Emergence of function in p450-proteins: A combined quantum mechanical/molecular mechanical and molecular dynamics study of the reactive species in the H₂O₂-dependent cytochrome P450_SPα and its regio- and enantioselective hydroxylation of fatty acids

This work uses combined quantum mechanical/molecular mechanical and molecular dynamics simulations to investigate the mechanism and selectivity of H₂O₂-dependent hydroxylation of fatty acids by the P450_SPα class of enzymes. H₂O₂ is found to serve as the surrogate oxidant for generating the principal oxidant, Compound I (Cpd I), in a mechanism that involves homolytic O-O bond cleavage followed by H-abstraction from the Fe-OH moiety. Our results rule out a substrate-assisted heterolytic cleavage of H₂O₂ en route to Cpd I. We show, however, that substrate binding stabilizes the resultant Fe-H₂O₂ complex, which is crucial for the formation of Cpd I in the homolytic pathway. A network of hydrogen bonds locks the HO· radical, formed by the O-O homolysis, thus directing it to exclusively abstract the hydrogen atom from Fe-OH, thereby forming Cpd I, while preventing the autoxoidative reaction, with the porphyrin ligand, and the substrate oxidation. The so formed Cpd I subsequently hydroxylates fatty acids at their α-position with S-enantioselectivity. These selectivity patterns are controlled by the active site: substrate's binding by Arg241 determines the α-regioselectivity, while the Pro242 residue locks the prochiral α-CH₂, thereby leading to hydroxylation of the pro-S C-H bond. Our study of the mutant Pro242Ala sheds light on potential modifications of the enzyme's active site in order to modify reaction selectivity. Comparisons of P450_SPα to P450_BM3 and to P450_BSβ reveal that function has evolved in these related metalloenzymes by strategically placing very few residues in the active site.