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bioRxiv · 10.64898/2026.05.22.727096

Stochastic spin selection in the mechanism of semiquinone formation at the ubiquinol oxidation Qo site of cytochrome bc1

Bujnowicz, Łukasz·Pietras, R.·Wojcik-Augustyn, A.·Osyczka, A.·Sarewicz, M.
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Abstract

Cytochrome bc1 is one of the key enzymes of biological energy-conserving systems. In its catalytic Q cycle, the central reaction is the oxidation of quinol (QH2), 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 bL), 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 bc1. We detect SQ spin-coupled to the reduced 2Fe2S cluster (2Fe2Sred), whose population markedly exceeds that of reduced heme bL and forms exclusively in sites containing oxidized heme. We also identify that the lowest-energy configuration corresponds to the state with reduced heme bH (adjacent to heme bL), oxidized heme bL and SQ-2Fe2Sred. 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 Qo that are observed spectroscopically.

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Bujnowicz, Łukasz, Pietras, R., Wojcik-Augustyn, A., Osyczka, A., Sarewicz, M.. 2026-05-26. Stochastic spin selection in the mechanism of semiquinone formation at the ubiquinol oxidation Qo site of cytochrome bc1. https://doi.org/10.64898/2026.05.22.727096

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