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.

Involvement of lysine-88 of spinach ferredoxin-NADP+ reductase in the interaction with ferredoxin / A. Aliverti, M.E. Corrado, G. Zanetti. - In: FEBS LETTERS. - ISSN 0014-5793. - 343:3(1994 May 02), pp. 247-250.

Involvement of lysine-88 of spinach ferredoxin-NADP+ reductase in the interaction with ferredoxin

A. Aliverti
Primo
;
G. Zanetti
Ultimo
1994

Abstract

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.
Electrostatic interaction; Ferredoxin; Ferredoxin-NADP+ reductase; Flavoprotein; Protein engineering; Protein-protein interaction
Settore BIO/10 - Biochimica
Settore BIO/11 - Biologia Molecolare
Settore BIO/04 - Fisiologia Vegetale
2-mag-1994
http://www.sciencedirect.com/science/article/pii/0014579394805652
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/191959
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