| Abstract
| - P450BM3 is a bacterial fusion protein between a cytochrome P450fatty acid hydroxylase(CYP102) and an FAD- and FMN-containing flavoprotein homologous toNADPH:cytochrome P450reductase. It has been shown that incubation of P450BM3 with NADPHin the absence of a fatty acidsubstrate results in inhibition of hydroxylase activity [Narhi, L. O.,& Fulco, A. J. (1986) J. Biol. Chem.261, 7160−7169]. We show that laurate-dependentoxidation of NADPH and oxygen consumption arealso inhibited under those conditions. The inhibited enzyme isunable to transfer electrons to the hemeiron, but reduces artificial electron acceptors such as cytochromec, 2,6-dichlorophenolindophenol, orferricyanide. Incubation with these acceptors rapidly restoreshydroxylase activity of P450BM3. Theactive enzyme is able to catalyze the reduction of cytochromec and hydroxylation of laurate simultaneously.Cytochrome c has no effect on theKm and Vmax of lauratehydroxylation. Laurate and other substratesstimulate cytochrome c reduction by 50−70%. Carbonmonoxide inhibits hydroxylase activity, butstimulates cytochrome c reduction 3−4 fold and has noeffect on the Km for cytochrome c.This stimulationrequires binding of a substrate at the heme catalytic site.Laurate binding induces conformational changesin the flavoprotein domain as shown by a 2-fold increase of the flavinfluorescence. Inactivation ofP450BM3 by NADPH abolishes the stimulation of cytochrome creduction by laurate and CO. Completeinhibition of hydroxylase activity correlates with complete lack ofstimulation of cytochrome c reduction.The results suggest that a specific conformation of the twodomains is maintained in the active P450BM3,ensuring high hydroxylase activity. Cytochrome creductase and hydroxylase activities of P450BM3involve different sites of interaction with the flavoprotein domain,different catalytic intermediates, anddifferent rate-limiting steps.
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