The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase

Abstract

In the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN 1 -to-heme 2 ) electron transfer supports oxygenase activity in the BM3 dimer.

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.…”

Unusual Spectroscopic and Ligand Binding Properties of the Cytochrome P450-Flavodoxin Fusion Enzyme XplA

“…S5). XplA is thus different from the P450 BM3 (CYP102A1) P450-NADPH-cytochrome P450 reductase fusion enzyme, which is functional as a dimer with intermonomer electron transfer (67). However, the more recently characterized CYP116B1 phthalate dioxygenase reductase-P450 fusion enzyme was also shown to be monomeric (68).…”