Substitutions of the phylogenetically conserved phenylalanine residue F393 were constructed in flavocytochrome P450 BM3 from Bacillus megaterium. The high degree of conservation of this residue in the P450 superfamily and its proximity to the heme (and its ligand Cys400) implies an essential role in P450 activity. Extensive kinetic and thermodynamic characterisation of mutant enzymes F393A, F393H and F393Y highlighted significant differences from wild-type P450 BM3. All enzymes expressed to high levels and contained their full complement of heme. Whilst the reduction and subsequent treatment of the mutant P450s with carbon monoxide led to the formation of the characteristic P450 spectra in all cases, the absolute position of the Soret absorption varied across the series WT/F393Y (449 nm), F393H (445 nm) and F393A (444 nm). Steady-state turnover rates with both laurate and arachidonate showed the trend WT>F393Y>>F393H>F393A. Conversely, the trend in the pre-steady-state flavin-to-heme electron transfer was the reverse of the steady-state scenario, with F393A>F393H>>F393Y>wild-type. These data are consistent with the more positive substrate-free (-312 mV (F393A), -332 mV (F393H)) and substrate-bound (-151 mV (F393A), -176 mV (F393H)) reduction potentials of F393A and F393H heme domains, favouring the stabilisation 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 electron transfer to the heme. This results in a decrease in the driving force for oxygen reduction by the ferrous heme iron, hence an overall decline in the rate of turnover 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 trade-off is established between the rate of heme reduction and the rate at which the ferrous heme can bind and, subsequently, reduce molecular oxygen. The structural and spectroscopic characterisation of the mutants reveals the probable role of the phenylalanine to be to preserve the hydrophobic environment of the heme. This prevents unfavourable inter- and intra-molecular interactions with the heme or heme-ligand, which have been demonstrated to perturb this delicate thermodynamic balance. This study highlights the electronic influence the cysteine ligand has in dictating the characteristic reactivity of P450s, and its sensitivity to its chemical environment. Modulating the electron density of the Fe-S bond (introduction of intramolecular hydrogen bonds) has a direct effect on the ability of P450s to bind and activate molecular oxygen. This point is key to the high degree of conservation of this residue throughout the P450 superfamily ?—monooxygenation requires oxygen activation. This phenylalanine is conserved in every member which needs to perform this activation, for the small number that do not, the nature of this residue varies.