Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450BM3

Site-directed mutants of the phylogenetically conserved phenylalanine resid ue F393 were constructed in flavocytochrome P450 BM3 from Bacillus megateri um. The high degree of conservation of this residue in the P450 superfamily and its proximity to the heme (and its ligand Cys400) infers an essential role in P450 activity. Extensive kinetic and thermodynamic characterization of mutant enzymes F393A, F393H, and F393Y highlighted significant differen ces from wild-type P450 BM3. All enzymes expressed to high levels and conta ined their full complement of heme. While the reduction and subsequent trea tment of the mutant P450s with carbon monoxide led to the formation of the characteristic P450 spectra in all cases, the absolute position of the Sore t absorption varied across the series WT/F393Y (449 nm), F393H (445 nm), an d F393A (444 nm). Steady-state turnover rates with both laurate and arachid onate showed the trend WT > F393Y much greater than F393H > F393A. Converse ly, the trend in the pre-steady-state flavin-to-heme electron transfer was the reverse of the steady-state scenario, with rates varying F393A > F393H much greater than F393Y approximate to wild-type. These data are consistent with the more positive substrate-free [-312 mV (F393A), -332 mV (F393H)] a nd substrate-bound [-151 mV (F393A), -176 mV (F393H)] reduction potentials of F393A and F393H heme domains, favoring the stabilization of the ferrous- form in the mutant P450s relative to wild-type. Elevation of the heme iron reduction potential in the F393A and F393H mutants facilitates faster elect ron transfer to the heme. This results in a decrease in the driving force f or oxygen reduction by the ferrous heme iron, so explaining lower overall t urnover of the mutant P450s. We postulate that the nature of the residue at position 393 is important in controlling the delicate equilibrium observed in P450s, whereby a tradeoff is established between the rate of heme reduc tion and the rate at which the ferrous heme can bind and, subsequently, red uce molecular oxygen.