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.

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.

To identify amino acid residues that influence the assembly or stability of the manganese cluster in photosystem II, we have generated site-directed mutations in the D1 polypeptide of the cyanobacterium, Synechocystis sp. PCC 6803. Indirect evidence has suggested that the D1 polypeptide provides some of the ligands that are required for metal binding. Mutations at position 170 of D1 were selected for characterization, since an aspartate to asparagine mutation (DN170D1) at this position completely abolishes photoautotrophic growth, while retention of a carboxylic acid at this position (aspartate to glutamate, DE170D1) supports photoautotrophic growth. Photosystem II particles were purified from control, DE170D1, and DN170D1 cells by a procedure that retains high rates of oxygen evolution activity in control particles [Noren, G.H., Boerner, R.J., & Barry, B.A. (1991) Biochemistry 30, 3943-3950]. Spectroscopic analysis shows that the tyrosine radical, Z+, which normally oxidizes the manganese cluster, is rapidly reduced in the DE170D1 mutant, but not in the DN170D1 mutant. A possible explanation of this block or dramatic decrease in the rate of electron transfer between the manganese cluster and tyrosine Z is an alteration in the properties of the metal center. Quantitation of manganese in these particles is consistent with aspartate 170 influencing the stability or assembly of the manganese cluster, since the aspartate to asparagine mutation results in a decrease in the manganese content per reaction center. Photosystem II particles from DN170D1 show a 60% decrease in the amount of specifically bound manganese per reaction center, when compared to control particles. Also, we observe a 70% decrease in the amount of specifically bound manganese per reaction center in partially purified DN170D1 particles and at least an 80% decrease in the amount of hydroxylamine-reducible manganese in DN170D1 thylakoid membranes. Single-turnover fluorescence assays and steady-state EPR measurements demonstrate that the remaining, endogenous manganese does not rapidly reduce tyrosine Z+ in the DN170D1 mutant. Additional evidence that aspartate 170 influences the assembly or stability of the metal site comes from analysis of the DE170D1 mutant. Although this mutant assembles a functional manganese cluster, as assessed by oxygen evolution and spectroscopic assays, the properties of the manganese site are perturbed.

Photosystem II, the photosynthetic water oxidizing complex, contains two well characterized redox active tyrosines, D and Z. D forms a stable radical of unknown function. Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site. The vibrational difference spectra associated with the oxidation of tyrosines Z and D have been obtained through the use of infrared spectroscopy (MacDonald, G. M., Bixby, K.A., and Barry, B.A. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11024-11028). Here, we examine the effect of deuterium exchange on these vibrational difference spectra. While the putative C-O vibration of stable tyrosine radical D. downshifts in 2H2O, the putative C-O vibration of tyrosine radical Z. does not. This result is consistent with the existence of a hydrogen bond to the phenol oxygen of the D. radical; we conclude that a hydrogen bond is not formed to the Z. radical. In an effort to identify the amino acid residue that is the proton acceptor for Z, we have performed global 15N labeling. While significant 15N shifts are observed in the vibrational difference spectrum, substitution of a glutamine for a histidine that is predicted to lie in the environment of tyrosine Z has little or no effect on the difference infrared spectrum. There is also no significant change in the yield or lineshape of the Z. EPR signal under continuous illumination in this mutant. Our results are inconsistent with the possibility that this residue, histidine 190 of the D1 polypeptide, acts as the sole proton acceptor for tyrosine Z.