Cloning and Expression of Human Chromium-Reducing Enzymes

摘要

The cloning and expression in E. coli of three of the proteins of interest (FMO3, P450 reductase, and b5 reductase) were accomplished. Human cytochrome b5 and P450 reductase became available from commercial sources. Recombinant FMO3 had little to no NADPH dependent Cr(VI) reduction activity; similarly, when tested alone, none of the other proteins (cytochrome b5, P450 reductase, b5 reductase) had prominent Cr(VI) reductase activity. Efficient electron transfer from NADPH to cytochrome b5 was observed using proteoliposomes containing human recombinant cytochrome b5 and P450 reductase. When normalized to equivalent cytoclirome b5 concentrations, the NADPH-dependent Cr(VI) reduction rates mediated by these proteoliposomes were essentially identical to those for human microsomes. Trace amounts of iron (Fe) could dramatically stimulate Cr(VI) reduction by these proteoliposomes; this stimulation could be abolished by deferoxamine. The Fe(III) reduction rates were sufficient to account for the Fe-mediated stimulation of Cr(VI) reduction. Cr(V) was detected as a transient intermediate formed during NADPH-dependent Cr(VI) reduction mediated by these proteoliposomes. Iron also stimulated the subsequent reduction of Cr(V) by these proteoliposomes which would accelerate the formation of Cr(IV) , a highly reactive species. Under aerobic conditions, Cr(VI) reduction by these proteoliposomes resulted in the generation of hydroxyl radical (.OH), a highly damaging species. Overall, the interaction of cytochrome b5 with P450 reductase can account for: (1) essentially all of the NADPH-dependent Cr(VI) reduction seen with human microsomes; (2) the iron-mediated stimulation of Cr(VI) reduction; and (3) the generation of reactive species E.G., Cr(V), .OH which are likely involved in some of the cytotoxic and genotoxic effects associated with Cr(VI) exposure.