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 mutagene sis. The mutant enzyme was fully active as a diaphorase, but partially impaired in ferredoxin-dependent cytochrome c reductase activity. By steady-state kinetics, the K-m for ferredoxin of the K88Q enzyme was f ound to have increased 10-fold, whereas the k(cat) was unaffected by t he amino acid replacement. The interaction between oxidized ferredoxin and the enzyme forms was also studied by spectrofluorimetric titratio n: K-d 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 w ith ferredoxin, confirming previous cross-linking studies.