Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450_(BM3)(CYP102A1)

Fatty acid binding and oxidation kinetics for wild type P450_(BM3)(CYP102A1)from Bacillus megaterium have been found to display chain length-dependent homotropic behavior.Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths.Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate,pH 7.4,but sigmoidal kinetics(with an apparent intercept)in low ionic strength buffers and at physiological phosphate concentrations.In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites,with high cooperativity for the binding of the fourth palmitate molecule,and the full-length enzyme showing tighter palmitate binding than the heme domain.The first flavin-to-heme electron transfer is faster for laurate,myristate and palmitate in 100 mmol/L phosphate than in 50mmol/L Tris(pH 7.4),yet each substrate induces similar high-spin heme content.For palmitate in low phosphate buffer concentrations,the rate constant of the first electron transfer is much larger than kcat.The results suggest that phosphate has a specific effect in promoting the first electron transfer step,and that P450_(BM3) could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.