Journal
BIOCHEMISTRY
Volume 43, Issue 11, Pages 3152-3166Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi035915f
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- NIGMS NIH HHS [GM37300] Funding Source: Medline
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Isotope-edited FTIR difference spectroscopy was employed to determine if the C-terminal alpha-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 S-1 --> S-2 transition. Wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803 were propagated photoautotrophically in the presence of L-[1-C-13]alanine or unlabeled (C-12) L-alanine. In wild-type cells, both the C-terminal alpha-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 alpha-COO- group of the D1 polypeptide will not be labeled because this group is no longer provided by alanine. The resultant S2-minus-S, FTIR difference spectra of purified wild-type and mutant PSII particles showed that one symmetric carboxylate stretching mode that is altered during the S-1 --> S-2 transition is sensitive to L-[1-C-13]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 alpha-COO- group of the DI polypeptide, we assign the L-[l-C-13]alanine-sensitive symmetric carboxylate stretching mode to the alpha-COO- group of D1-Ala344. In unlabeled wild-type PSII particles, this mode appears at similar to1356 cm(-1) in the S-1 state and at similar to1339 or similar to1320 cm(-1) in the S-2 state. These frequencies are consistent with unidentate ligation of the (Mn)(4) cluster by the alpha-COO- group of D1-Ala344 in both the S-1 and S-2 states. The apparent 17-36 cm(-1) downshift in frequency in response to the S-1 --> S-2 transition is consistent with the alpha-COO- group of D1-Ala344 ligating a Mn ion whose charge increases during the S-1 --> S-2 transition. Accordingly, we propose that the alpha-COO- group of D1-Ala344 ligates the Mn ion that undergoes an oxidation during the S-1 --> S-2 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.
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