Reactivity of cytochromes c and f with mutant forms of spinach plastocyanin.
The reduction of plastocyanin by cytochromes c and f has been investigated with mutants of spinach plastocyanin in which individual, highly conserved surface residues have been modified. These include Leu-12 and Phe-35 in the 'northern' hydrophobic patch and Tyr-83 and Asp-42 in the 'eastern' acidic patch. The differences observed all involved binding rather than the intrinsic rates of electron transfer. The Glu-12 and Ala-12 mutants showed small but significant decreases in binding constant with cytochrome c, even though the cytochrome is not expected to make contact with the northern face of plastocyanin. These results, and small changes in the EPR parameters, suggested that these mutations cause small conformational changes in surface residues on the eastern face of plastocyanin, transmitted through the copper centre. In the case of cytochrome f, the Glu-12 and Ala-12 mutants also bound less strongly, but Leu12Asn showed a marked increase in binding constant, suggesting that cytochrome f can hydrogen bond directly to Asn-12 in the reaction complex. A surprising result was that the kinetics of reduction of Asp42Asn were not significantly different from wild type, despite the loss of a negative charge.
Modi
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Department of Biochemistry, University of Cambridge, UK.
Nordling
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M
Lundberg
L G
LG
Hansson
O
O
Bendall
D S
DS
D000595:Amino Acid Sequence; D001665:Binding Sites; D003574:Cytochrome c Group / Q000378:metabolism*; D003580:Cytochromes / Q000378:metabolism*; D045348:Cytochromes f; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010970:Plastocyanin / Q000235:genetics / Q000378:metabolism*
D003574:Cytochrome c Group; D003580:Cytochromes; D010970:Plastocyanin; D045348:Cytochromes f