Impact of D1-V185 on the Water Molecules That Facilitate O2 Formation by the Catalytic Mn4CaO5 Cluster in Photosystem II

The oxidations of the O-2-evolving Mn4CaO5 cluster in Photosystem II are coupled to the release of protons to the thylakoid lumen via one or more proton egress pathways. These pathways are comprised of extensive networks of hydrogen-bonded water molecules and amino acid side chains. The hydrophobic residue, D1-V185, is adjacent to numerous water molecules in one of these pathways. The D1-V185N mutation dramatically slows O-O bond formation. This impairment has been attributed to a disruption of the hydrogen-bonded water molecules that are crucial for proton egress or whose rearrangement is required for catalysis. In this study, Fourier transform infrared spectroscopy was employed to characterize the impact of the D1-V185N mutation on the carboxylate groups and water molecules that form a network of hydrogen bonds in this putative proton egress pathway. By analyzing carboxylate stretching modes, carbonyl stretching modes of hydrogen-bonded carboxylic acids, O-H stretching modes of hydrogen-bonded water molecules, and D-O-D bending modes, we obtain evidence that the D1-V185N mutation perturbs the extensive network of hydrogen bonds that extends from Y-Z to D1-D61 to a greater extent than any mutation yet examined but does not alter the water molecules that interact directly with D1-D61. The mutation also alters the environments of the carboxylate groups whose pK(a) values change in response to the S-1 to S-2 and S-2 to S-3 transitions. Finally, the mutation alters the environment of the water molecule whose bending mode vanishes during the S-2 to S-3 transition, consistent with assigning the Ca2+-bound W3 as the water molecule that deprotonates and joins oxo bridge O5 during the S-2 to S-3 transition, possibly as the second substrate water molecule for O-2 formation.