title: Evidence from directed mutagenesis that aspartate 170 of the D1 polypeptide influences the assembly and/or stability of the manganese cluster in the photosynthetic water-splitting complex.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Minnesota, St. Paul 55108.
authors.forename: R J
authors.initials: RJ
authors.lastname: Boerner
authors.forename: A P
authors.initials: AP
authors.lastname: Nguyen
authors.forename: B A
authors.initials: BA
authors.lastname: Barry
authors.forename: R J
authors.initials: RJ
authors.lastname: Debus
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D002734:Chlorophyll / Q000032:analysis / Q000378:metabolism; D000077194:Chlorophyll A; D000458:Cyanobacteria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004237:Diuron / Q000494:pharmacology; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes; D046911:Macromolecular Substances; D008345:Manganese / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010788:Photosynthesis / Q000187:drug effects; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence
chemical_list: D045342:Light-Harvesting Protein Complexes; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D002734:Chlorophyll; D001224:Aspartic Acid; D008345:Manganese; D004237:Diuron; D000077194:Chlorophyll A