Some properties of mitochondrial adrenodoxin associated with its nonconventional electron donor function toward rabbit liver microsomal cytochrome P450 2B4

Mitochondrial adrenodoxin (Adx) was found to cross-react with microsomal cy tochrome P450 2B4 (CYP2B4) as the terminal electron acceptor. When compared with NADPH-cytochrome P450 reductase (P450R), the natural redox partner of CUP2B4, Adx was less efficient both in transferring the first electron and in coupling the system. The ferredoxin yielded an unusual reverse type I s pectral change with low-spin CYP2B4, which underwent transformation to a ty pical type I optical perturbation upon deletion of the signal anchor sequen ce (Delta 2-27) of the hemoprotein. Truncation of CYP2B4 slightly fostered electron transfer from Adx, but was deleterious to reduction of the enginee red isozyme by P450R. Addition of manganese-substituted cytochrome b(5), wh ich 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 t he ferredoxin-dependent reduction kinetics through causing a proportional r ise in both K-m and V-max Conservative replacement of Asp-76 with glutamate in the Adx molecule was associated with a drastic drop in reductive effici ency 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 cons ervation of surface regions participating in contacts with heterologous don or proteins. (C) 1999 Academic Press.