Heme-Peroxo Intermediate: A Powerful Oxidant in Cytochrome P450 that is Controlled by the Proton Channel of the Protein

Cytochrome P450 enzymes catalyze numerous biosynthetic and metabolic transformations. While the high-valent oxo–iron(IV) porphyrin π-radical cation (so-called Compound I) species has generally been accepted to be the principal oxidant of P450s, the ferric peroxo species has been invoked as an alternative oxidant, particularly for P450-catalyzed C–C cleavage reactions during steroid metabolism. However, the active species and the mechanisms for these P450-mediated C–C bond cleavages have been highly controversial according to previous experimental and computational studies. To address these yet unsettled issues, we report here comparative MD simulation and QM/MM studies on reactivities of both the Fe(III)-peroxo and Compound I (Cpd I) species in P450 17A1 vs P450 51A1. For P450 17A1, our study demonstrates that the Fe(III)-peroxo species is incapable of mediating the C17–C20 cleavage of 17α-hydroxy pregnenolone (17-OH PREG). Instead, a water channel facilitates the conversion of Fe(III)-peroxo to the active oxo-iron species (Cpd I), which can trigger the C–C cleavage via the H-abstraction from the C17–OH of 17-OH PREG. For P450 51A1, we found that the oxidant choice is controlled by the aldehyde vs gem-diol forms of the substrate bound in the active site. The aldehyde substrate disrupts the subsequent protonation of peroxo species, but enables an efficient nucleophilic attack by the Fe(III)-peroxo species. By contrast, the gem-diol substrate maintains the proton channel, promoting the efficient generation of Cpd I, which in turn triggers the C–C bond cleavage. This study reveals the critical role of proton channels in determining the reactivity and fate of peroxo species in P450s.