Obligatory Intermolecular Electron-Transfer from FAD to FMN in Dimeric P450BM-3

Abstract

Cytochromes P450 typically catalyze the monooxygenation of hydrophobic compounds resulting in the insertion of one atom of dioxygen into the organic substrate and the reduction of the other oxygen atom to water. The two electrons required for the reaction are normally provided by another redox active protein, for example cytochrome P450 reductase (CPR) in mammalian endoplasmic reticulum membranes. P450BM-3 from Bacillus megaterium is a widely studied P450 cytochrome in which the P450 is fused naturally to a diflavin reductase homologous to CPR. From the original characterization of the enzyme by Fulco's laboratory, the enzyme was shown to have a nonlinear dependence of reaction rate on enzyme concentration. In recent experiments we observed enzyme inactivation upon dilution, and the presence of substrate can diminish this inactivation. We therefore carried out enzyme kinetics, cross-linking experiments, and molecular weight determinations that establish that the enzyme is capable of dimerizing in solution. The dimer is the predominant form at higher concentrations under most conditions and is the only form with significant activity. Further experiments selectively knocking out the activity of individual domains with site-directed mutagenesis and measuring enzyme activity in heterologous dimers establish that the electron-transfer pathway in P450BM-3 passes through both protein molecules in the dimer during a single turnover, traversing from the FAD domain of one molecule into the FMN domain of the other molecule before passing to the heme domain. Analysis of our results combined with other analyses in the literature suggests that the heme domain of either monomer may accept electrons from the reduced FMN domain.

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

“…5D and 7A), where sigmoidal kinetics was also observed, showed similar behavior that was satisfactorily modeled by the 4-site equation. The derived parameters were also similar (Table 5) Although multiple palmitate binding sites were revealed for the heme domain, these could in principle be irrelevant for fulllength P450 BM3 , which is an obligate dimer in its functional form (Neeli et al, 2005;Kitazume et al, 2007). However, titrations with the full-length protein in 50 mmol/L Tris showed virtually identical behavior to the heme domain (Fig.…”

“…It is conceivable that external phosphate could have a comparable effect. Electron transfer in the functional dimeric form of the enzyme occurs from one molecule to the other (Neeli et al, 2005;Kitazume et al, 2007). Phosphate could participate in inter-domain interactions to induce conformational changes that promote electron transfer.…”

“…The final concentration of P450 BM3 in activity assays was 0.1 μmol/L. The following steps were taken to minimize monomer formation (Neeli et al, 2005;Kitazume et al, 2007). For activity assays in 10 mmol/L and 25 mmol/L phosphate, the enzyme was eluted from the PD-10 column with 50 mmol/L phosphate to maintain it in the dimeric form.…”

“…Monooxygenase activity is negligible in the monomeric form of the enzyme (Neeli et al, 2005;Kitazume et al, 2007;Girvan et al, 2011). Electrons are supplied by NADPH to one monomer and subsequently transferred to the other monomer.…”

“…Electrons are supplied by NADPH to one monomer and subsequently transferred to the other monomer. Detailed understanding of the properties and function of P450 BM3 is further complicated by the fact that enzyme activity is strongly dependent on buffer composition and ionic strength (Kitazume et al, 2007). The heme spin state of the Npalmitoylglycine-bound enzyme varies significantly with temperature (Jovanovic et al, 2005).…”