, C = 0.385 mm s–1 for 57Fe3+ were obtained from linear regression between the measured isomer shifts and OLYP-calculated electron densities at Fe nuclei of 17 Fe2+,2.5+ complexes (31 total Fe sites) and 19 Fe2.5+,3+,3.5+,4+ complexes (30 total Fe sites), respectively.

The electric field gradient (EFG) at the Fe nucleus obtained from the all-electron/all-TZP single-point energy calculation is used to calculate the Mössbauer quadrupole splitting (ΔEQ) property. Normally, the EFG tensors V are diagonalized and the eigenvalues are reordered such that |V| ≥ |V| ≥ |V|. The asymmetry parameter η is defined as

Then, the ΔEQ for 57Fe of the nuclear excited state (I = 3/2) can be calculated aswhere e is the electrical charge of a positive electron, and Q is the nuclear quadrupole moment of Fe. Recently, the ADF software package determines the ΔEQ value using eQ = 0.16 electron-barn.[48] We will report the ADF-calculated ΔEQ values in the current study.

A few comments outlining how different state energies are compared should prove helpful. Using the ADF code, state energies are defined with respect to a sum of reference fragment energies (reference atoms in the present case). Then, these electronic energies E can be directly compared when the states have the same atom compositions (types and numbers of atoms). Such states are isomers (tautomers). When there are proton transfers between tautomers, ΔE can be defined by adding the ΔZPE difference between states (see below). Further, when considering net deprotonation/protonation, the H+ reference energy in the gas phase and in aqueous solution must be included, as discussed next. Systems differing in the total number of water molecules cannot be directly compared in energies because an H2O (in bulk aqueous solvent) reference energy is also needed. This H2O gas phase and H2O in solvent energy reference was presented in our earlier works (refs (17) and (42)) but is not a focus in this paper.

To determine the protonation state of the oxygen species residing between the Fe and Cu sites and of the Tyr237 side chain, we performed pKa calculations for certain states using[23,42]where E(A–) and E(AH) are the calculated energies of the deprotonated and protonated states, ΔGsol(H+,1 atm) is the solvation free energy of a proton at 1 atm pressure. We use the “best available” experimental value of −264.0 kcal mol–1 for this term based on analysis of cluster-ion solvation data.[49−51] Unlike in ref (23), where E(H+) was calculated as the energy of a proton (in gas-phase with OLYP potential) with respect to a spin-restricted hydrogen atom, here, we take the empirically corrected E(H+) = 12.71 eV = 293.1 kcal mol–1 based on experimental standard hydrogen electrode energy and the proton solvation free energy (see appendix in ref (42)). The translational entropy contribution to the gas-phase free energy of a proton is taken as −TΔSgas(H+) = −7.8 kcal mol–1 at 298 K and 1 atm pressure.[52] (5/2)RT = 1.5 kcal mol–1 includes the proton translational energy (3/2)RT and PV = RT.[52] The term ΔZPE is the zero point energy difference for the deprotonated state (A–) minus the protonated state (AH), and it was estimated as ΔZPE = −7.6 and −7.9 kcal mol–1 for the OH–/H2O and the Tyr237–/Tyr237 systems, respectively, by only optimizing the geometries (and then performing frequency calculations) of an OH– and an H2O molecule and the linked His233–Tyr237 side chains in both neutral and anionic His233–Tyr237– forms within the COSMO solvation model (with ε = 18.5 and all-electron/all-TZP basis set). Similarly, we also obtained ΔZPE = −8.1 kcal mol–1 for the His376 side chain to estimate its pKa in section .

Results and Discussion of Fe2+/Cu+ Reduced State DNC Model Calculations

Fe2+···Cu+ DNC Calculations—Models Taken from 3EH5, 3EH3, and 1EHK. Feasible Structure of the Second Observed 57Fea32+ Component?

No substantial electron density was observed directly between the Fe and Cu sites in the DNC’s of the chemically reduced X-ray crystal structures of 3EH5 and 3EH3.[12] To see if such a DNC will yield the Mössbauer properties observed for the reduced Tt ba3 CcO, we first built up our Fe2+···Cu+ DNC model (similar to Figure , but without the oxygen species between the Fe and Cu sites) based on the 3EH5 Cartesian coordinates. Both the Fe2+···Cu+(Y237) (180 atoms with neutral Tyr237 side chain) and Fe2+···Cu+(Y237–) (179 atoms with deprotonated anionic Tyr237– side chain) clusters were geometry optimized with Fe2+ in LS, IS, and HS states. The calculated properties of these DNC models, including the main geometric parameters (Å), energies (E, off set by −25100 kcal mol–1), Mulliken net spin polarizations on Fe2+ and Cu+, and the 57Fe2+ Mössbauer isomer shifts (δ) and quadrupole splittings (ΔEQ) are given in Table (in the 3EH5 section).

Table 2

Calculated Properties for the Reduced Fe2+···Cu+(Y237/Y237–) DNC Models Generated Starting from the DNCs of 3EH5, 3EH3, and 1EHK X-ray Crystal Structuresa

		geometry									net spin		57Fe2+ Mössbauer
starting structure	modelsb	Fe–N (H384)	Fe···Cu	Cu–N (H233)	Cu–N (H282)	Cu–N (H283)	O···O (Y237)	E	pKa (Y237)	Q	Fe2+	Cu+	δ	ΔEQ
3EH5	Fe2+,LS···Cu+(Y237)	1.96	5.74	1.99	2.08	2.03	3.34	–41.8	7.0	0	0.00	0.00	0.64	2.43
	Fe2+,IS···Cu+(Y237)	2.38	5.48	2.00	2.07	2.04	3.43	–50.3	9.0	0	2.31	0.00	0.73	2.02
	Fe2+,HS···Cu+(Y237)	2.22	5.62	2.00	2.08	2.04	3.33	–49.6	7.8	0	3.90	0.00	0.92	2.21
	Fe2+,LS···Cu+(Y237–)	1.95	5.82	1.99	2.08	2.04	2.82	–47.1		–1	0.00	0.00	0.64	2.41
	Fe2+,IS···Cu+(Y237–)	2.34	5.55	1.99	2.08	2.04	2.80	–52.9		–1	2.32	0.00	0.73	2.08
	Fe2+,HS···Cu+(Y237–)	2.21	5.73	1.99	2.09	2.04	2.80	–53.8		–1	3.91	0.00	0.91	2.16
3EH3c	Fe2+,HS···Cu+(Y237–)	2.39	5.06	2.03	2.28	1.99	2.75	–38.6		–1	3.85	0.00	0.92	2.44
1EHKd	Fe2+,HS···Cu+(Y237–)(1)	3.00	5.09	2.03	2.08	2.03	2.67	–31.5		–1	3.86	0.00	0.90	2.90
	Fe2+,HS···Cu+(Y237–)(2)	2.71	5.36	2.02	2.10	2.03	2.68	–37.8		–1	3.87	0.00	0.94	2.67
	Fe2+,HS···Cu+(Y237–)(3)	2.45	5.59	2.02	2.11	2.04	2.70	–42.4		–1	3.88	0.00	0.96	2.43
	Fe2+,HS···Cu+(Y237–)(4)	2.33	5.74	2.02	2.10	2.04	2.70	–44.4		–1	3.89	0.00	0.96	2.34

The properties include geometries (Å), electronic energies (E, offset by −25100 kcal mol–1) obtained from the all-electron/all-TZP single-point energy calculations on the optimized geometries, pKa’s, the net charge (Q) of the clusters, Mulliken net spin polarizations for the Fe and Cu sites, and the 57Fe Mössbauer isomer shifts (δ, mm s–1) and quadrupole splittings (ΔEQ, mm s–1).

LS, low-spin; IS, intermediate-spin; HS, high-spin.

The Cartesian coordinates of N(His384), Fe, and Cu were fixed during geometry optimization.

Here, we deleted the bridging oxygen atom from the 1EHK structure, and geometry optimized the DNC cluster to see how the Fe···N(His384) distance and the Mössbauer properties will change. The Fe2+,HS···Cu+(Y237–)(1), Fe2+,HS···Cu+(Y237–)(2), and Fe2+,HS···Cu+(Y237–)(3) are three points along the geometry optimization pathway. Fe2+,HS···Cu+(Y237–)(4) is the lowest energy structure obtained.

For both the Fe2+···Cu+(Y237) and Fe2+···Cu+(Y237–) cases, the Fe2+,HS and Fe2+,IS states yield similar electronic energies, which are much lower than the corresponding Fe2+,LS state energy. Further, the calculated Mössbauer properties of δ = 0.92 mm s–1 and ΔEQ = 2.21 mm s–1 for the Fe2+,HS···Cu+(Y237) state and δ = 0.91 mm s–1 and ΔEQ = 2.16 mm s–1 for the Fe2+,HS···Cu+(Y237–) state agree very well with the observed Mössbauer properties (δ = 0.92 mm s–1, ΔEQ = 2.34 mm s–1) of the second 57Fea32+ component (Figure ). Conversely, the calculated Mössbauer isomer shifts (0.73 mm s–1) for both the 57Fe2+,IS sites are too small compared with the observed δ = 0.92/0.95 mm s–1 values. Therefore, our Mössbauer property calculations support that the 57Fea32+ species observed in the Mössbauer experiments are in high-spin states. The OLYP potential may underestimate the energy gap between the Fe2+,IS and Fe2+,HS states. However, although not included in the present calculations, both vibrational entropy and vibrational zero-point-energy terms most likely favor the Fe2+,HS state over the Fe2+,IS state. The Fe2+ site of the reduced DNC of ba3 CcO in Tt then most likely exists only in the high-spin state at the Thermus optimum growth temperature of 50–80 °C.[53] We will therefore only report the results of the Fe2+,HS state for the rest of the reduced-DNC calculations in the current study.

For comparison, the experimental Mössbauer δ and ΔEQ values for the reduced 57Fea32+ sites in c1aa3 from Tt(54) and aa3 from a bovine heart[55] are also given in Table . The observed δ values for all Fea32+ sites are similar (0.92–0.96 mm s–1) and are >0.9 mm s–1, indicating they all are in the high-spin state. The ΔEQ value for the 57Fea32+ in c1aa3Tt (2.05 mm s–1) is close to the second ΔEQ (2.34 mm s–1) observed in ba3Tt. These two may have very similar DNC structural features.

From the Mössbauer property calculations, one cannot tell under the conditions of the Mössbauer experiments whether the Tyr237 side chain is in a neutral or deprotonated anionic state. Taking E[Fe2+,HS···Cu+(Y237–)] and E[Fe2+,HS···Cu+(Y237)] from Table for the E(A–) and E(AH) terms in eq , we then obtained the pKa(Y237) = 7.8. Because this calculated pKa value is close to 7, considering the uncertainty of DFT calculations, the limited size of the quantum cluster model, and the fact that the pKa calculations are for the equilibrium state at room temperature, it is not certain whether the Tyr237 side chains of the Fe2+···Cu+-type DNC’s are in a neutral or anionic state.

The experimental H-bonding O···O distance between the Tyr237 side chain and the geranyl −OH of the a3-heme is only 2.61 Å in the X-ray crystal structure of 3EH5 (2.8 Å resolution). The DFT OLYP calculations predict 2.80 Å and a longer 3.33 Å for this O···O distance in the Fe2+,HS···Cu+(Y237–) and Fe2+,HS···Cu+(Y237) states, respectively. Therefore, the geometry optimization calculations support that the Tyr237 side chain in the X-ray crystal structure of 3EH5 is in anionic deprotonated state.

So far, our calculations show that the DNC of the second (minority) Fea32+ component observed in the Mössbauer experiment may have the Fe2+,HS···Cu+(Y237–/Y237) structure. Now the question is, what is the DNC structure of the first (majority) Fea32+ component observed in the Mössbauer experiment?

We noticed that the DNC of 3EH3 is similar to that of 3EH5 but with a longer Fe–N(His384) distance (2.39 Å in 3EH3 relative to 2.10 Å in 3EH5 and 2.21 Å in the OLYP-optimized Fe2+,HS···Cu+(Y237–) structure). Next, we would examine how a longer Fe–N(His384) and a shorter Fe2+,HS···Cu+ distance (5.06 Å in 3EH3 relative to 5.73 Å in the OLYP-optimized Fe2+,HS···Cu+(Y237–)) will affect the 57Fe2+ Mössbauer spectra, and if such a Fe2+,HS···Cu+ DNC in 3EH3 would represent the first Fea32+ component observed in the Mössbauer experiment.

A new Fe2+···Cu+(Y237–) model was then constructed based on the DNC coordinates in 3EH3. Because we wanted to keep the Fe–N(His384) (2.39 Å in 3EH3) and Fe2+,HS···Cu+ (5.06 Å in 3EH3) distances, the atom positions of N(His384), Fe, and Cu were fixed during geometry optimization. Then, the Mössbauer property calculation on this partially optimized 3EH3-Fe2+,HS···Cu+(Y237–) model yielded δ = 0.92 mm s–1 and ΔEQ = 2.44 mm s–1 (also given in Table , 3EH3 section), which is similar to the Mössbauer parameters of δ = 0.91 mm s–1 and ΔEQ = 2.16 mm s–1 obtained for the 3EH5-Fe2+,HS···Cu+(Y237–) model. Because the experimentally observed component 1 of 57Fea32+ has a smaller ΔEQ = 1.43 mm s–1,[18] whereas the model 3EH3-Fe2+,HS···Cu+(Y237–) yields an even larger ΔEQ = 2.44 mm s–1, this 3EH3-Fe2+,HS···Cu+(Y237–) model does not represent the DNC structure of the first Fea32+ component observed in the Mössbauer experiment.

As mentioned earlier in 1EHK, the axial His384 side chain was farther away from the Fe atom with the Fe···N(His384) distance of 3.31 Å. An oxygen species (probably originating from the oxidized state) was found between the Fe and Cu sites. To see if this long Fe···N(His384) distance will remain and how it affects the 57Fe2+ Mössbauer properties when the bridging oxygen species is omitted, we took the DNC model (Figure ) from 1EHK, deleted the bridging oxygen atom, and optimized the geometry in the Fe2+,HS···Cu+(Y237–) state. The geometric and Mössbauer properties of the three structures Fe2+,HS···Cu+(Y237–)(1–3) along the optimization trajectory and the final optimized structure Fe2+,HS···Cu+(Y237–)(4) are also shown in Table (in the 1EHK section). Without the bridging oxygen species, the Fe2+,HS and the Cu+ sites move away from each other during the geometry optimization. The Fe···N(His384) distance is gradually shortened, and because the His384 side chain cannot move freely (Hlink is fixed during geometry optimization), Fe2+ moves toward the N(His384) atom. The final structure Fe2+,HS···Cu+(Y237–)(4) is 9.4 kcal mol–1 higher in energy than that of the 3EH5-Fe2+,HS···Cu+(Y237–) optimized geometry.

The calculated Mössbauer properties on Fe2+,HS···Cu+(Y237–)(1–4) show that when the Fe···N(His384) distance is very long (3.0 Å), the calculated Mössbauer isomer shift is relatively smaller (δ = 0.90 mm s–1), and the quadrupole splitting value is very large (ΔEQ = 2.90 mm s–1). As the Fe···N(His384) distance gets shorter, the calculated δ gets larger and ΔEQ becomes smaller. Therefore, the Fe2+,HS···Cu+(Y237–) model with a long Fe···N(His384) distance does not represent the DNC structure of the experimentally observed component one, which has a large δ = 0.95 mm s–1 and a small ΔEQ = 1.43 mm s–1.[18]

Next, we study whether the bridging oxygen species in 1EHK is H2O or OH– and if the Fe2+-H2O-Cu+/Fe2+-OH–-Cu+ structures will yield the Mössbauer properties of the first 57Fea32+ component observed in the Mössbauer experiment.

Bridging H2O (Fe2+-H2O-Cu+) or OH– (Fe2+-OH–-Cu+) in the DNC of 1EHK?

From the X-ray crystal structure, the Tyr237 side chain in 1EHK is also likely in the deprotonated anionic state because the H-bonding O···O distance between the Tyr237 side chain and the geranyl −OH of the a3-heme is only 2.52 Å. Therefore, we optimized the geometry of the 1EHK-Fe2+,HS-H2O-Cu+(Y237–) DNC cluster (Figure , starting with bridging H2O) with the deprotonated Tyr237– side chain. The calculated geometrical parameters of the DNC, the energy (E), and the 57Fe2+ Mössbauer properties are given in Table .

Table 3

Calculated Properties of the Reduced Fe2+,HS-H2O-Cu+(Y237–) Model with Bridging H2O and Fe2+,HS-OH–-Cu+(Y237–/Y237) Models with Bridging OH– Generated from the DNC of the 1EHK X-ray Crystal Structurea

	geometry													net spin		57Fe2+ Mössbauer
state	Fe–N (H384)	Fe–O	Cu–O	∠Fe–O–Cu	Fe···Cu	Cu–N (H233)	Cu–N (H282)	Cu–N (H283)	O···O (Y237)	E	pKa (Y237)	pKa (H2O)	Q	Fe2+	Cu+	δ	ΔEQ
Fe2+,HS-H2O-Cu+(Y237–)	3.15	2.41	3.57	122	5.26	2.01	2.10	2.04	2.72	–357.7		17.3	–1	3.86	0.00	0.90	2.60
Fe2+,HS-OH–-Cu+(Y237–)	4.07	1.95	2.31	135	3.95	2.11	2.15	2.16	2.73	–349.2			–2	3.90	0.05	0.80	1.82
Fe2+,HS-OH–-Cu+(Y237)	4.17	1.94	2.39	138	4.05	2.10	2.14	2.14	3.15	–352.3	13.1		–1	3.88	0.03	0.80	1.89
exp. (1EHK)b	3.31	2.26	2.31	148	4.40	2.10	2.21	2.13	2.52

The properties include geometries (Å, °), electronic energies (E, offset by −25100 kcal mol–1) obtained from the all-electron/all-TZP single-point energy calculations on the optimized geometries, pKa’s, the net charge (Q) of the clusters, Mulliken net spin polarizations for the Fe and Cu sites, and the 57Fe Mössbauer isomer shifts (δ, mm s–1) and quadrupole splittings (ΔEQ, mm s–1).

A bridging oxygen species was found in between the Fe and Cu sites in the DNC of the radiation-reduced X-ray crystal structure of 1EHK (2.4 Å resolution).[10] Here we study if this bridging oxygen species is a H2O molecule or a hydroxide anion.

In 1EHK, the Fe···Cu distance is very short (4.40 Å); the distance of Fe–O (2.26 Å) is similar to the distance of Cu–O (2.31 Å), and the bridging O is close to the line connecting the Fe and Cu sites with ∠Fe-O-Cu = 148°. During DFT geometry optimization, the proposed bridging H2O molecule gradually moves away from both the Fe2+ and Cu+ sites, and finally detaches from them. The Fe2+ and Cu+ sites also move apart from each other. The final Fe···O, Cu···O, and Fe···Cu distances are 2.41, 3.57, and 5.26 Å, respectively. Therefore, the H2O molecule does not bind with either the Fe2+,HS or Cu+ site. The bridging oxygen species in 1EHK is therefore probably not a water molecule.

The central portion of the optimized Fe2+,HS-H2O-Cu+(Y237–) geometry is shown in Figure S1 in the Supporting Information. Although the H2O molecule is 2.41 Å away from the Fe2+,HS site, it prevents the Fe2+,HS from moving toward the His384 side chain. Without this H2O molecule (see section and Fe2+,HS···Cu+(Y237–)(4) in Table ), Fe2+,HS would bind to His384, and the calculated Fe···N(His384) distance would be shortened to 2.33 Å. This H2O molecule only moderately affects the Mössbauer properties of 57Fe2+,HS. From 3EH5-Fe2+,HS···Cu+(Y237–) (see Table ) to the current 1EHK-Fe2+,HS-H2O-Cu+(Y237–), the calculated isomer shift decreases from 0.91 to 0.90 mm s–1, and the quadrupole splitting increases from 2.16 to 2.60 mm s–1. Overall, the δ = 0.90 mm s–1 and ΔEQ = 2.60 mm s–1 values are still in reasonable agreement with the experimental results (the second 57Fea32+ component) of δ = 0.92 mm s–1 and ΔEQ = 2.34 mm s–1. It is not clear if there is a water molecule trapped in the DNC of the enzyme under the conditions of the Mössbauer experiments.

To test if an OH– would stay in the bridging position of the oxygen species found in the DNC of 1EHK, we also geometry optimized the 1EHK-Fe2+,HS-OH–-Cu+(Y237–) model. The OH– clearly stays in the bridging position. The optimized structure is given in Figure S2 in the Supporting Information. The calculated Fe–O (1.95 Å) and Cu–O (2.31 Å) distances in this model (see Table ) are much closer to the corresponding distances (2.26 and 2.31 Å) in 1EHK than in the calculated Fe2+,HS-H2O-Cu+(Y237–) model. Therefore, the bridging oxygen species in 1EHK is better represented by a hydroxide anion than by a water molecule.

The optimized Fe2+,HS-OH–-Cu+(Y237–) structure has a shorter Fe···Cu distance and a longer Fe···N(His384) distance compared to the X-ray crystal structure (1EHK). The radiolyticly reduced protein is likely not in an equilibrium state. The structure 1EHK may represent a mixture of the Fe2+,HS-OH–-Cu+(Y237–) and the open Fe2+,HS···Cu+(Y237–) states (Table ). A minority contribution from Fe2+,HS-H2O-Cu+(Y237–) to 1EHK is also possible.

We further optimized the geometry of the Fe2+,HS-OH–-Cu+(Y237) state with neutral Tyr237; its calculated properties are provided in Table . Because the calculated pKa for the Tyr237 side chain is 13.1, the Tyr237 energetically favors the neutral protonated form in the equilibrium state at room temperature. Further, comparing tautomers, the energy of Fe2+,HS-H2O-Cu+(Y237–) is 5.4 kcal mol–1 lower than that of the Fe2+,HS-OH–-Cu+(Y237) state, indicating that Fe2+,HS-H2O-Cu+(Y237–) is energetically more stable than the Fe2+,HS-OH–-Cu+(Y237) state, and therefore also more stable than the Fe2+,HS-OH–-Cu+(Y237–) state. This conclusion is also supported by the calculated pKa value of 17.3 for the H2O molecule in Fe2+,HS-H2O-Cu+(Y237–). Therefore, the radiolyticly reduced DNC of Fe2+,HS-OH–-Cu+(Y237–) in 1EHK is not in an equilibrium state. The bridging OH– is likely trapped, originating from 2e– reduction of the oxidized Fe3+-OH–-Cu2+(Y237–) state at low temperature with an effective kinetic barrier to proton transfer from the K-path.

The Fe2+,HS-OH–-Cu+(Y237–) and Fe2+,HS-OH–-Cu+(Y237) clusters yield essentially the same Mössbauer isomer shift (0.80 mm s–1) and quadrupole splitting (1.82/1.89 mm s–1) properties. Compared with the open Fe2+,HS···Cu+(Y237–/Y237) structures in Table , the bridging OH– in Fe2+,HS-OH–-Cu+(Y237–/Y237) lowers both the 57Fe2+,HS Mössbauer isomer shift and quadrupole splitting values. Because the calculated δ = 0.80 mm s–1 is well below the observed values of 0.92/0.95 mm s–1, the radiolyticly reduced Fe2+,HS-OH–-Cu+(Y237–) DNC in 1EHK does not represent either Fea32+ component observed in the Mössbauer experiments.

Terminal H2O or OH– in the DNC of 3EH4?

As mentioned earlier, in the radiolyticly reduced X-ray crystal structures 3EH4 and 1XME (Table ), the oxygen species found in the DNC is much closer to the Cu+ site. We now start from the DNC of 3EH4 to study whether a water molecule or an OH– can be terminally bound to the Cu+ site. We geometry optimized the Fe2+,HS···H2O-Cu+(Y237–/Y237) and Fe2+,HS···OH–-Cu+(Y237–/Y237) DNC clusters in both anionic Tyr237– and neutral Tyr237 states and also calculated the 57Fe2+,HS Mössbauer properties. The calculated results are given in Table .

Table 4

Calculated Properties of the Reduced Fe2+,HS···H2O-Cu+(Y237–/Y237) and Fe2+,HS···OH–-Cu+(Y237–/Y237) DNC Models Generated from the DNC of the X-ray Crystal Structure of 3EH4a

	geometry													net spin		57Fe2+ Mössbauer
state	Fe–N (H384)	Fe···O	Cu–O	∠Fe–O–Cu	Fe···Cu	Cu–N (H233)	Cu–N (H282)	Cu–N (H283)	O···O (Y237)	E	pKa (Y237)	pKa (H2O)	Q	Fe2+	Cu+	δ	ΔEQ
Fe2+,HS···H2O-Cu+(Y237–)	2.17	4.05	3.30	100	5.65	1.99	2.11	2.01	2.81	–366.5		27.4	–1	3.88	0.00	0.91	2.31
Fe2+,HS···H2O-Cu+(Y237)	2.18	3.93	3.25	104	5.68	2.00	2.09	2.04	3.29	–366.8	11.1	25.0	0	3.88	0.00	0.91	2.20
Fe2+,HS···OH–-Cu+(Y237–)	2.22	4.01	1.95	113	5.10	2.18	2.07	2.29	2.89	–344.2			–2	3.83	0.28	0.93	1.01
Fe2+,HS···OH–-Cu+(Y237)	2.22	3.82	1.93	116	4.98	2.31	2.02	2.42	3.29	–347.7	13.4		–1	3.84	0.23	0.92	1.20
exp. (3EH4)b	2.39	3.08	2.20	126	4.73	1.96	2.03	1.97	2.47

The properties include geometries (Å, °), electronic energies (E, offset by −25100 kcal mol–1) obtained from the all-electron/all-TZP single-point energy calculations on the optimized geometries, pKa’s, the net charge (Q) of the clusters, Mulliken net spin polarizations for the Fe and Cu sites, and the 57Fe Mössbauer isomer shifts (δ, mm s–1) and quadrupole splittings (ΔEQ, mm s–1).

An oxygen species between the Fe2+ and Cu+ sites but much closer to the Cu+ site was observed in the DNC of the X-ray crystal structure of 3EH4 (2.9 Å resolution).[12] Here we study if this oxygen species is a terminally bound (to Cu+) H2O molecule or a hydroxide anion.

As shown in Figure , during geometry optimizations, the Cu+-terminally bound H2O molecule in both Fe2+,HS···H2O-Cu+(Y237–/Y237) models dissociated from the Cu+ site. It stayed in between Fe2+ and Cu+ but did not bind to either site. Therefore, the oxygen species observed in the DNC of 3EH4 cannot be a water molecule.

Figure 3

Left: The central portion of the initial Fe2+,HS···H2O-Cu+(Y237–/Y237) structure, which was taken from the DNC of the 3EH4 X-ray crystal structure. Right: During geometry optimizations, the H2O molecule dissociated from the Cu+ site. Distances (Å) on the right are taken from the optimized Fe2+,HS···H2O-Cu+(Y237) structure.

The optimized Fe2+,HS···H2O-Cu+(Y237–/Y237) clusters yield very similar 57Fe2+,HS Mössbauer properties (δ = 0.91 mm s–1, ΔEQ = 2.31/2.20 mm s–1), which are very close to the calculated 57Fe2+,HS Mössbauer properties (δ = 0.91/0.92 mm s–1, ΔEQ = 2.16/2.21 mm s–1) for the open Fe2+,HS···Cu+(Y237–/Y237) structures calculated for the 3EH5 DNC (see Table ) and are also consistent with the second 57Fea32+ component (δ = 0.92 mm s–1, ΔEQ = 2.34 mm s–1) observed in the Mössbauer experiments.[18]

Although this dangling H2O molecule between the Fe2+ and Cu+ sites has little effect on the 57Fe2+,HS Mössbauer properties, it does affect the pKa of the Tyr237 side chain. The Fe2+···Cu+ distance is very similar with or without the weak H2O bridge. Here, with a water molecule in the DNC, the pKa of the Tyr237 side chain is estimated as 11.1, which is a 3.3-unit increase from the corresponding calculated pKa (7.8) for the 3EH5 open DNC Fe2+,HS···Cu+(Y237–/Y237) models in Section (Table ). The sensitivity of the pKa of this unique Tyr237 residue in response to this structural and component change in the DNC is suggestive of a potential role in proton transfer and proton pumping during the catalytic cycle. For example, proton transfer from Tyr237 toward the reaction center or above it could be promoted in the “open” form.

By contrast, during geometry optimizations, the terminally bound OH– in both Fe2+,HS···OH–-Cu+(Y237–/Y237) clusters remained tightly bound to the Cu+ site (see Figure ). In these two optimized geometries, the Fe···O and Fe···Cu distances are longer and the Fe–N(His384) and Cu–O distances are shorter than the corresponding values in the radiolyticly reduced X-ray crystal structure 3EH4. It is possible that the DNC of 3EH4 represents a mixture of the structures Fe2+,HS···OH–-Cu+(Y237–), Fe2+,HS···Cu+(Y237–), and/or Fe2+,HS-OH–-Cu+(Y237–) (bridging OH–).

Figure 4

Central portion of the optimized Fe2+,HS···OH–-Cu+(Y237) structure with OH– binding with Cu+. Labeled distances are in Å.

The Fe2+,HS···OH–-Cu+(Y237–/Y237) states also yield very similar Mössbauer properties (δ = 0.93/0.92 mm s–1, ΔEQ = 1.01/1.20 mm s–1), which are so far the closest calculated results to the majority first 57Fea32+ component property (δ = 0.95 mm s–1, ΔEQ = 1.43 mm s–1) observed in Mössbauer experiments.[18]

The calculated pKa (27.4/25.0) of the H2O molecule in the DNC and the pKa (11.1/13.4) of the Tyr237 side chain indicate that, when there is an oxygen species in between the Fe2+,HS and Cu+ sites, the Fe2+,HS···Cu+ DNC strongly favors a H2O molecule at thermodynamic equilibrium rather than a hydroxide anion, and the Tyr237 side chain energetically prefers the protonated charge-neutral state. In 3EH4, the O···O distance between the Tyr237 side chain and the geranyl −OH of the a3-heme is very short (2.47 Å). It is likely that the Tyr237 side chain in 3EH4 is in the deprotonated anionic state. Therefore, the Fe2+···OH–-Cu+(Y237–) DNC structure of 3EH4 probably originates from the oxidized state. That is, if the oxidized as-isolated DNC has a structure like Fe3+···OH–-Cu2+(Y237–) when it is radiolyticly reduced by the X-ray beam at low temperature with limited proton mobility, the Cu-bound OH– is then trapped in the reduced state, and the Tyr237 side chain remains in the anionic state.

However, comparing the optimized geometries of the Fe2+,HS···OH–-Cu+(Y237–) and Fe2+,HS···OH–-Cu+(Y237) states (in Table ), we notice that, in Fe2+,HS···OH–-Cu+(Y237), both the Cu–O and Cu–N(H282) distances are shorter and both the Cu–N(H233) and Cu–N(H283) distances are longer than the corresponding Cu-ligand distances in Fe2+,HS···OH–-Cu+(Y237–). In other words, upon protonation of the Tyr237 side chain, the OH– ligand will bind even stronger with Cu+. Therefore, even when Tyr237 is protonated in the Fe2+,HS···OH–-Cu+ state, because of the proton transfer barrier from neutral Tyr237 to the OH–-Cu+, the Fe2+,HS···OH–-Cu+(Y237) state may still be trapped at low temperature in the Mössbauer experimental conditions.

Changing of the Cu+ Coordination State and Feasible DNC Structure of the first 57Fea32+ Component Observed in the Mössbauer Experiments

The overall charge of the Fe2+,HS···OH–-Cu+(Y237) quantum cluster is −1, whereas the total charge of the Fe2+,HS···H2O-Cu+(Y237) state is 0. It is also possible that the Fe2+,HS···OH–-Cu+(Y237) state is stabilized by an extra proton in the proton loading site (PLS), which is close to the DNC. It is still not known where the PLS exactly locates in the ba3 CcO from Tt. For simplicity, Fee et al. protonated the His376 side chain (the positive imidazolium form) to represent having a proton at the PLS.[17,42] According to Gennis and co-workers’ mutagenesis and kinetics studies of proton pumping in ba3 CcO from Tt, His376 is probably close to the proton exit path but not absolutely required.[56] However, to compare how the energy changes when an extra proton exists in the Fe2+,HS···OH–-Cu+(Y237) cluster, starting from the optimized Fe2+,HS···OH–-Cu+(Y237) structure, we also protonated the His376 side chain and performed geometry optimization calculations. This His376-protonated cluster is named Fe2+,HS···OH–-Cu+(Y237)(H376H+). Interestingly, we observed that during the geometry optimization the His283 side chain gradually tilted up and dissociated from the Cu+ site, and the Cu+ changed from 4-coordinate (4c) state to 3-coordinate (3c) configuration. In the meantime, the His233 side chain also slowly moved away from the Cu+, and both the OH– ligand and the His282 side chain were closer to the Cu+. Finally, the His233 side chain also dissociated from Cu+, and the optimized structure ended with a 2-coordinate (2c) Cu+ site with a near-linear OH–-Cu+2c-N(His282) configuration (see Figure ). The calculated properties of this structure are given in the first row of Table (footnote b). Its relative energy (−364 kcal mol–1) is very close to that (−366.8 kcal mol–1) of the Fe2+,HS···H2O-Cu+(Y237) state (in Table ). These two tautomers are therefore close in energy but differ strongly in Cu+ coordination geometry and proton location.

Figure 5

Optimized structure of the Fe2+,HS···OH–-Cu+2c(Y237)(H376H+) cluster with a neutral Tyr237 and a cationic protonated His376+ side chain. During the geometry optimization process, the Cu+ site changes from 4-coordinate to 2-coordinate (2c) configuration with both His283 and His233 side chains gradually dissociating from the Cu+.

Table 5

Calculated Properties for the DNC Models with a 3- or 2- Coordinate (3c/2c) Cu+ Site in which His233/His283 Dissociated from the Cu+a

	geometry												net spin		57Fe2+ Mössbauer
state	Fe–N (H384)	Fe···O	Cu–O	∠Fe–O–Cu	Fe···Cu	Cu–N (H233)	Cu–N (H282)	Cu–N (H283)	∠O–Cu–N (H282)	E	pKa (H376H+)	Q	Fe2+	Cu+	δ	ΔEQ
Fe2+,HS···OH–-Cu+2c(Y237)(H376H+)b	2.20	3.95	1.85	101	4.66	3.02	1.90	4.03	176	–364.0	10.3	0	3.89	0.00	0.91	2.17
Fe2+,HS···OH–-Cu+2c(Y237)c	2.19	4.19	1.86	97	4.78	3.09	1.90	4.25	176	–364.6		–1	3.89	0.00	0.91	2.28
Fe2+,HS···OH–-Cu+3c(Y237)(H376H+)(1)d	2.21	3.86	1.92	113	4.94	2.32	2.00	2.60	160	–350.7	11.2	0	3.85	0.20	0.92	1.32
Fe2+,HS···OH–-Cu+3c(Y237)(H376H+)(2)d	2.20	3.86	1.91	111	4.89	2.36	1.97	2.83	163	–352.8	11.2	0	3.86	0.15	0.92	1.51
Fe2+,HS···OH–-Cu+3c(Y237)(H376H+)(3)d	2.21	3.82	1.89	110	4.80	2.44	1.95	3.09	166	–355.3	12.1	0	3.87	0.11	0.92	1.75
Fe2+,HS···OH–-Cu+2c(Y237)(H376H+)(4)d	2.21	3.78	1.89	108	4.71	2.64	1.92	3.30	172	–356.9	10.8	0	3.88	0.04	0.91	2.02
Fe2+,HS···OH–-Cu+3c(Y237)(1)e	2.22	3.85	1.91	114	4.94	2.32	2.00	2.60	159	–350.0		–1	3.85	0.20	0.92	1.31
Fe2+,HS···OH–-Cu+3c(Y237)(2)e	2.21	3.85	1.90	112	4.89	2.36	1.97	2.83	162	–352.1		–1	3.86	0.15	0.92	1.52
Fe2+,HS···OH–-Cu+3c(Y237)(3)e	2.21	3.83	1.89	109	4.80	2.44	1.95	3.09	166	–353.4		–1	3.87	0.11	0.92	1.75
Fe2+,HS···OH–-Cu+2c(Y237)(4)e	2.21	3.77	1.87	108	4.71	2.64	1.92	3.30	171	–356.8		–1	3.88	0.04	0.92	2.06
Fe2+,HS···H2O-Cu+3c(Y237)f	2.19	4.19	2.35	106	5.35	1.99	1.94	4.45	132	–368.9		0	3.89	0.00	0.91	2.31
Fe2+,HS···H2O···Cu+2c(Y237)f	2.20	4.76	3.13	95	5.92	1.92	1.91	5.40		–378.7		0	3.88	0.00	0.91	2.24

The properties include geometries (Å, °), electronic energies (E, offset by −25100 kcal mol–1) obtained from the all-electron/all-TZP single-point energy calculations on the optimized geometries, pKa’s, the net charge (Q) of the clusters, Mulliken net spin polarizations for the Fe and Cu sites, and the 57Fe Mössbauer isomer shifts (δ, mm s–1) and quadrupole splittings (ΔEQ, mm s–1).

Both His233 and His283 side chains dissociated from the Cu+ site during the geometry optimization.

After deleting the H+ of His376+ in Fe2+,HS···OH–-Cu+2c(Y237)(H376H+), and geometry optimizing the structure, the Cu+ site remained in the 2c configuration.

Four structures (1–4) with a 3c or 2c Cu+ site, which were taken from the geometry optimization trajectory of the Fe2+,HS···OH–-Cu+2c(Y237)(H376H+) state.

See footnote d above. These are the four corresponding structures of the Fe2+,HS···OH–-Cu+(Y237) state.

When protonating the OH– ligand in Fe2+,HS···OH–-Cu+2c(Y237) and optimizing the geometry, the Cu+ coordination state changes from Fe2+,HS···H2O-Cu+2c(Y237) (left in Figure ) to Fe2+,HS···H2O-Cu+3c(Y237) (middle in Figure ) and to Fe2+,HS···H2O···Cu+2c(Y237) (right in Figure ).

Figure 7

Changing of the Cu+ coordination state during the geometry optimization calculation after manually protonating the OH– ligand in Fe2+,HS···OH–-Cu+2c(Y237) (second row of Table ). Left: The initial linear H2O-Cu+2c-N(His282) structure when adding a proton to the OH– ligand. Other parts of the model cluster are similar to those in Figure but with a neutral His376 side chain. Middle: During the geometry optimization, the O–Cu+ distance lengthens, the His233 side chain rebinds with Cu+, and a 3-coordinate Cu+ configuration is formed (see Fe2+,HS···H2O-Cu+3c(Y237) with footnote f in Table ). Right: The H2O ligand eventually dissociates from the Cu+ site, and a 2-coordinate N(His233)-Cu+2c-N(His282) configuration is obtained (see Fe2+,HS···H2O···Cu+2c(Y237) in Table ).

We then deleted the added proton on His376+ and optimized the geometry to obtain the Fe2+,HS···OH–-Cu+2c(Y237) structure with the 2-coordinated Cu+. The calculated properties of the optimized Fe2+,HS···OH–-Cu+2c(Y237) cluster are given in the second row of Table (footnote c). During the geometry optimization, the Fe···Cu, Cu···N(His233), and Cu···N(His283) distances are lengthened further. However, overall, the geometries of the optimized Fe2+,HS···OH–-Cu+2c(Y237)(H376H+) and Fe2+,HS···OH–-Cu+2c(Y237) structures are very similar. In both structures, the angle of OH––Cu–N(His282) is 176°, which is close to a linear structure. The relative energy of the Fe2+,HS···OH–-Cu+2c(Y237) state is ∼17 kcal mol–1 lower than that of its corresponding 4-coordinate Cu+ state Fe2+,HS···OH–-Cu+(Y237), as given in Table . Therefore, with OH– binding to Cu+, the DNC favors the 2-coordinate Cu+ structure. The calculated Mössbauer properties of both the Fe2+,HS···OH–-Cu+2c(Y237)(H376H+) and Fe2+,HS···OH–-Cu+2c(Y237) states (δ = 0.91 mm s–1, ΔEQ = 2.17/2.28 mm s–1) are now similar to those of the open Fe2+,HS···Cu+(Y237–/Y237) structures given in Table and the structure with a dangling H2O molecule (in Table ) between the Fe2+,HS and Cu+ sites. In other words, the Fe2+,HS···OH–-Cu+2c(Y237)(H376H+) and Fe2+,HS···OH–-Cu+2c(Y237) states with 2-coordinate Cu+ do not represent the DNC of the first (majority) 57Fea32+ component observed in the Mössbauer experiments[18] but are entirely compatible with the observed second (minority) 57Fea32+ Mössbauer experimental parameters (δ = 0.92 mm s–1, ΔEQ = 2.34 mm s–1).

Reviewing the X-ray crystal structure of 3EH4, we found that the side chain of Trp229 is very close (with the closest atom to atom distance being 3.4 Å) and parallel to the side chain of His283 (see Figure ). During the Cu+4c → Cu+2c transition in the geometry optimization of the Fe2+,HS···OH–-Cu+(Y237)(H376H+) state, the side chain of His283 tilted up and away from the Cu+ and moved toward the side chain of Trp229 (not included in the current model calculation). Therefore, we propose that because of the steric interactions and the potential energy barriers encountered upon moving the His233 and His283 side chains away from Cu+, the OH–-Cu+ site is frequently trapped in the Cu+4c or Cu+3c configurations, particularly under the low temperature in Mössbauer experiments.

Figure 6

Taken from the X-ray crystal structure of 3EH4, which shows that the side chain of Trp229 is very close (with the closest atom to atom distance being 3.4 Å) and parallel to the side chain of His283. The steric interaction between the two side chains prevents His283 from freely moving away from the Cu+ site.

To test this idea, we picked four structures with a “maximum gradient” of <0.005 au/Å along the geometry optimization trajectory of Fe2+,HS···OH–-Cu+(Y237)(H376H+) to represent the middle structures of the Cu+4c → Cu+2c transition. The calculated properties of the four Fe2+,HS···OH–-Cu+3c/2c(Y237)(H376H+)(1–4) structures from all-electron/all-TZP single-point energy calculations are also given in Table (rows 3–6 with footnote d). We noticed that the structural and coordination state of the Cu+ site indeed influences the electric field at the Fe2+ site and therefore affects the 57Fe2+ Mössbauer, especially the quadruple splitting property. The Cu+4c structure of Fe2+,HS···OH–-Cu+(Y237–) in Table yields a large calculated isomer shift (0.93 mm s–1) and a small quadrupole splitting value (1.01 mm s–1). Next, in the Cu+4c structure of Fe2+,HS···OH–-Cu+(Y237) in Table , the Cu–N(His233) and Cu–N(His283) distances are increased and the Cu–O, Cu–N(282), and Fe···Cu distances are decreased; meanwhile, the calculated 57Fe2+ Mössbauer isomer shift (0.92 mm s–1) is decreased, and its quadruple splitting value (1.20 mm s–1) is increased. Further, the trend continues in the four Fe2+,HS···OH–-Cu+3c/2c(Y237)(H376H+)(1–4) structures in Table , that is, with a decrease in the Cu+ coordination number and the Fe2+···Cu+ distance, the calculated 57Fe2+ Mössbauer isomer shift decreases and its quadrupole splitting value increases. In particular, the ΔEQ values (1.32 and 1.51 mm s–1) of Fe2+,HS···OH–-Cu+3c(Y237)(H376H+)(1) and Fe2+,HS···OH–-Cu+3c(Y237)(H376H+)(2) structures are very close to the observed ΔEQ = 1.43 mm s–1 of the first 57Fea32+ Mössbauer component.[18] It is therefore probable that the DNC structure of the first (majority) 57Fea32+ component observed in the Mössbauer experiment is trapped at a structure similar to the ones in Fe2+,HS···OH–-Cu+3c(Y237)(H376H+)(1)/(2) at low temperature.

It is not evident whether the His376 side chain is typically protonated or not. To see how the protonation state of His376 affects the Mössbauer properties of the 57Fe2+ site, we deleted the added proton on His376+ in Fe2+,HS···OH–-Cu+3c/2c(Y237)(H376H+)(1–4) and optimized the geometries with fixed atom positions of Fe, Cu, N(His233), N(His283), and N(His283). The calculated properties of these four partially optimized DNC structures, Fe2+,HS···OH–-Cu+3c/2c(Y237)(1–4) are also given in Table (rows 7–10 with footnote e). The calculated net spin populations on Fe2+ and Cu+, and the Mössbauer isomer shifts and quadrupole splittings of the four Fe2+,HS···OH–-Cu+3c/2c(Y237)(1–4) structures, are essentially the same as the corresponding ones of Fe2+,HS···OH–-Cu+3c/2c(Y237)(H376H+)(1–4). Therefore, the protonation state of the His376 side chain does not affect the Mössbauer properties of the 57Fe2+ site.

The calculated pKas of His376-H+ are larger than 10, which indicates that the system favors this charge neutral state. Without including the carboxylate group of the Glu126B side chain (which is within a strong H-bonding distance of Nε2 of the His376 side chain) in the DNC quantum cluster model, it is not clear whether the His376 side chain would be protonated in a realistic protein environment.

If we use the energies of Fe2+,HS···OH–-Cu+2c(Y237) (second row of Table ) and Fe2+,HS···H2O-Cu+(Y237) (second row of Table ) to calculate the pKa of the H2O molecule, we obtain a value of 12.7. These structures differ very significantly in Cu+ coordination geometry. We then protonated the OH– ligand in the optimized structure Fe2+,HS···OH–-Cu+2c(Y237) (see the left structure in Figure ) and performed geometry optimization to see if the linear H2O-Cu+2c-N(His282) configuration would persist. However, during the geometry optimization, the H2O–Cu+ distance was gradually lengthened and the Cu+–N(His233) distance shortened, and the Cu+ site became 3-coordinate with ligands His233, His282, and H2O (see the middle structure in Figure ).

The relative energy of a middle Fe2+,HS···H2O-Cu+3c(Y237) state (−368.9 kcal mol–1) (footnote f in Table ) is similar to that (−366.8 kcal mol–1) of the Fe2+,HS···H2O-Cu+(Y237) state (with three His ligands at the Cu+ site and a weak Cu+···H2O interaction), as given in Table . As the geometry optimization continued, the H2O molecule and the Cu+ center continued moving away from each other, and finally the H2O dissociated from the Cu+ site and H-bonded with the His283 side chain. The 2-coordinate His233-Cu+2c-His282 center is shown on the right side of Figure , and the calculated properties of this model cluster are given in the last row of Table . This structure has a very long Fe···Cu distance of 5.92 Å and is ∼10 kcal mol–1 lower in energy than the middle Fe2+,HS···H2O-Cu+3c(Y237) state. We do not know if such an Fe2+,HS···H2O···Cu+2c(Y237) state is feasible during the catalytic cycle given other protein geometric constraints. For example, both Fe···Cu (5.92 Å) and Cu–N(H283) (5.40 Å) distances are very long. However, our calculations indeed show the mobility of the His233 and His283 side chains, which can be potential proton loading sites during the catalytic cycle and proton pumping.

We note that the calculated Mössbauer properties of both the Fe2+,HS···H2O-Cu+3c(Y237) and Fe2+,HS···H2O···Cu+2c(Y237) states (δ = 0.91 mm s–1, ΔEQ = 2.31/2.24 mm s–1) are also similar to those of the open Fe2+,HS···Cu+(Y237–/Y237) structures given in Table and are consistent with the observed Mössbauer properties for the second 57Fea32+ component. Therefore, up until now, one cannot draw an exclusive conclusion about the DNC structure of the second observed 57Fea32+ component in the Mössbauer experiments.[18]

Fe2+-(HO2)−-Cu+ DNC Models from 3S8G

In ref (23), on the basis of a series of structural calculations and comparisons, we proposed that the dioxygen species found in the DNC of 3S8G is HO2– with the majority species having the proton of the HO2– residing on the oxygen atom that is closer to the Fe2+ site in the Fe2+-(HO-O)−-Cu+ conformation. Details of the optimized 3S8G-Fe2+,LS/IS/HS-(HO2)−-Cu+(Y237–/Y237) DNC structures are given in Tables 4–8 of ref (23). We have now performed Mössbauer property calculations on those structures. It turns out that the calculated Mössbauer properties of the 3S8G-Fe2+,LS/IS/HS-(HO2)−-Cu+(Y237–/Y237) DNC models, which are given in Tables S1 and S2 in the Supporting Information, are not consistent with either of the observed 57Fea32+ Mössbauer properties. This also supports the conclusion that the HO2– species found in the 3S8G X-ray crystal structure arises from the recombination of two radiation produced HO· radicals (product of water radiolysis) formed very near or even between the two metal sites,[15] or comes from the photoreaction of the H2O/OH– between the as-isolated oxidized Fe3+ and Cu2+ sites in the X-ray beam.[23] Next, we will examine if Fe3+-OH–···H2O-Cu2+- and Fe3+-H2O···OH–-Cu2+-type model clusters represent the DNC structures observed in the Mössbauer experiments for the oxidized ba3 CcO from Tt.

Results and Discussion on the Fe3+/Cu2+ Oxidized State DNC Model Calculations

The Fe3+-OH–···H2O-Cu2+ and Fe3+-H2O···OH–-Cu2+ DNC Models from 3S8G

The initial model structures in this subsection are constructed by replacing the dioxygen atoms observed in the DNC of 3S8G (Figures 1 and 2 of ref (23)) with the OH–···H2O/H2O···OH– species. The central structures of the Fe3+-OH–···H2O-Cu2+ and Fe3+-H2O···OH–-Cu2+ model clusters are shown in Figures and 9, respectively.

Figure 8

Detailed central portion of the Fe3+-OH–···H2O-Cu2+ cluster generated by modifying the oxidized 3S8G DNC model. The rest of the model cluster is shown in Figures 1 and 2 of ref (23).

Figure 9

Detailed central portion of the Fe3+-H2O···OH–-Cu2+ cluster generated by modifying the oxidized 3S8G DNC model. The rest of the model cluster is shown in Figures 1 and 2 of ref (23).

We have optimized the geometries and calculated the Mössbauer properties of the Fe3+-OH–···H2O-Cu2+ and Fe3+-H2O···OH–-Cu2+ model clusters with the Tyr237 side chain in either the deprotonated (Y237–) or neutral protonated (Y237) state and with Fe3+ in either the LS, IS, or HS state, which is F- or AF-coupled to the Cu2+ site. The calculated properties are given in Tables and 7, respectively, for the Fe3+-OH–···H2O-Cu2+ and Fe3+-H2O···OH–-Cu2+ model clusters.

Table 6

Calculated Properties of the Oxidized Fe3+-OH–···H2O-Cu2+(Y237–/Y237) Clusters Generated from the DNC of the X-ray Crystal Structure 3S8Ga

	geometry												net spin		57Fe3+ Mössbauer
stateb	Fe–N (H384)	Fe–O1	Cu–O2	O1···O2	Fe···Cu	Cu–N (H233)	Cu–N (H282)	Cu–N (H283)	O···O (Y237)	Ec	pKa (Y237)	Q	Fe3+	Cu2+	δ	ΔEQ
Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F)	2.09	1.84	2.36	2.66	5.47	2.05	2.09	2.04	3.04	–58.8		0	1.04	0.33	0.19	2.53
Fe3+,LS-OH–···H2O-Cu2+(Y237–)(AF)	2.09	1.85	2.32	2.63	5.40	2.05	2.09	2.04	3.02	–58.3		0	0.95	–0.35	0.21	2.16
Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F)	2.43	2.00	2.20	2.51	5.31	2.07	2.09	2.06	3.08	–56.1		0	2.71	0.38	0.39	1.57
Fe3+,IS-OH–···H2O-Cu2+(Y237–)(AF)d										–56.3		0	2.69	–0.38	0.39	1.64
Fe3+,HS-OH–···H2O-Cu2+(Y237–)(F)	2.42	1.95	2.23	2.59	5.24	2.08	2.07	2.05	3.01	–51.1		0	4.10	0.38	0.36	0.21
Fe3+,HS-OH–···H2O-Cu2+(Y237–)(AF)e										–53.0		0	4.10	–0.37	0.36	0.21
Fe3+,LS-OH–···H2O-Cu2+(Y237)(F)	2.08	1.83	2.28	2.68	5.45	2.06	2.10	2.03	3.29	–49.7	5.0	1	1.28	0.39	0.14	2.07
Fe3+,LS-OH–···H2O-Cu2+(Y237)(AF)	2.09	1.85	2.13	2.58	5.41	2.07	2.08	2.02	3.39	–48.1	3.4	1	0.85	–0.46	0.19	2.25
Fe3+,HS-OH–···H2O-Cu2+(Y237)(F)	2.38	1.99	2.05	2.48	5.26	2.07	2.05	2.02	3.42	–43.8	5.5	1	4.12	0.51	0.39	0.32
Fe3+,HS-OH–···H2O-Cu2+(Y237)(AF)	2.42	1.97	2.06	2.49	5.25	2.07	2.06	2.02	3.50	–45.5	5.4	1	4.10	–0.50	0.36	0.39

The properties include geometries (Å), electronic energies (E, offset by −28200 kcal mol−1 in this table and in Table ) obtained from the all-electron/all-TZP single-point energy calculations on the optimized geometries, pKa’s, the net charge (Q) of the clusters, Mulliken net spin polarizations for the Fe and Cu sites, and the 57Fe Mössbauer isomer shifts (δ, mm s–1) and quadrupole splittings (ΔEQ, mm s–1).

F stands for ferromagnetically coupled and AF for antiferromagnetically coupled.

The energies calculated for the AF-coupled states are broken-symmetry state energies.

A broken-symmetry single-point energy calculation on the Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F) optimized geometry.

A broken-symmetry single-point energy calculation on the Fe3+,HS-OH–···H2O-Cu2+(Y237–)(F) optimized geometry.

Table 7

Calculated Properties of the Oxidized Fe3+-H2O···OH–-Cu2+(Y237–/Y237) Clusters Generated from the DNC of the X-ray Crystal Structure 3S8Ga

	geometry												net spin		57Fe3+ Mössbauer
stateb	Fe–N (H384)	Fe–O1	Cu–O2	O1···O2	Fe···Cu	Cu–N (H233)	Cu–N (H282)	Cu–N (H283)	O···O (Y237)	Ec	pKa (Y237)	Q	Fe3+	Cu2+	δ	ΔEQ
Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F)	2.27	2.29	1.94	2.58	5.48	2.09	2.06	2.06	2.93	–58.7		0	2.74	0.50	0.40	2.42
Fe3+,IS-H2O···OH–-Cu2+(Y237–)(AF)	2.26	2.28	1.94	2.57	5.48	2.08	2.06	2.06	2.91	–58.4		0	2.74	–0.50	0.39	2.42
Fe3+,HS-H2O···OH–-Cu2+(Y237–)(F)	2.20	2.31	1.93	2.60	5.53	2.09	2.05	2.05	3.02	–54.1		0	4.06	0.51	0.52	1.49
Fe3+,HS-H2O···OH–-Cu2+(Y237–)(AF)	2.19	2.29	1.93	2.58	5.53	2.09	2.05	2.05	2.99	–54.0		0	4.07	–0.52	0.50	1.40
Fe3+,IS-H2O···OH–-Cu2+(Y237)(F)	2.27	2.29	1.93	2.59	5.49	2.10	2.07	2.05	3.39	–51.9	6.6	1	2.75	0.52	0.39	2.49
Fe3+,IS-H2O···OH–-Cu2+(Y237)(AF)	2.27	2.27	1.93	2.57	5.47	2.11	2.06	2.05	3.44	–52.3	6.9	1	2.74	–0.52	0.40	2.47
Fe3+,HS-H2O···OH–-Cu2+(Y237)(F)	2.21	2.24	1.93	2.57	5.51	2.11	2.06	2.05	3.46	–48.8	7.0	1	4.11	0.52	0.47	0.88
Fe3+,HS-H2O···OH–-Cu2+(Y237)(AF)d										–48.9	7.2	1	4.11	–0.52	0.47	0.88

The properties include geometries (Å), electronic energies (E, offset by −28200 kcal mol−1 in this table and in Table ) obtained from the all-electron/all-TZP single-point energy calculations on the optimized geometries, pKa’s, the net charge (Q) of the clusters, Mulliken net spin polarizations for the Fe and Cu sites, and the 57Fe Mössbauer isomer shifts (δ, mm s–1) and quadrupole splittings (ΔEQ, mm s–1).

F stands for ferromagnetically coupled and AF for antiferromagnetically coupled.

The energies calculated for the AF-coupled states are broken-symmetry state energies.

A broken-symmetry single-point energy calculation on the optimized Fe3+,HS-H2O···OH–-Cu2+(Y237)(F) geometry.

In Table , we failed to obtain the optimized Fe3+,IS-OH–···H2O-Cu2+(Y237–)(AF) and Fe3+,HS-OH–···H2O-Cu2+(Y237–)(AF) structures, but instead, the geometry optimizations of these two states led to the lower-energy Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F) and Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F) states, respectively. The properties given for Fe3+,IS-OH–···H2O-Cu2+(Y237–)(AF) and Fe3+,HS-OH–···H2O-Cu2+(Y237–)(AF) states were obtained from the broken-symmetry single-point energy calculations on the optimized Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F) and Fe3+,HS-OH–···H2O-Cu2+(Y237–)(F) geometries, respectively. We did not obtain the optimized Fe3+,IS-OH–···H2O-Cu2+(Y237)(F/AF) structures (neutral Tyr237) either because the proton on the Cu2+-bound H2O ligand, which originally H-bonded to the Fe3+,IS-bound OH– ligand, spontaneously transferred to the OH– during the geometry optimizations, and the Fe3+,IS-H2O···OH–-Cu2+(Y237)(F/AF) states were obtained.

The calculated pKa values (3.4–5.5) in Table show that the Tyr237 side chain favors the deprotonated anionic state (Y237–) when the DNC has the Fe3+-OH–···H2O-Cu2+-type structure no matter whether Fe3+ is in LS or HS state and F- or AF-coupled with Cu2+.

The Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F) and Fe3+,LS-OH–···H2O-Cu2+(Y237–)(AF) states have very similar energies (with only 0.5 kcal mol–1 difference), and they are 2–3 kcal mol–1 and >5 kcal mol–1 lower in energy than the Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F/AF) and Fe3+,HS-OH–···H2O-Cu2+(Y237–)(F/AF) states, respectively. Therefore, if the oxidized DNC has the Fe3+-OH–···H2O-Cu2+-type structure at 4.2 K, it is expected to be in the Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F/AF) state. The calculated ΔEQ = 2.53/2.16 mm s–1 for the Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F/AF) states are in good agreement with the experimentally observed value ΔEQ = 2.24 mm s–1 for the “low-spin” 57Fea33+ species at 4.2 K < T < 190 K.[18] The calculated δ = 0.19/0.21 mm s–1 of these two states are ∼0.1 mm s–1 smaller than, but are still reasonably consistent with, the corresponding observed value of δ = 0.29 mm s–1 (see Figure ).

For the Fe3+-H2O···OH–-Cu2+ DNC model calculations in Table , during geometry optimizations starting from the Fe3+,LS-H2O···OH–-Cu2+(Y237–)(F/AF) clusters (see Figure ), the proton on the Fe3+,LS-bound H2O ligand, which originally H-bonded to the Cu2+-bound OH– ligand, transferred to the OH–. As a result, the Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F/AF) states (in Table and Figure ) were obtained. Therefore, Table shows only the Fe3+,IS-H2O···OH–-Cu2+ and Fe3+,HS-H2O···OH–-Cu2+ state calculations; these forms are stable under geometry optimization.

The calculated pKas of the Tyr237 side chain for the Fe3+,IS-H2O···OH–-Cu2+ and Fe3+,HS-H2O···OH–-Cu2+ clusters are 6.6–7.2 (Table ). Considering the uncertainty of DFT calculations and the fact that the pKa calculations are for the equilibrium state at room temperature, it is not certain whether the Tyr237 side chains of the Fe3+,IS/HS-H2O···OH–-Cu2+-type DNCs are in the neutral or anionic state under the Mössbauer experimental conditions. The very short O···O distance (2.66 Å) between the Tyr237 side chain and the geranyl side chain of a3-heme in 3S8G implies that the Tyr237 is in the deprotonated anion form in this radiolyticly reduced X-ray crystal structure.[23] However, in ref (23), our calculations have shown that Tyr237 energetically favors the neutral protonated form in the reduced Fe2+,LS/IS/HS-(HO2)−-Cu+ DNC. Therefore, Tyr237 is likely in the anionic state in the oxidized protein before the X-ray irradiation and is trapped at very low temperature after the irradiation.

The Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) states are 4.3–4.7 kcal mol–1 lower in energy than the Fe3+,HS-H2O···OH–-Cu2+(Y237–)(F/AF) states (Table ). In addition, the energies of the Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) states (Table ) are very similar to the energies of the Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F/AF) states (Table ). The calculated quadrupole splittings for the Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) states (2.42/2.42 mm s–1) are also similar to those for the Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F/AF) states (2.53/2.16 mm s–1). Further, the average (avg) of the four calculated isomer shifts and quadrupole splittings (δavg = 0.30 mm s–1, ΔEQavg = 2.38 mm s–1) of the Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) and Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F/AF) states agrees very well with the experimentally observed spectra (δ = 0.29 mm s–1, ΔEQ = 2.24 mm s–1), which was suggested as a “low-spin” 57Fea33+ species at 4.2 K < T < 190 K.[18] We therefore propose that this “low-spin” 57Fea33+ species observed in the Mössbauer experiment at 4.2 K < T < 190 K is a mixture of the Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) and Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F/AF) states.

It was reported that this “low-spin” 57Fea33+ component underwent a transition to a “high-spin” species with ΔEQ ≈ 1 mm s–1 when the temperature was increased above 190 K, and the transition was complete at 245 K.[18] The isomer shift of this new “high-spin” 57Fea33+ species was not given in ref (18). We notice that the calculated ΔEQ values of the Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F/AF) (in Table ) and Fe3+,HS-H2O···OH–-Cu2+(Y237–)(F/AF) (in Table ) states are very similar (1.4–1.6 mm s–1) and are close to the observed ΔEQ ≈ 1 mm s–1. It is likely that when the temperature is increased the Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F/AF) and Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) states start to transform to the Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F/AF) and Fe3+,HS-H2O···OH–-Cu2+(Y237–)(F/AF) states, respectively. Qualitatively, one expects the vibrational entropies to increase (ΔSvib > 0) when Fe3+,LS → Fe3+,IS and Fe3+,IS → Fe3+,HS. The vibrational entropy differences (ΔGvib = −TΔSvib) are not in our current models. They require careful frequency calculations on fairly large models.

When the Fea33+ site is in the intermediate-spin state, the Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) DNC clusters (in Table and Figure ) are 2–3 kcal mol–1 lower in energy than the Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F/AF) (in Table and Figure ) clusters. We have mentioned above that, during geometry optimizations of the Fe3+,IS-OH–···H2O-Cu2+(Y237)(F/AF) structures (neutral Tyr237), the proton on the Cu2+-bound H2O ligand transferred to the Fe3+-bound OH– ligand, and the Fe3+,IS-H2O···OH–-Cu2+(Y237)(F/AF) states are obtained. Therefore, the proton transfer barrier for the transition of Fe3+,IS-OH–···H2O-Cu2+(Y237)(F/AF) → Fe3+,IS-H2O···OH–-Cu2+(Y237)(F/AF) is small. With a further increase in temperature, the Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F/AF) states (anionic tyrosine) also likely undergo proton transfer and change to the Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) states, which then transform to the Fe3+,HS-H2O···OH–-Cu2+(Y237–)(F/AF) states. Therefore, all these transformations will end in Fe3+,HS-H2O···OH–-Cu2+(Y237–)(F/AF) states at 245 K. This proposed transformation process is shown schematically in Figure .

Figure 10

We propose that the so-called “low-spin” 57Fea33+ species observed in the Mössbauer experiments at 4.2 K < T < 190 K is a mixture of the Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F/AF) and Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) states, which transform to the Fe3+,HS-H2O···OH–-Cu2+(Y237–)(F/AF) states with increasing temperature.

To estimate the proton transfer barrier in the transformation of Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F) → Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F) (energy difference ΔE = −2.6 kcal mol–1), starting from the optimized geometry of Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F), we gradually moved the proton on O2 (see Figure ) toward the oxygen atom O1 along the H···O1 direction, then optimized the cluster with the position of this proton (labeled as H in Figure ) fixed. The electronic energies of these optimized clusters with different fixed-H positions are very similar to the originally optimized Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F) cluster. The highest energy structure we obtained is only 2.0 kcal mol–1 higher than the energy of the lowest-energy Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F) structure. The central portion of this highest-energy structure is shown in Figure in which the proton H is at equal distance (1.20 Å) with O1 and O2. The distances of Fe–O1 and Cu–O2 are also the same (2.06 Å). Because of the size of the system, we did not perform transition state search and frequency calculations. According to the features of this structure, it is reasonable to assume that it is close to the transition state of the proton transfer. Then, the barrier for this proton transfer should also be ∼2 kcal mol–1, which is smaller than the energy differences (3.3/5.0 kcal mol–1) between the Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F/AF) and Fe3+,HS-OH–···H2O-Cu2+(Y237–)(F/AF) states (Table ). Therefore, the Fe3+,IS-OH–···H2O-Cu2+(Y237–)(F/AF) states will transform more easily to the Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) states than to the Fe3+,HS-OH–···H2O-Cu2+(Y237–)(F/AF) states with increasing temperature. Such a proton transfer from the water molecule ligating the Cu2+ ion to the hydroxyl ligand of Fe3+ was also obtained in DFT calculations on the oxidized states from two aa3 CcO DNCs (bacterial, Paracoccus denitrificans; and mammalian, bovine) by Sharma et al.[57]

Figure 11

To estimate the energy barrier of the proton (H) transfer of Fe3+,IS-OH−···H2O-Cu2+(Y237−)(F) → Fe3+,IS-H2O···OH−-Cu2+(Y237−)(F) from the position shown in Figure to the position in Figure , we gradually moved the proton along the H···O1 direction toward the O1 atom and then optimized the cluster with the fixed position of this proton. This figure shows the detailed central portion of the highest-energy DNC structure obtained among these H-fixed optimized clusters.

In addition, a high-spin 57Fea33+ species with (δ = 0.41 mm s–1, ΔEQ = 0.7 mm s–1, see Figure ) was also observed in the Mössbauer experiments on Tt, which did not change through the temperature range from 4.2 to 245 K.[18] Next, we present our studies on DNC models generated from the X-ray crystal structures 3EH4 and 1EHK to see if this high-spin 57Fea33+ species could be represented by the DNC structure containing a Cu2+-terminally bound OH– or bridging OH– between Fe3+ and Cu2+.

Fe3+···OH–-Cu2+ DNC Models from 3EH4

Our calculations in sections and 3.4 imply that the Fe2+,HS···OH–-Cu+ DNC in 3EH4 is probably trapped from the earlier oxidized Fe3+,HS···OH–-Cu2+ (or Fe3+,HS-H2O···OH–-Cu2+) structure after X-ray irradiation. We now geometry optimize the Fe3+,HS···OH–-Cu2+(Y237–/Y237)(F/AF) clusters. However, the calculations for the Fe3+,HS···OH–-Cu2+(Y237–/Y237)(AF) states lead instead to Fe3+,IS···OH–-Cu2+(Y237–/Y237)(F) states. Therefore, we present the calculated properties of both Fe3+,IS/HS···OH–-Cu2+(Y237–/Y237)(F) states in Table . In these models (and all the Fe3+···Cu2+ DNC models in the current paper), the three histidine ligands of Cu2+ bind strongly with typical Cu2+–N(His) distances of 2.02–2.15 Å.

Table 8

Calculated Properties of the Oxidized Fe3+···OH–-Cu2+(Y237–/Y237) Models Generated from the DNC of the X-ray Crystal Structure 3EH4a

	geometry										net spin		57Fe3+ Mössbauer
stateb	Fe–N	Fe···O	Cu–O	Fe···Cu	Cu–N (H233)	Cu–N (H282)	Cu–N (H283)	E	pKa (Y237)	Q	Fe3+	Cu2+	δ	ΔEQ
Fe3+,IS···OH–-Cu2+(Y237–)(F)	2.22	4.08	1.91	5.13	2.08	2.07	2.06	–165.4		0	2.62	0.52	0.41	2.38
Fe3+,HS···OH–-Cu2+(Y237–)(F)	2.10	4.29	1.91	5.32	2.08	2.07	2.06	–163.1		0	4.02	0.53	0.57	1.93
Fe3+,IS···OH–-Cu2+(Y237)(F)	2.19	4.11	1.91	5.18	2.11	2.07	2.05	–158.8	6.1	1	2.72	0.53	0.39	2.47
Fe3+,HS···OH–-Cu2+(Y237)(F)	2.08	4.25	1.91	5.33	2.11	2.07	2.05	–155.1	5.0	1	4.11	0.53	0.49	0.34

The properties include geometries (Å), electronic energies (E, offset by −25100 kcal mol–1) obtained from the all-electron/all-TZP single-point energy calculations on the optimized geometries, pKa’s, the net charge (Q) of the clusters, Mulliken net spin polarizations for the Fe and Cu sites, and the 57Fe Mössbauer isomer shifts (δ, mm s–1) and quadrupole splittings (ΔEQ, mm s–1).

F stands for ferromagnetically coupled.

The calculated pKa values for the Tyr237 side chain show that the Fe3+,IS/HS···OH–-Cu2+(Y237–)(F) states with deprotonated anionic Tyr237– are energetically more stable than the corresponding Fe3+,IS/HS···OH–-Cu2+(Y237)(F) states. Further, the Fe3+,IS···OH–-Cu2+(Y237–)(F) state is 2.3 kcal mol–1 lower in energy than the Fe3+,HS···OH–-Cu2+(Y237–)(F) state. Therefore, if the Fe3+···OH–-Cu2+(Y237–)-type DNC exists, it will be in the Fe3+,IS···OH–-Cu2+(Y237–) state at low temperature and will likely transform to the Fe3+,HS···OH–-Cu2+(Y237–) state as the temperature increases, a feature similar to the Fe3+,IS-H2O···OH–-Cu2+(Y237–) → Fe3+,HS-H2O···OH–-Cu2+(Y237–) transition proposed in the previous section. The calculated Mössbauer properties (δ = 0.41/0.57 mm s–1, ΔEQ = 2.38/1.93 mm s–1) of the Fe3+,IS/HS···OH–-Cu2+(Y237–)(F) states are also fairly similar to the corresponding properties (δ = 0.40/0.52 mm s–1, ΔEQ = 2.42/1.49 mm s–1) of the Fe3+,IS/HS-H2O···OH–-Cu2+(Y237–)(F) states. Therefore, the Fe3+,HS···OH–-Cu2+(Y237–) DNC model does not represent the high-spin 57Fea33+ species observed with δ = 0.41 mm s–1 and ΔEQ = 0.7 mm s–1 through 4.2–245 K in the Mössbauer experiments.[18]

Fe3+-OH–-Cu2+ DNC Models from 1EHK

The 1EHK-Fe3+-OH-Cu2+ DNC model shown in Figure was geometry optimized with Fe3+ in LS, IS, and HS states F- or AF-coupled with the Cu2+ site and with Tyr237 side chain in neutral or an anionic state. The main calculated properties of these 1EHK-Fe3+-OH-Cu2+ cluster models are given in Table . Note that we did not obtain the optimized structures for the Fe3+,LS-OH–-Cu2+(Y237–)(F) and Fe3+,IS-OH–-Cu2+(Y237–)(F) states. Calculations for these two states led to the Fe3+,IS-OH–-Cu2+(Y237–)(AF) and Fe3+,HS-OH–-Cu2+(Y237–)(AF) states, respectively.

Table 9

Calculated Properties of the Oxidized Fe3+-OH–-Cu2+(Y237–/Y237) Clusters Generated from the DNC of the 1EHK X-ray Crystal Structurea

	geometry											net spin		57Fe3+ Mössbauer
stateb	Fe–N (H384)	Fe–O	Cu–O	Fe···Cu	Cu–N (H233)	Cu–N (H282)	Cu–N (H283)	O···O (Y237)	Ec	pKa (Y237)	Q	Fe3+	Cu2+	δ	ΔEQ
Fe3+,LS-OH–-Cu2+(Y237–)(AF)	3.72	1.81	2.72	4.31	2.02	2.08	2.04	2.80	–157.3		0	0.99	–0.29	0.24	3.03
Fe3+,IS-OH–-Cu2+(Y237–)(AF)	3.78	1.98	2.15	3.93	2.06	2.11	2.15	2.77	–163.0		0	2.65	–0.32	0.37	2.02
Fe3+,HS-OH–-Cu2+(Y237–)(F)	4.05	1.91	2.31	3.97	2.05	2.09	2.09	2.83	–166.1		0	4.09	0.34	0.39	0.54
Fe3+,HS-OH–-Cu2+(Y237–)(AF)	4.08	1.91	2.30	3.97	2.05	2.10	2.09	2.82	–167.5		0	4.08	–0.29	0.38	0.51
Fe3+,HS-OH–-Cu2+(Y237)(F)	4.13	1.94	2.19	3.90	2.05	2.06	2.04	3.08	–150.6	–0.4	1	4.09	0.49	0.41	0.42
Fe3+,HS-OH–-Cu2+(Y237)(AF)	4.11	1.93	2.21	3.91	2.05	2.07	2.05	3.05	–153.4	0.6	1	4.04	–0.43	0.39	0.40

The properties include geometries (Å), electronic energies (E, offset by −25100 kcal mol–1) obtained from the all-electron/all-TZP single-point energy calculations on the optimized geometries, pKa’s, the net charge (Q) of the clusters, Mulliken net spin polarizations for the Fe and Cu sites, and the 57Fe Mössbauer isomer shifts (δ, mm s–1) and quadrupole splittings (ΔEQ, mm s–1).

F stands for ferromagnetically-coupled and AF for antiferromagnetically-coupled.

The energies calculated for the AF-coupled states are broken-symmetry state energies.

The calculated pKa(Y237) values (−0.4 and 0.6) for the Fe3+,HS-OH–-Cu2+(Y237)(F/AF) models clearly show that the Fe3+,HS-OH–-Cu2+-type DNC energetically favors the anionic form of the Tyr237 side chain.

Both Fe3+,HS-OH–-Cu2+(Y237–)(F) and Fe3+,HS-OH–-Cu2+(Y237–)(AF) states yield very similar geometric and Mössbauer properties. The Fe3+,HS-OH–-Cu2+(Y237–)(AF) state yields the lowest energy, which is 10.2, 4.5, and 1.4 kcal mol–1 lower in energy than the Fe3+,LS-OH–-Cu2+(Y237–)(AF), Fe3+,IS-OH–-Cu2+(Y237–)(AF), and Fe3+,HS-OH–-Cu2+(Y237–)(F) states, respectively. Therefore, if the oxidized DNC exists in the Fe3+-OH–-Cu2+ form with a bridging hydroxo, it would stay in the Fe3+,HS-OH–-Cu2+(Y237–)(AF) state. Further, we noticed that the calculated Mössbauer properties (δ = 0.38 mm s–1, ΔEQ = 0.51 mm s–1) of this Fe3+,HS-OH–-Cu2+(Y237–)(AF) state are in very good agreement with the observed Mössbauer spectra (δ = 0.41 mm s–1, ΔEQ = 0.7 mm s–1).[18] Therefore, it is very likely that the Fe3+,HS-OH–-Cu2+(Y237–)(AF) DNC structure represents the observed (δ = 0.41 mm s–1, ΔEQ = 0.7 mm s–1) 57Fea33+ high-spin species, which exists through the 4.2–245 K range in the the Mössbauer experiments.[18]

Conclusions

The X-ray crystal structures (PDB entries: 1EHK,[10]1XME,[11]3EH3,[12]3EH4,[12]3EH5,[12]3S8F,[15] and 3S8G(15)) of the reduced ba3 cytochrome c oxidase (CcO) from Thermus thermophilus (Tt) showed different Fea32+···CuB+ dinuclear center (DNC) structures. Mössbauer experiments on the ba3 CcO from Tt also revealed DNC structural heterogeneities in both oxidized and reduced states. Both radiolytic reduction and chemical reduction[10−12,15] with dithionite[58] will typically generate high-energy reduced state intermediates compared to the physiological donor cytochrome c, which has a midpoint redox potential of approximately +0.20 to +0.25 V relative to SHE. By contrast, the dithionite midpoint potential is quite negative and depends also on the dithionite initial (and final) concentrations. Use of the dithionite chemical reduction data from Zimmermann et al.,[18] and from Liu et al.[12] in combination with Mayhew’s[58] analysis, yields estimated dithionite midpoint potentials of about −0.50 and −0.45 V, respectively. So there is an additional reductive driving force of about 0.7 to 0.8 V using dithionite relative to cytochrome c. Radiolytic reduction of the X-ray structures in the synchrotron X-ray beam is also highly reducing[10−12,15] although less predictable. The resulting unstable or metastable intermediates can be trapped at low temperatures (the Cryogenic X-ray structures are at ∼100 K). The X-ray structures in Table (particularly 3EH3 and 3EH5) are consistent with the view that chemical reduction prior to freezing in liquid N2 and X-ray irradiation yield more geometrically and energetically relaxed structures.

Using DFT OLYP potential, we have performed geometric, energetic, and Mössbauer property calculations on the DNCs of the X-ray crystal structures listed above. Our calculations support that the 57Fea32+ sites observed in the Mössbauer experiments are in high-spin states. The oxygen species between the Fe2+ and Cu+ sites found in 1EHK,[10]1XME,[11] and 3EH4,[12] is better represented by a hydroxide anion (OH–). The first (majority) 57Fea32+ component observed in the Mössbauer experiment is best represented by the DNC in the Fe2+,HS···OH–-Cu+ state, where OH– terminally binds with Cu+ and the Cu+ is in a 4- or 3-coordinate configuration. With OH– as a ligand, the Cu+ site energetically favors a linear OH–-Cu+-N(His282) 2-coordinate (2c) configuration with the His233 and His283 side chains dissociating from the Cu+ site. However, steric interactions, especially from the side chain of Trp229 (not included in the current model calculations), which is beside the His283 side chain, the potential energy barriers for geometry rearrangement, and the low temperature under the experimental conditions may prevent the formation of the Fe2+,HS···OH–-Cu+2c state. Further, a reduced DNC with OH– between the Fe2+,HS and the Cu+ sites are not in the equilibrium state. The system strongly favors a water molecule (if there is solvent oxygen species) rather than a hydroxide anion in the DNC. Therefore, the reduced Fe2+,HS···OH–-Cu+/Fe2+,HS-OH–-Cu+ DNCs found in the Mössbauer experiments/X-ray crystal structures should arise from the earlier oxidized state with structures like Fe3+,HS···OH–-Cu2+/Fe3+,HS-OH–-Cu2+ and be trapped at very low temperature.

Our calculations indeed have shown that the DNC structure of Fe3+,HS-OH–-Cu2+(Y237–)(AF), in which a hydroxo bridges the AF-coupled high-spin Fe3+ and Cu2+ sites, yields very similar Mössbauer properties (δ = 0.38 mm s–1, ΔEQ = 0.51 mm s–1) to the observed 57Fea33+ high-spin species (δ = 0.41 mm s–1, ΔEQ = 0.7 mm s–1) in the temperature range of 4.2–245 K. We have also found that the DNC structure of the observed “low-spin” 57Fea33+ species at 4.2–190 K with (δ = 0.29 mm s–1, ΔEQ = 2.24 mm s–1)[18] is likely a mixture of both the Fe3+,LS-OH–···H2O-Cu2+(Y237–)(F/AF) and Fe3+,IS-H2O···OH–-Cu2+(Y237–)(F/AF) states, and they gradually transition to the Fe3+,HS-H2O···OH–-Cu2+(Y237–)(F/AF) states with increasing temperature (see Figure ), which explains the observed transition of “low-spin” 57Fea33+ to high-spin 57Fea33+ in the Mössbauer experiment when the temperature was increased from 190 to 245 K.[18]

Previous resonance Raman experiments on ba3 CcO from Tt (at ambient temperature) also discovered two high-spin Fea33+ species with one in an in-plane (6-coordinated) and another in an out-of-plane (5-coordinated) configuration.[59] The high-spin Fea33+ DNCs we have proposed here, Fe3+,HS-H2O···OH–-Cu2+(Y237–)(F/AF) and Fe3+,HS-OH–-Cu2+(Y237–)(AF), are indeed in 6- and 5- coordinated configurations, respectively. The average dihedral angle between Fe3+ and three of the four ligating interior N atoms in the Fe3+,HS-H2O···OH–-Cu2+(Y237–)(F/AF) structures is 5°, whereas the corresponding value in Fe3+,HS-OH–-Cu2+(Y237–)(AF) is −20°. The Fe3+ ion in the Fe3+,HS-OH–-Cu2+(Y237–)(AF) structure is certainly in the out-of-plane position toward the bridging OH–.

Very recently, Sharma et al. presented their computational study of a metastable but activated ferric/cupric state (OH in their notation) in the catalytic cycle of aa3 CcO.[57] The experimental evidence suggests that the DNC in the catalytically relevant OH state may have high-spin Fea33+ with a strongly H-bonded distal hydroxide ligand and CuB2+ with a raised redox potential.[57] On the basis of their DFT calculations, Sharma et al. have proposed that the high energy and high redox potential of CuB in the OH state may stem from a 3-coordinated near-planar geometry of CuB2+ (first alternative).[57] As a second alternative, they have also proposed that the DNC may be compact in state OH with a very short Fe–Cu distance and a bridging μ-hydroxo between the two metals.[57] Note that this proposed second alternative OH structure has a very similar DNC structure to our calculated Fe3+,HS-OH–-Cu2+(Y237–)(AF) state.

To date, several DNC structures in our calculations yield very similar 57Fe2+,HS Mössbauer properties, which are very close to the properties of the second 57Fea32+ component observed in the Mössbauer experiments (Figure ).[18] These structures include: (1) the open Fe2+,HS···Cu+ DNC structure as observed in the X-ray crystal structures 3EH3 and 3EH5,[12] where no oxygen species is between the Fe2+,HS and Cu+ sites, and the Cu+ binds with all three His233, His282, and His283 side chains; (2) similar structures to the open Fe2+,HS···Cu+ structure, but with one (or more) water molecule(s) staying between but not binding with the Fe2+,HS and Cu+ sites; (3) the Fe2+,HS···OH–-Cu+2c-His282 DNC structure, where the His233 and His283 side chains dissociate from Cu+ and the 2-coordinate Cu+ site linearly binds with OH– and the His282 side chain; (4) the Fe2+,HS···H2O-Cu+3c-type structure, in which the 3-coordinate Cu+ site binds with an H2O and the His233 and His282 side chains; and (5) the Fe2+,HS···His233-Cu+2c-His282 structure with the H2O ligand in structure (4) dissociating from the Cu+ site and the Cu+ binding with only the His233 and His282 side chains.

It is not known if the low coordination Cu+2c state exists in the ba3 CcO from Tt during its catalytic cycle. However, the variation of the Cu+ coordination state and the mobility of the His233 and His283 side chains shown in our calculations do suggest that the His233 and His283 side chains can be potential proton loading sites during the catalytic cycle and in the proton pumping process. Considering the high optimal growth temperature of Thermus thermophilus, such variation of the Cu+ coordination state is possible. If His283 side chain dissociates and is then protonated to form a His283+ cation, the nearby Trp229 side chain could potentially stabilize this state through a cation-π interaction,[60] but the relative His283-Trp229 geometry must then be nearly optimal. Further calculations with larger DNC models, including the Trp229 side chain, are planned in the near future.