Purification and catalytic properties of a cross-linked complex between adrenodoxin reductase and adrenodoxin.
A stable covalent complex was prepared by cross-linking adrenodoxin reductase with adrenodoxin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was purified extensively until free components were removed completely. The major component of the complex had a molecular weight of 63 kDa, which corresponds to a 1:1 stoichiometric complex between adrenodoxin reductase and adrenodoxin. NADPH-cytochrome c reduction activity of the covalent complex was comparable to that of an equimolar mixture of adrenodoxin reductase and adrenodoxin (native complex), and the NADPH-ferricyanide reduction activity of the complex was equal to that of the native one. In contrast to the native complex, the covalent complex produced much less superoxide upon NADPH-oxidation, and the covalent complex was found to be more stable than the native complex, suggesting that the complex state is more favorable for catalysis. From these results, we conclude that the adrenodoxin molecule does not need to dissociate from the complex during electron transfer from NADPH to cytochrome c.
Hara
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Department of Chemistry, Wayne State University, Detroit, Michigan 48202.
Kimura
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T
D000325:Adrenodoxin / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide; D005287:Ferredoxin-NADP Reductase / Q000302:isolation & purification* / Q000378:metabolism; D005292:Ferricyanides / Q000378:metabolism; D005415:Flavins / Q000032:analysis; D006358:Hot Temperature; D066298:In Vitro Techniques; D007501:Iron / Q000032:analysis; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis / Q000302:isolation & purification; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D013455:Sulfur / Q000032:analysis; D013481:Superoxides / Q000378:metabolism
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D005292:Ferricyanides; D005415:Flavins; D013481:Superoxides; D000325:Adrenodoxin; C007931:hexacyanoferrate III; D013455:Sulfur; D007501:Iron; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D005022:Ethyldimethylaminopropyl Carbodiimide