Several site-directed photosystem II mutants with substitutions at Asp-170 of the D1 polypeptide were characterized by noninvasive methods in vivo. In several mutants, including some that evolve oxygen, a significant fraction of photosystem II reaction centers are shown to lack photooxidizable Mn ions. In this fraction of reaction centers, either the high-affinity site from which Mn ions rapidly reduce the oxidized secondary electron donor, YZ+, is devoid of Mn ions or the Mn ion(s) bound at this site are unable to reduce YZ+. It is concluded that the Mn clusters in these mutants are unstable or are assembled inefficiently in vivo. Mutants were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803. The in vivo characterization procedures employed in this study involved measuring changes in the yield of variable chlorophyll a fluorescence following a saturating flash or brief illumination given in the presence of the electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, or following each of a series of saturating flashes given in the absence of this inhibitor. These procedures are easily applied to mutants that evolve little or no oxygen, facilitate the characterization of mutants with labile oxygen-evolving complexes, permit photosystem II isolation efforts to be concentrated on mutants having the stablest Mn clusters, and guide systematic spectroscopic studies of isolated photosystem II particles to mutants of particular interest.
To identify amino acid residues that ligate the manganese and calcium ions of photosystem II or that are otherwise crucial to water oxidation, site-directed mutations were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803 at all conserved carboxylate, histidine, and tyrosine residues in the lumenal interhelical domains of the D1 polypeptide. Mutants with impaired photoautotrophic growth or oxygen evolution were characterized in vivo by measuring changes in the yield of variable chlorophyll a fluorescence after a saturating flash or brief illumination given in the presence of an electron-transfer inhibitor or following each in a series of saturating flashes given in the absence of inhibitor [Chu, H.-A., Nguyen, A. P., & Debus, R. J. (1994) Biochemistry 33, 6137-6149]. Mutants were also characterized after propagation in media having other cations substituted for calcium. We conclude that Asp-59 and Asp-61 may ligate calcium, that Asp-59, Asp-61, Glu-65, and His-92 influence the properties of the manganese cluster without significantly affecting its stability or ability to assemble, that Glu-189 plays an important structural role in maintaining the catalytic efficiency of the Mn cluster and partly influences the cluster's stability or ability to assemble, that His-92 and Glu-189 influence the binding of calcium, and that His-190 strongly influences the redox properties of the secondary electron donor, YZox, and either ligates manganese or serves as a crucial base or hydrogen bond donor. In addition, we conclude that Asp-170 may ligate manganese, but that its replacement with Val, Leu, or Ile causes structural perturbations that partly compensate for the loss of the carboxylate moiety.
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-(13)C]alanine or unlabeled ((12)C) 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 S(2)-minus-S(1) 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-(13)C]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 D1 polypeptide, we assign the L-[1-(13)C]alanine-sensitive symmetric carboxylate stretching mode to the alpha-COO(-) group of D1-Ala344. In unlabeled wild-type PSII particles, this mode appears at approximately 1356 cm(-1) in the S(1) state and at approximately 1339 or approximately 1320 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.
To identify amino acid residues that ligate the manganese and calcium ions of photosystem II or are otherwise crucial to water oxidation, site-directed mutations were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803 at all conserved carboxylate and histidine residues in the carboxy-terminal domain of the D1 polypeptide. Mutants with impaired photoautotrophic growth or oxygen evolution were characterized in vivo by measuring changes in the yield of variable chlorophyll a fluorescence after a saturating flash or brief illumination given in the presence of an electron-transfer inhibitor or following each in a series of saturating flashes given in the absence of inhibitor [Chu, H.-A., Nguyen, A. P., & Debus, R.J. (1994) Biochemistry 33, 6137-6149]. Mutants were also characterized after propagation in media having other cations substituted for calcium. We conclude that His-332 Glu-333, His-337, and Asp-342 influence the assembly and/or stability of the manganese cluster, that His-332, Glu-333, and His-337 may ligate manganese, that Asp-342 may ligate manganese, calcium, or both, that Glu-333 and Asp-342 may play important structural roles, and that His-332, Glu-333, and His-337 influence the binding of calcium, although Glu-333 is unlikely to ligate Ca2+ directly. Several His-332, Glu-333, His-337, and Asp-342 mutants were very light sensitive, possibly because toxic activated oxygen species were released from altered or partly assembled manganese clusters. Finally, mutations at Asp-342 do not prevent posttranslational cleavage of the carboxy-terminal extension of the D1 polypeptide's precursor form in vivo.
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