Hydroxylation of benzene by immobilized cytochrome c in an organic solvent

Abstract

Recently we showed that cytochrome c (cyt.c) has some cytochrome P450 (P450)-like substrate oxidation activity, which was enhanced by immobilization on poly-γ-methyl-L-glutamate. We now report that immobilized cyt.c catalysed hydroxylation of benzene when benzene containing a small amount of water was used as the reaction solvent, whereas free cyt.c did not catalyse the reaction. The EPR spectrum of immobilized cyt.c showed that the immobilization had caused a change in the protein conformation, and this might have led cyt.c to acquire the new activity. The results of the reaction using [¹⁸O]m-chloroperbenzoic acid (mCPBA) or [¹⁸O] molecular dioxygen showed that 59% of the oxygen atom of the product, phenol, was derived from the oxidant, and 33% from molecular dioxygen. When [¹⁸O]mCPBA was used as an oxidant, the value of deuterium isotope effect (k_H/k_D)was 1.08 when ¹⁸O was incorporated into the product, and 1.48 when ¹⁸O was not incorporated. These results indicate that immobilized cyt.c catalysed hydroxylation of benzene via two major pathways, of which one resembles that of P450.