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Involvement of lysine‐88 of spinach ferredoxin‐NADP + reductase in the interaction with ferredoxin
Author(s) -
Aliverti Alessandro,
Corrado Mario Ermanno,
Zanetti Giuliana
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)80565-2
Subject(s) - ferredoxin , ferredoxin—nadp(+) reductase , ferredoxin thioredoxin reductase , spinach , reductase , biochemistry , chemistry , enzyme , mutant , glutamine synthetase , amino acid , glutamine , thioredoxin reductase , glutathione , gene
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.