Evidence that the C-Terminus of the D1 Polypeptide of Photosystem II Is Ligated to the Manganese Ion that Undergoes Oxidation during the S₁ to S₂ Transition: An Isotope-Edited FTIR Study

Isotope-edited FTIR difference spectroscopy was employed to determine if the C-terminal α-COO^- group of the D1 polypeptide ligates the (Mn)₄ cluster in photosystem II (PSII) and, if so, if it ligates the Mn ion that undergoes an oxidation during the S₁ → S ₂ transition. Wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803 were propagated photoautotrophically in the presence of L-[1-¹³C]alanine or unlabeled (¹²C) L-alanine. In wild-type cells, both the C-terminal α-COO^- group of the D1 polypeptide at D1-Ala344 and all alanine-derived peptide carbonyl groups will be labeled. In D1-A344G and D1-A344S mutant cells, the C-terminal α-COO^- group of the D1 polypeptide will not be labeled because this group is no longer provided by alanine. The resultant S ₂-minus-S₁ FTIR difference spectra of purified wild-type and mutant PSII particles showed that one symmetric carboxylate stretching mode that is altered during the S₁ → S₂ transition is sensitive to L-[1-¹³C]alanine-labeling in wild-type PSII particles but not in D1-A344G and D1-A344S PSII particles. Because the only carboxylate group that can be labeled in the wild-type PSII particles but not in the mutant PSII particles is the C-terminal α-COO^- group of the D1 polypeptide, we assign the L-[1-¹³C]alanine-sensitive symmetric carboxylate stretching mode to the α-COO^- group of D1-Ala344. In unlabeled wild-type PSII particles, this mode appears at ∼1356 cm ^-1 in the S₁ state and at ∼1339 or ∼1320 cm ^-1 in the S₂ state. These frequencies are consistent with unidentate ligation of the (Mn)₄ cluster by the α-COO ^- group of D1-Ala344 in both the S₁ and S₂ states. The apparent 17-36 cm^-1 downshift in frequency in response to the S₁ → S₂ transition is consistent with the α-COO^- group of D1-Ala344 ligating a Mn ion whose charge increases during the S₁ → S₂ transition. Accordingly, we propose that the α-COO^- group of D1-Ala344 ligates the Mn ion that undergoes an oxidation during the S₁ → S₂ transition. Control experiments were conducted with Mn-depleted wild-type PSII particles. These experiments showed that tyrosine Y_D may be structurally coupled to the carbonyl oxygen of an alanine-derived peptide carbonyl group.