The use of a specific fluorescence probe to study the interaction of adrenodoxin with adrenodoxin reductase and cytochrome P-450scc.
The single free cysteine at residue 95 of bovine adrenodoxin was labeled with the fluorescent reagent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS). The modification had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc, suggesting that the AEDANS group at Cys-95 was not located at the binding site for these molecules. Addition of adrenodoxin reductase, cytochrome P-450scc, or cytochrome c to AEDANS-adrenodoxin was found to quench the fluorescence of the AEDANS in a manner consistent with the formation of 1:1 binary complexes. Förster energy transfer calculations indicated that the AEDANS label on adrenodoxin was 42 A from the heme group in cytochrome c, 36 A from the FAD group in adrenodoxin reductase, and 58 A from the heme group in cytochrome P-450scc in the respective binary complexes. These studies suggest that the FAD group in adrenodoxin reductase is located close to the binding domain for adrenodoxin but that the heme group in cytochrome P-450scc is deeply buried at least 26 A from the binding domain for adrenodoxin. Modification of all the lysines on adrenodoxin with maleic anhydride had no effect on the interaction with either adrenodoxin reductase or cytochrome P-450scc, suggesting that the lysines are not located at the binding site for either protein. Modification of all the arginine residues with p-hydroxyphenylglyoxal also had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc. These studies are consistent with the proposal that the binding sites on adrenodoxin for adrenodoxin reductase and cytochrome P-450scc overlap, and that adrenodoxin functions as a mobile electron carrier.
D000325:Adrenodoxin / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D004735:Energy Transfer; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D005456:Fluorescent Dyes; D006639:Histidine / Q000378:metabolism; D006736:Horses; D007527:Isoenzymes / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008433:Mathematics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009282:Naphthalenesulfonates
D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D007527:Isoenzymes; D009282:Naphthalenesulfonates; D000325:Adrenodoxin; D006639:Histidine; C006421:1,5-I-AEDANS; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D008239:Lysine