COMPARATIVE STRUCTURAL AND IMMUNOCHEMICAL CHARACTERIZATION OF RECOMBINANT AND NATURAL CYTOCHROME P450SCC (CYPXIAI)

Optimization of the conditions for heterologous expression of recombin ant cytochrome P450scc in E, coli provided an expression level of abou t 420 nmoles of cytochrome P450scc per liter of bacterial culture. A n ew procedure for purification of recombinant protein in substrate-boun d high-spin and substrate-free low-spin form is described. Highly puri fied electrophoretically homogeneous recombinant cytochrome P450scc co ntains 12.3 and 16.7 nmoles heme per mg protein for substrate-free and substrate-bound forms, respectively. The recombinant and natural cyto chromes P450scc from bovine adrenocortical mitochondria were compared functionally and immunochemically. The dissociation constants for the complexes of cytochrome P450scc with cholesterol and adrenodoxin, the efficiency of enzymatic reduction in the reconstituted system (NADPH-a drenodoxin reductase-adrenodoxin), and cholesterol side-chain cleavage activity were determined. It was found that limited proteolysis of th e recombinant cytochrome P450scc with trypsin forms two main fragments which are electrophoretically and immunochemically identical with the fragments F1 (29.8 kD) and F2 (26.6 kD) formed during proteolysis of bovine adrenocortical cytochrome P450scc. The quantitative values of t he studied parameters are practically identical in natural and substra te-bound recombinant cytochrome P450scc, while there were great differ ences between substrate-bound and substrate-free forms of recombinant cytochrome P450scc both of functional (decrease of cholesterol side-ch ain cleavage activity, of efficiency of enzymatic reduction in the rec onstituted system, and of affinity to adrenodoxin for substrate-free c ytochrome P450scc) and of structural (increase in accessibility to exo genous and endogenous proteolysis) character. The identity of the fold ing process for recombinant and natural proteins as well as the nature of a stabilizing and activating effect of cholesterol on cytochrome P 450scc are discussed.