Evidence from FTIR difference spectroscopy that D1-Asp61 influences the water reactions of the oxygen-evolving Mn4CaO5 cluster of photosystem II.

Understanding the mechanism of photosynthetic water oxidation requires characterizing the reactions of the water molecules that serve as substrate or that otherwise interact with the oxygen-evolving Mn4CaO5 cluster. FTIR difference spectroscopy is a powerful tool for studying the structural changes of hydrogen bonded water molecules. For example, the O-H stretching mode of water molecules having relatively weak hydrogen bonds can be monitored near 3600 cm(-1), the D-O-D bending mode can be monitored near 1210 cm(-1), and highly polarizable networks of hydrogen bonds can be monitored as broad features between 3000 and 2000 cm(-1). The two former regions are practically devoid of overlapping vibrational modes from the protein. In Photosystem II, water oxidation requires a precisely choreographed sequence of proton and electron transfer steps in which proton release is required to prevent the redox potential of the Mn4CaO5 cluster from rising to levels that would prevent its subsequent oxidation. Proton release takes place via one or more proton egress pathways leading from the Mn4CaO5 cluster to the thylakoid lumen. There is growing evidence that D1-D61 is the initial residue of one dominant proton egress pathway. This residue interacts directly with water molecules in the first and second coordination spheres of the Mn4CaO5 cluster. In this study, we explore the influence of D1-D61 on the water reactions accompanying oxygen production by characterizing the FTIR properties of the D1-D61A mutant of the cyanobacterium, Synechocystis sp. PCC 6803. On the basis of mutation-induced changes to the carbonyl stretching region near 1747 cm(-1), we conclude that D1-D61 participates in the same extensive networks of hydrogen bonds that have been identified previously by FTIR studies. On the basis of mutation-induced changes to the weakly hydrogen-bonded O-H stretching region, we conclude that D1-D61 interacts with water molecules that are located near the Cl(-)(1) ion and that deprotonate or participate in stronger hydrogen bonds as a result of the S1 to S2 and S2 to S3 transitions. On the basis of the elimination of a broad feature between 3100 and 2600 cm(-1), we conclude that the highly polarizable network of hydrogen bonds whose polarizability or protonation state increases during the S1 to S2 transition involves D1-D61. On the basis of the elimination of features in the D-O-D bending region, we conclude that D1-D61 forms a hydrogen bond to one of the H2O molecules whose H-O-H bending mode changes in response to the S1 to S2 transition. The elimination of this H2O molecule in the D1-D61A mutant provides one rationale for the decreased efficiency of water oxidation in this mutant. Finally, we discuss reasons why the recent conclusion that a substrate-containing cluster of five water molecules accepts a proton from the Mn4CaO5 cluster during the S1 to S2 transition and deprotonates during subsequent S state transitions should be reassessed.