Effect of CO binding to P450 BM3 F393 mutants on electron density distribution in the heme cofactor

Resonance Raman spectroscopy has been performed on a set of cytochrome P450 BM3 heme domains in which mutation of the highly conserved Phe393 induces significant variation in heme iron reduction potential. In previous work [Chen, Z., Ost, T.W.B., and Schelvis, J.P.M. (2004) Biochemistry 43, 1798–1808], a correlation between heme vinyl conformation and the heme iron reduction potential indicated a steric control by the protein over the distribution of electron density in the reduced heme cofactor. The current study aims to monitor changes in electron density on the ferrous heme cofactor following CO binding. In addition, ferric-NO complexes have been studied to investigate potential changes to the proximal Cys400 thiolate. We find that binding of CO to the ferrous heme domains results in a reorientation of the vinyl groups to a largely out-of-plane conformation, the extent of which correlates with the size of the residue at position 393. We conclude that FeII dπ back bonding to the CO ligand largely takes away the need for conjugation of the vinyl groups with the porphyrin ring to accommodate FeII dπ back bonding to the porphyrin ligand. The ferrous-CO and ferric-NO data are consistent with a small decrease in σ-electron donation from the proximal Cys400 thiolate in the F393A mutant and, to a lesser extent, the F393H mutant, potentially due to a small increase in hydrogen bonding to the proximal ligand. Phe393 seems strategically placed to preserve robust σ-electron donation to the heme iron and to fine-tune its electron density by limiting vinyl group rotation. Resonance Raman spectroscopy of cytochrome P450 BM3 F393 mutants reveal how the protein uses steric hinderance of heme vinyl group conformation to provide additional control over FeII dπ back bonding, heme iron reduction potential and chemical reactivity. [Display omitted] •Regulation of electron density in cytochrome P450 BM3 by steric hinderance of vinyl group.•Increase in hydrogen bonding to proximal ligand in F393A P450 BM3.•P450 BM3 uses steric hinderance by Phe393 as one tool to control reactivity.