Dithionite-supported hydroxylation of palmitic acid by cytochrome P450BM-3

The ability of dithionite, an inexpensive reducing agent routinely used to produce the ferrous-carbonyl form of P450, to support P450BM-3-catalyzed hydroxylation of palmitate was studied. The hydroxylation products in the presence of dithionite were 15, 14, and 13-hydroxyhexadecanoate, with relative distributions similar to those observed with NADPH. The hydroxylation reaction was carried out in two separate steps, anaerobic reduction and subsequent oxidation of P450BM-3 by oxygen bubbling. The reduction step was much slower than the oxidation step, thus limiting the overall rate of hydroxylation. Upon addition of dithionite, the reductase domain of P450BM-3 seemed to be reduced before significant reduction of the heme domain occurred. The discovery of new reducing agents for P450-catalyzed reaction raises the possibility of replacing NADPH in specialty chemical hydroxylation catalyzed by P450s, especially catalytically self-sufficient P450s, such as P450BM-3 or recombinant fusion proteins of P450 covalently linked to a reductase.