Stochastic Spin Selection in the Mechanism of Semiquinone Formation at the Ubiquinol Oxidation Qo Site of Cytochrome bc 1

Cytochrome bc1 is a pivotal enzyme in biological energy conservation systems. The central reaction in its catalytic Q cycle is the oxidation of quinol (QH2), during which electrons are channeled into two distinct cofactor chains. However, the molecular mechanism underlying this reaction remains elusive. The canonical model, which assumes a series 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 electron bifurcation derived from quantum mechanical calculations─EMET (EMergent Electron Transfer) (10.1021/acsomega.5c13233). These two models indicate fundamentally different microstates of the enzyme, specifically related to the formation of semiquinone (SQ) at the catalytic site (Qo) and the prediction of different lowest energy configurations. Here, we validate these predictions through electron paramagnetic resonance spectroscopy of 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 is found in sites containing oxidized heme. Furthermore, we ascertain that the lowest energy configuration corresponds to the state with reduced heme bH (adjacent to heme bL), oxidized heme bL, and SQ-2Fe2Sred. Notably, these two features are not predicted 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 lead to electron stoichiometry among hemes b, the 2Fe2S cluster, and SQ at Qo that are detected spectroscopically.