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 K-m for ferredoxin of the K88Q enzyme was found to have increased 10-fold, whereas the k(cat) was unaffected by the amino acid replacement. The interaction between oxidized ferredoxin and the enzyme forms was also studied by spectrofluorimetric titration: 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 with ferredoxin, confirming previous cross-linking studies.