I helix Mediates the Allosteric Regulation in Cytochrome P450cam

Abstract

Cytochrome P450cam, a key monooxygenase in the P450 superfamily, is pivotal in metabolic and industrial processes. Despite extensive studies, a unified mechanism governing its conformational heterogeneity, substrate-dependent allostery, and multi-substrate binding remains elusive. Here, integrating molecular dynamics simulations, NMR pseudocontact shift (PCS) analysis, and crystallographic data, we identify the I-helix (I) as the central regulator of P450cams allostery. Its intrinsic flexibility, dictated by glycine residues (G248 and G249), orchestrates enzyme conformational dynamics. Specifically, I transitions between straight and kinked conformations, modulating the opening and closing of substrate access channels (channel-1 and channel-2) and mediating allosteric communication between active and allosteric sites. Substrate binding stabilizes the straight conformation, promoting channel closure and enhancing allosteric regulation. This I-helix-based mechanism reconciles 125 crystallographic poses, spanning straight-to-kinked I conformations. Notably, the kink-inducing glycine G249 is evolutionarily conserved across species, including humans, underscoring Is fundamental role in enzyme function and broader significance within the P450 superfamily. NMR PCS measurements align with the kinked and straight conformations in the substrate-free and substrate-bound states, with Q-scores of 0.108 and 0.061, respectively. Leveraging this mechanistic insight, we designed proof-of-concept P450cam mutants locked in either constitutively open or closed conformations for the first time. By shifting the focus from the traditional FG-helix-centric view to an I-helix-centric framework, this study provides a comprehensive blueprint for conformational and allosteric regulation, paving the way for engineering tailored P450 variants.