Stochastic spin selection in the mechanism of semiquinone formation at the ubiquinol oxidation Q _o site of cytochrome bc ₁

Cytochrome bc ₁ is one of the key enzymes of biological energy-conserving systems. In its catalytic Q cycle, the central reaction is the oxidation of quinol (QH ₂ ), upon which electrons are directed to two separate cofactor chains. The molecular mechanism of this reaction remains elusive. The canonical model, assuming a sequence of reactions dictated by the equilibrium redox midpoint potentials of cofactors (the 2Fe2S cluster and heme b _L ), has recently been challenged by a new model of EB derived from quantum mechanical (QM) calculations – EMET (EMergent Electron Transfer) ( https://doi.org/10.1021/acsomega.5c13233 ). These two models predict fundamentally different microstates of the enzyme in which semiquinone (SQ) is formed in the catalytic site (Q _o ) and also predict different lowest-energy configurations. Here, we test these predictions using EPR spectroscopy on highly concentrated preparations of isolated bacterial cytochrome bc ₁ . We detect SQ spin-coupled to the reduced 2Fe2S cluster (2Fe2S ^red ), whose population markedly exceeds that of reduced heme b _L and forms exclusively in sites containing oxidized heme. We also identify that the lowest-energy configuration corresponds to the state with reduced heme b _H (adjacent to heme b _L ), oxidized heme b _L and SQ-2Fe2S ^red . These two features are precluded by the canonical model but are consistent with EMET. We conclude that EMET, unlike the canonical EB model, satisfactorily describes the occurrence of stochastic, spin-selective processes that result in electron stoichiometry among hemes b , the 2Fe2S cluster, and SQ at Q _o that are observed spectroscopically.