Site-directed mutagenesis of conserved histidines in the helix VIII domain of PsaB impairs assembly of the photosystem I reaction center without altering spectroscopic characteristics of P700.
The chloroplast psaB gene encodes one of the polypeptides of the photosystem I reaction center heterodimer that coordinates the electron transfer components P700, A0, and A1. Histidine residues in the most highly conserved region of the PsaB protein are predicted to coordinate the P700 reaction center chlorophyll(s) and the initial electron acceptor, A0. Oligonucleotide-mediated site-directed mutagenesis and chloroplast transformation of Chlamydomonas reinhardtii have been used to determine the importance of these conserved histidines in photosystem I reaction center biogenesis and function. It is demonstrated that these histidine residues are essential for stable accumulation of the photosystem I reaction center. Protein pulse-labeling shows that changing the histidine residues impairs a post-translational step in reaction center assembly. Photosystem I complexes from the mutants have been characterized by Electron Nuclear Double Resonance and Electron Spin Echo Envelope Modulation spectroscopy to determine the impact of any mutations on P700+. In all cases we determine that spectroscopic characteristics of P700+ remain unchanged. The implications of these results to current models of the photosystem I reaction center and related bacterial reaction centers are discussed.
Department of Botany, Arizona State University, Tempe 85287-1601.
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Cui
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Bingham
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Kuhn
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Käss
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Lubitz
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Webber
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002696:Chlamydomonas / Q000737:chemistry*; D002736:Chloroplasts / Q000737:chemistry* / Q000235:genetics; D017124:Conserved Sequence; D015342:DNA Probes; D004578:Electron Spin Resonance Spectroscopy; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics; D045331:Photosystem I Protein Complex; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D006639:Histidine