Some properties of mitochondrial adrenodoxin associated with its nonconventional electron donor function toward...

Mitochondrial adrenodoxin (Adx) was found to cross-react with microsomal cytochrome P450 2B4 (CYP2B4) as the terminal electron acceptor. When compared with NADPH-cytochrome P450 reductase (P450R), the natural redox partner of CYP2B4, Adx was less efficient both in transferring the first electron and in coupling the system. The ferredoxin yielded an unusual reverse type I spectral change with low-spin CYP2B4, which underwent transformation to a typical type I optical perturbation upon deletion of the signal anchor sequence (Delta2-27) of the hemoprotein. Truncation of CYP2B4 slightly fostered electron transfer from Adx, but was deleterious to reduction of the engineered isozyme by P450R. Addition of manganese-substituted cytochrome b5, which failed to serve as an electron donor to CYP2B4, augmented the amount of hemoprotein existing in form of a low-spin complex with Adx and affected the ferredoxin-dependent reduction kinetics through causing a proportional rise in both Km and Vmax. Conservative replacement of Asp-76 with glutamate in the Adx molecule was associated with a drastic drop in reductive efficiency toward CYP2B4, while spectral binding of the mutant to the hemoprotein was marginally changed. The results support the concept of an evolutionary relationship between the various cytochrome P450 forms as regards the conservation of surface regions participating in contacts with heterologous donor proteins.