Preparation and properties of a cross-linked complex between ferredoxin--NADP+ reductase and flavodoxin.
The electrostatically stabilized complex between Anabaena variabilis ferredoxin--NADP+ reductase and Azotobacter vinelandii flavodoxin has been covalently cross-linked by treatment with 1-ethyl-3-(3-dimethylaminopropyl)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. Immunochemical 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 Km 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.
Pueyo
J J
JJ
Department de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain.
Sancho
J
J
Edmondson
D E
DE
Gómez-Moreno
C
C
D001395:Azotobacter / Q000378:metabolism; D002234:Carbodiimides / Q000494:pharmacology*; D000458:Cyanobacteria / Q000201:enzymology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D005418:Flavodoxin / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism*; D005779:Immunodiffusion; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D013053:Spectrophotometry
D002234:Carbodiimides; D005418:Flavodoxin; D005420:Flavoproteins; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D005022:Ethyldimethylaminopropyl Carbodiimide