Mutagenesis of photosystem I in the region of the ferredoxin cross-linking site: modifications of positively charged amino acids.
The psaD gene isolated from the cyanobacterium Synechocystis sp. PCC 6803 has been mutated in the region encoding a cross-linking site for ferredoxin. A glucose tolerant strain of Synechocystis 6803 was first deleted for psaD, and the resulting PS-I was characterised by EPR and flash absorption spectroscopy. The major modification related to the absence of the PsaD subunit is the disappearance of the first order reduction of ferredoxin which is replaced by a second order reaction. Reconstitution of the deleted PS-I with the purified PsaD polypeptide restored 80% of the fast photoreduction of ferredoxin. The deletion of PsaD has no apparent effect on the main biochemical features of the resulting depleted PS-I complex, with the exception of minor modifications to the FA/FB centers. The deleted strain was transformed by a series of psaD genes mutated at three conserved residues, all located close to the ferredoxin cross-linking site. The resulting photosystem I complexes were extensively studied by flash absorption spectroscopy. Unexpectedly, the change of Lys 106 involved in the cross-linking of ferredoxin for an uncharged amino acid has almost no effect (mutation K106A). However, the functional consequences of more drastic substitutions of either Lys 106 or Arg 111 indicate a role for these two basic amino acids in the binding and submicrosecond reduction of ferredoxin. Various mutations of the unique His at position 97 show that this amino acid is involved in the increased affinity of PS-I for ferredoxin when the pH is lowered. This histidine could be central in regulating in vivo the rate of ferredoxin reduction as a precise sensor of the local proton concentration.
Hanley
J
J
Département de Biologie Cellulaire et Moléculaire, CEA-Service de Bioénergétique/CNRS-URA 1290, Gif sur Yvette, France.
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Bottin
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D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry; D000458:Cyanobacteria / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000737:chemistry*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics; D045331:Photosystem I Protein Complex; D010940:Plant Proteins / Q000235:genetics; D017384:Sequence Deletion
D000596:Amino Acids; D003432:Cross-Linking Reagents; D005288:Ferredoxins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D010940:Plant Proteins; C070290:PsaD protein, cyanobacteria