The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1⅐CO and CYP11B1⅐CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4 -108), Adx-(4 -114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/ S112W, and Y82S/S112W (the last four mutants are ⌬113-128) are presented. The rate

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... ants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone. Cytochromes P450 (CYP11A1, CYP11B1, and CYP11B2) 1 of the inner mitochondrial membrane catalyze various hydroxylation steps in the biosynthesis of steroid hormones (mineralocorticoids, glucocorticoids, and androgens) in vertebrates (1, 2). The electrons required for these hydroxylation reactions are provided by NADPH and are transferred to the cytochromes P450, which mediate the final oxygen activation, via a small electron transport chain. Comparable with the components of the soluble bacterial cytochrome P450 systems, such as P450 cam (CYP101) and P450 terp (CYP108), the proteins of this electron transport system are an FAD-containing NAD(P)H-de-