The subzero temperature stabilized oxyferro complex of purified cytochrome P450 SCC

Abstract

Purified preparations of bovine adrenocortical mitochondrial cytochrome P450, capable of catalyzing the side-chain cleavage of cholesterol have been obtained [ 1,2]. The spectral, biochemical and immunological characteristics of this hemoprotein are now known [3,4], and the multistep nature of this bioconversion leading through enzyme-bound intermediates to pregnenolone formation, established [5-71. The first step in the activation of molecular oxygen by cytochrome P450 involves the formation of an oxyferro complex [a]. Although oxyferro intermediates of a number of cytochrome P45Os have been described [9] no information is available about this form of cytochrome P450,. The development of subzero temperature techniques [lo-l 31 together with our studies on proton activity, dielectric constants and viscosity in fluid hydroorganic media, enabled the stabilization and detection of intermediates in various biological reactions [ 14-161. Among these are the oxyferro species of cytochrome P450, from Pseudomonas putida [ 171 and the highly purified microsomal cytochrome P450 (LM) from rabbit liver [ 181. Here we describe the formation and stabilization at low temperature of the oxyferro intermediate of cytochrome P450, isolated from bovine adrenocortical mitochondria.