HYDROXYLATION OF BENZENE BY IMMOBILIZED CYTOCHROME-C IN AN ORGANIC-SOLVENT

Recently we showed that cytochrome c (cyt.c) has some cytochrome P450 (P450)-like substrate oxidation activity, which was enhanced by immobilization on poly-gamma-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 [O-18]m-chloroperbenzoic acid (mCPBA) or [O-18]molecular dioxygen showed that 59% of the oxygen atom of the product, phenol, was derived from the oxidant, and 33% from molecular dioxygen. When [O-18]mCPBA was used as an oxidant, the value of deuterium isotope effect (k(H)/k(D)) was 1.08 when O-18 was incorporated into the product, and 1.48 when O-18 was not incorporated. These results indicate that immobilized cyt.c catalysed hydroxylation bf benzene via two major pathways, of which one resembles that of P450.