The surface-exposed tyrosine residue Tyr83 of pea plastocyanin is involved in both binding and electron transfer reactions with cytochrome f.
Site-directed mutants of the pea plastocyanin gene in which the codon for the surface-exposed Tyr83 has been changed to codons for Phe83 and Leu83 have been expressed in transgenic tobacco plants. The mutant proteins have been purified to homogeneity and their conformations shown not to differ significantly from the wild-type plastocyanin by 1H-NMR and CD. Overall rate constants for electron transfer (k2) from cytochrome f to plastocyanin have been measured by stopped-flow spectrophotometry and rate constants for binding (ka) and association constants (KA) have been measured from the enhanced Soret absorption of cytochrome f on binding plastocyanin. These measurements allow the calculation of the intrinsic rate of electron transfer in the binary complex. An 8-fold decrease in the overall rate of electron transfer to the Phe83 mutant is due entirely to a decreased association constant for cytochrome f, whereas the 40-fold decrease in the overall rate of electron transfer to the Leu83 mutant is due to weaker binding and a lower intrinsic rate of electron transfer. This indicates that Tyr83 is involved in binding to cytochrome f and forms part of the main route of electron transfer.
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Cambridge Centre for Molecular Recognition, University of Cambridge, UK.
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Bendall
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Gray
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D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003580:Cytochromes / Q000378:metabolism*; D045348:Cytochromes f; D003854:Deoxyribonucleotides; D004583:Electrons; D007887:Fabaceae / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D030821:Plants, Genetically Modified / Q000235:genetics; D010946:Plants, Medicinal; D010957:Plasmids; D010970:Plastocyanin / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000235:genetics*
D003580:Cytochromes; D003854:Deoxyribonucleotides; D014443:Tyrosine; D010970:Plastocyanin; D045348:Cytochromes f