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.