The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450(scc)) and CYP11B1 (cytochrome P450(11 beta)) - Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin

The kinetics of protein-protein interaction and heme reduction between adre nodoxin 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 CYP11A1(.)CO and CYP11B1(.)CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4-108), Adx-(4-114), T54S, T54A, and S112W, an d the double mutants Y82F/S112W, Y82L/ S112W, and Y82S/S112W (the last four mutants are Delta 113-128) are presented. The rate constants observed diff er by a factor of up to 10 among the respective adrenodoxin mutants for CYP 11A1 but not for CYP11B1. According to their apparent rate constants for CY P11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutant s forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone co nversion. This strong correlation suggests that C-terminal truncation of ad renodoxin in combination with the introduction of a C-terminal tryptophan r esidue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a va riation of the adrenodoxin structure resulted in a mutant protein (S112W) s howing a 100-fold increased efficiency in conversion of cholesterol to preg nenolone.