Complex formation in vesicle-reconstituted mitochondrial cytochrome P450 systems (CUP11A1 and CYP11B1) as evidenced by rotational diffusion experiments using EPR and ST-EPR

Rotational diffusion measurements using EPR and saturation transfer EPR wer e applied to analyze complex formation between the electron-transfer compon ents of the mitochondrial steroid-hydroxylating cytochrome P450 systems (CY P11A1 and CYP11B1) in phosphatidylcholine/phosphatidylethanolamine/cardioli pin Vesicles prepared by octyl glucoside dialysis/adsorption. Octyl glucosi de reconstitution of P450SCC results in large vesicles, which have an advan tage over small vesicles in that vesicle tumbling does not contribute to me asured rotational diffusion rates. Immobilization of spin-labeled adrenodox in by both P450SCC and adrenodoxin reductase indicates equimolar complexati on between P450SCC and adrenodoxin as well as between adrenodoxin reductase and adrenodoxin. Combination of rotational diffusion and antibody cross-li nking confirmed the complexation of adrenodoxin with P450SCC and for the fi rst time provided direct evidence of a complex between P450SCC and P45011 b eta in the membrane. In contrast, no evidence was found for the existence o f adrenodoxin reductase-P450SCC complexes or a ternary complex of all three proteins. Thus, these experiments confirm the shuttle mechanism of electro n transfer to vesicle-reconstituted P450SCC and P45011 beta.