Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 280, Issue 18, Pages 17634-17644Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M413826200
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Flavocytochrome P450 BM3 is a member of the diflavin reductase enzyme family. Members include cytochrome P450 reductase, nitric-oxide synthase, methionine synthase reductase, and novel oxidoreductase 1. These enzymes show a strong preference for NADPH over NADH as reducing coenzyme. An aromatic residue stacks over the FAD isoalloxazine ring in each enzyme, and in some cases it is important in controlling coenzyme specificity. In P450 BM3, the aromatic residue inferred from sequence alignments to stack over the FAD is Trp-1046. Mutation to Ala-1046 and His-1046 effected a remarkable coenzyme specificity switch. P450 BM3 W1046A/W106H FAD and reductase domains are efficient NADH-dependent ferricyanide reductases with selectivity coefficients (k(cat)/K-m(NADPH)/k(cat)/K-m(NADH)) of 1.5, 67, and 8571 for the W1046A, W1046H, and wild-type reductase domains, respectively. Stopped-flow photodiode array absorption studies indicated a charge-transfer intermediate accumulated in the W1046A FAD domain (and to a lesser extent in the W1046H FAD domain) and was attributed to formation of a reduced FADH(2)-NAD(P)(+) charge-transfer species, suggesting a relatively slow rate of release of NAD(P)(+) from reduced enzymes. Unlike wild-type enzymes, there was no formation of the blue semiquinone species observed during reductive titration of the W0146A/W146H FAD and reductase domains with dithionite or NAD(P) H. This was a consequence of elevation of the semiquinone/hydroquinone couple of the FAD with respect to the oxidized/semiquinone couple, and a concomitant similar to 100-mV elevation in the 2-electron redox couple for the enzyme-bound FAD (-320, -220, and -224 mV in the wild-type, W1046A, and W1046H FAD domains, respectively).
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