Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b5 via either NADPH–cytochrome P450 reductase or NADH–cytochrome b5 reductase, and the presence of cytochrome b5 stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b5, we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b5 reductase, cytochrome b5, and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b5 was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b5 by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b5 was necessary for the stimulation. Addition of cytochrome b5 reductase to the cytochrome b5/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b5, but b5 reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b5 reductase nor cytochrome b5 alone could support CYP2E1 activity. These results demonstrate that the cytochrome b5 reductase/cytochrome b5 pathway can support CYP2E1 activity in bacterial cells.