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 ( α I) as the central regulator of P450cam’s allostery. Its intrinsic flexibility, dictated by glycine residues (G248 and G249), orchestrates enzyme conformational dynamics. Specifically, α 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 α I conformations. Notably, the kink-inducing glycine G249 is evolutionarily conserved across species, including humans, underscoring α I’s 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.