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Preparation and properties of a cross-linked complex between ferredoxin - NADP+ reductase and flavodoxin

AutorPueyo, José Javier ; Sancho, Javier ; Edmondson, Dale E.; Gómez-Moreno, Carlos
Palabras claveFerredoxin
Fecha de publicación1989
CitaciónEuropean Journal of Biochemistry 183:539-544 (1989)
ResumenThe electrostatically stabilized complex between Anahaena variabilis ferredoxin - NADP' reductase and Azotobacter vinelandii flavodoxin has been covalently cross-linked by treatment with l-ethyl-3-(3-dimethylaminopropy1) carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1 : 1 stoichiometry of the two flavoproteins. lmmunochemical cross-reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH - ferricyanide diaphorase activity although the K,,, for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH -cytochrome-c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1-2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome-c reductase activity is observed on adding ferredoxin to the cross-linked complex. Stopped-flow data show that covalent cross-linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP' show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin - NADP' reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross-linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of semiquinone and quinol forms.
Descripción6 pages, figures and tables statistics.
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