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Functional interactions in cytochrome P450BM3. Fatty acid substrate binding alters electron-transfer properties of
1Department of Entomology, University of Arizona, Tucson 85721, USA. marat@ccit.arizona.edu
Abstract:
P450BM3 is a bacterial fusion protein between a cytochrome P450 fatty acid hydroxylase (CYP102) and an FAD- and FMN-containing flavoprotein homologous to NADPH: cytochrome P450 reductase. It has been shown that incubation of P450BM3 with NADPH in the absence of a fatty acid substrate results in inhibition of hydroxylase activity [Narhi, L. O., & Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169]. We show that laurate-dependent oxidation of NADPH and oxygen consumption are also inhibited under those conditions. The inhibited enzyme is unable to transfer electrons to the heme iron, but reduces artificial electron acceptors such as cytochrome c, 2,6-dichlorophenolindophenol, or ferricyanide. Incubation with these acceptors rapidly restores hydroxylase activity of P450BM3. The active enzyme is able to catalyze the reduction of cytochrome c and hydroxylation of laurate simultaneously. Cytochrome c has no effect on the K(m) and Vmax of laurate hydroxylation. Laurate and other substrates stimulate cytochrome c reduction by 50-70%. Carbon monoxide inhibits hydroxylase activity, but stimulates cytochrome c reduction 3-4 fold and has no effect on the K(m) for cytochrome c. This stimulation requires binding of a substrate at the heme catalytic site. Laurate binding induces conformational changes in the flavoprotein domain as shown by a 2-fold increase of the flavin fluorescence. Inactivation of P450BM3 by NADPH abolishes the stimulation of cytochrome c reduction by laurate and CO. Complete inhibition of hydroxylase activity correlates with complete lack of stimulation of cytochrome c reduction. The results suggest that a specific conformation of the two domains is maintained in the active P450BM3, ensuring high hydroxylase activity. Cytochrome c reductase and hydroxylase activities of P450BM3 involve different sites of interaction with the flavoprotein domain, different catalytic intermediates, and different rate-limiting steps.