Tryptophan-96 in cytochrome P450 BM3 plays a key role in enzyme survival

Abstract

Flavocytochrome P450 from Bacillus megaterium (P450_BM3) is a natural fusion protein containing reductase and heme domains. In the presence of NADPH and dioxygen the enzyme catalyzes the hydroxylation of long-chain fatty acids. Analysis of the P450_BM3 structure reveals chains of closely spaced tryptophan and tyrosine residues that might serve as pathways for high-potential oxidizing equivalents to escape from the heme active site when substrate oxidation is not possible. Our investigations of the total number of enzyme turnovers before deactivation have revealed that replacement of selected tryptophan and tyrosine residues with redox inactive groups leads to a twofold reduction in enzyme survival time. Tryptophan-96 is critical for prolonging enzyme activity, suggesting a key protective role for this residue.

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