No evidence from FTIR difference spectroscopy that glutamate-189 of the D1 polypeptide ligates a Mn ion that undergoes oxidation during the S₀ to S₁, S₁ to S₂, or S₂ to S ₃ transitions in photosystem II

In the recent X-ray crystallographic structural models of photosystem II, Glu189 of the D1 polypeptide is assigned as a ligand of the oxygen-evolving Mn₄ cluster. To determine if D1-Glu189 ligates a Mn ion that undergoes oxidation during one or more of the S₀ → S ₁, S₁ → S₂, and S₂ → S₃ transitions, the FTIR difference spectra of the individual S-state transitions in D1-E189Q and D1-E189R mutant PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 were compared with those in wild-type PSII particles. Remarkably, the data show that neither mutation significantly alters the mid-frequency regions (1800-1200 cm^-1) of any of the FTIR difference spectra. Importantly, neither mutation eliminates any specific symmetric or asymmetric carboxylate stretching mode that might have been assigned to D1-Glu189. The small spectral alterations that are observed are similar in amplitude to those that are observed in wild-type PSII particles that have been exchanged into FTIR analysis buffer by different methods or those that are observed in D2-H189Q mutant PSII particles (the residue D2-His189 is located >25 Å from the Mn₄ cluster and accepts a hydrogen bond from Tyr Y_D). The absence of significant mutation-induced spectral alterations in the D1-Glu189 mutants shows that the oxidation of the Mn₄ cluster does not alter the frequencies of the carboxylate stretching modes of D1-Glu189 during the S₀ → S₁, S₁ → S₂, or S₂ → S₃ transitions. One explanation of these data is that D1-Glu189 ligates a Mn ion that does not increase its charge or oxidation state during any of these S-state transitions. However, because the same conclusion was reached previously for D1-Asp170, and because the recent X-ray crystallographic structural models assign D1-Asp170 and D1-Glu189 as ligating different Mn ions, this explanation requires that (1) the extra positive charge that develops on the Mn₄ cluster during the S₁ → S₂ transition be localized on the Mn ion that is ligated by the α-COO^- group of D1-Ala344 and (2) any increase in positive charge that develops on the Mn₄ cluster during the S₀ → S₁ and S₂ → S₃ transitions be localized on the one Mn ion that is not ligated by D1-Asp170, D1-Glu189, or D1-Ala344. An alternative explanation of the FTIR data is that D1-Glu189 does not ligate the Mn₄ cluster. This conclusion would be consistent with earlier spectroscopic analyses of D1-Glu189 mutants, but would require that the proximity of D1-Glu189 to manganese in the X-ray crystallographic structural models be an artifact of the radiation-induced reduction of the Mn₄ cluster that occurred during the collection of the X-ray diffraction data.