Journal of Inorganic Biochemistry ENVIRONMENTAL BIOINORGANIC CHEMISTRY 379

008 Azo Reduction Catalyzed by Cytochrome P450 1A2 and

NADPH-Cytochrome P450 Reductase

Makoto Umeno, Kazutaka Yanagita, lkuko Sagami, and Toru Shimizu

Institute for Chemical Reaction Science, Tohoku University, Sendai 980-77, Japan

Azo compounds are toxic and carcinogenic. Azo reductions have been intensively studied with liver

microsomes (SCHEME I). It has been suggested that the azo reduction is caused by cytochrome P450.

In the present study, we first carefully examined the azo reduction of methyl red in the reconstituted

system composed of purified cytochrome P450 1A2, NADPH-cytochrome P450 reductase, NADPH and

membrane under various conditions. Methyl red was not degraded by P450 1A2 alone, while it was

degraded by the reconstituted system under the aerobic conditions. Surprisingly the azo reduction is

solely caused by the reductase and NADPH, whereas P450 1A2 decelerated the azo reduction under the

aerobic conditions. A Glu318Ala mutation at the heme distal site of P450 1A2 further decelerated the

azo reduction under the aerobic conditions. A Thr319Ala mutation did not influence the P450 1A2

effect on the azo reduction. In contrast, under anaerobic conditions, the azo reduction of methyl red was

enhanced by P450 1A2. The Glu318Ala mutation further enhanced the azo reduction under the

anaerobic conditions. The Thr319Ala mutation did not influence the azo reduction assisted by P450

1A2. From those findings together with other results, it is suggested here that the azo reduction hitherto

reported to be caused by P450 is actually caused by the reductase under the aerobic conditions. Under

anaerobic conditions, P450 1A2 facilitates the azo reduction. Based on those results, we discuss the

role of the putative distal amino acids in the azo reduction of methyl red.

COOH COOH

N---'N N ~-- NH 2 + H2N \

SCHEME I : Methyl red azo reduction with P450 1A2 and the reductase