Network of Hydrogen Bonds near the Oxygen-Evolving Mn4CaO5 Cluster of Photosystem II Probed with FTIR Difference Spectroscopy

We previously provided experimental evidence that an extensive network of hydrogen bonds exists near the oxygen-evolving Mn4CaO5 cluster in photosystem II and that elements of this network form part of a dominant proton-egress pathway leading from the Mn4CaO5 cluster to the thylakoid lumen. The evidence was based on (i) the elimination of the same nu(C=O) mode of a protonated carboxylate group in the S-2-minus-S-1 FTIR difference spectrum of wild-type PSII core complexes from the cyanobacterium Synechocystis sp. PCC 6803 by the mutations D1-E65A, D2-E312A, and D1-E329Q and (ii) the substantial decrease in the efficiency of the S-3 to S-0 transition caused by the mutations D1-D61A, D1-E65A, and D2-E312A. The eliminated nu(C=O) mode corresponds to an unidentified carboxylate group whose pK(a) value decreases in response to the increased charge that develops on the Mn4CaO5 cluster during the S-1 to S-2 transition. In the current study, we have extended our work to include the nu(C=O) regions of other Sn+1-minus-S-n FTIR difference spectra and to additional mutations of residues inferred to participate in networks of hydrogen bonds near the Mn4CaO5 cluster or leading from the Mn4CaO5 cluster to the thylakoid lumen. Our data suggest that a different carboxylate group has its pK(a) value increased during the S-2 to S-3 transition and that a third carboxylate group experiences a change in its environment during the S-0 to S-1 transition. The pK(a) values that shift during the S-1 to S-2 and S-2 to S-3 transitions appear to be restored during the S-3 to S-0 transition. The D1-R334A mutation decreases or eliminates the same nu(C=O) modes from the S-2-minus-S-1 and S-3-minus-S-2 spectra as mutations D1-E65A, D2-E312A, and DI-E329Q and substantially decreases the efficiency of the S-3 to S-0 transition. We conclude that Dl-R334 participates in the same dominant proton-egress pathway that was identified in our previous study. The D1-Q165E mutation leaves the nu(C=O) region of the S-2-minus-S-1 FTIR difference spectrum intact, but it eliminates a mode from this region of the S-3-minus-S-2 spectrum. We conclude that D1-Q165 participates in an extensive network of hydrogen bonds that that extends across the Mn4CaO5 cluster to the D1-E65/D2-E312 dyad and that includes D1-E329 and several water molecules including the W2 and W3 water ligands of the Mn4CaO5 cluster's dangling Mnm and Ca ions, respectively. The D2-E307Q D2-D308N, D2-E310Q, and D2-E323Qmutations alter the nu(C=O) regions of none of the FTIR difference spectra. We conclude that these four residues are located far from the three unidentified carboxylate groups that give rise to the nu(C=O) features observed in the FT1R difference spectra.