Involvement of lysine-88 of spinach ferredoxin-NADP+ reductase in the interaction with ferredoxin.
A mutant of spinach ferredoxin-NADP+ reductase, in which Lys-88 has been changed to glutamine, has been obtained by site-directed mutagenesis. The mutant enzyme was fully active as a diaphorase, but partially impaired in ferredoxin-dependent cytochrome c reductase activity. By steady-state kinetics, the Km for ferredoxin of the K88Q enzyme was found to have increased 10-fold, whereas the kcat was unaffected by the amino acid replacement. The interaction between oxidized ferredoxin and the enzyme forms was also studied by spectrofluorimetric titration: Kd values of 110 and 10 nM were determined for the mutant and wild-type proteins, respectively. These data point out the importance of a positive charge at position 88 of the reductase for the interaction with ferredoxin, confirming previous cross-linking studies.
Aliverti
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Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Corrado
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ME
Zanetti
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D001483:Base Sequence; D003432:Cross-Linking Reagents; D004247:DNA; D004926:Escherichia coli; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014675:Vegetables / Q000201:enzymology*
D003432:Cross-Linking Reagents; D005288:Ferredoxins; D011994:Recombinant Proteins; D005973:Glutamine; D004247:DNA; D005287:Ferredoxin-NADP Reductase; D008239:Lysine