Molecular Principles of Proton-Coupled Quinone Reduction in the Membrane-Bound Superoxide Oxidase

Reactive oxygen species (ROS) are physiologically harmful radical species generated as byproducts of aerobic respiration. To detoxify ROS, most cells employ superoxide scavenging enzymes that disproportionate superoxide (O₂^·-) to oxygen (O₂) and hydrogen peroxide (H₂O₂). In contrast, the membrane-bound superoxide oxidase (SOO) is a minimal 4-helical bundle protein that catalyzes the direct oxidation of O₂^·- to O₂ and drives quinone reduction by mechanistic principles that remain unknown. Here, we combine multiscale hybrid quantum/classical (QM/MM) free energy calculations and microsecond molecular dynamics simulations with functional assays and site-directed mutagenesis experiments to probe the mechanistic principles underlying the charge transfer reactions of the superoxide-driven quinone reduction. We characterize a cluster of charged residues at the periplasmic side of the membrane that functions as a O₂^·- collecting antenna, initiating electron transfer via two <i>b</i> hemes to the active site for quinone reduction at the cytoplasmic side. Based on multidimensional QM/MM string simulations, we find that a proton-coupled electron transfer (PCET) reaction from the active site heme <i>b</i> and nearby histidine residues (H87, H158) results in quinol (QH₂) formation, followed by proton uptake from the cytoplasmic side of the membrane. The functional relevance of the identified residues is supported by site-directed mutagenesis and activity assays, with mutations leading to inhibition of the O₂^·--driven quinone reduction activity. We suggest that the charge transfer reactions could build up a proton motive force that supports the bacterial energy transduction machinery, while the PCET machinery provides unique design principles of a minimal oxidoreductase.