Electron sweep across four b-hemes of cytochrome bc1 revealed by unusual paramagnetic properties of the Qi semiquinone intermediate

Dimeric cytochromes be are central components of photosynthetic and respiratory electron transport chains. In their catalytic core, four hemes b connect four quinone (Q) binding sites. Two of these sites, Q(i) sites, reduce quinone to quinol (QH(2)) in a step-wise reaction, involving a stable semiquinone intermediate (SQ(i)). However, the interaction of the SQ(i) with the adjacent hemes remains largely unexplored. Here, by revealing the existence of two populations of SQ(i) differing in paramagnetic relaxation, we present a new mechanistic insight into this interaction. Benefiting from a clear separation of these SQ(i) species in mutants with a changed redox midpoint potential of hemes b, we identified that the fast-relaxing SQ(i) (SQ(iF)) corresponds to the form magnetically coupled with the oxidized heme hi, (the heme b adjacent to the Q(i) site), while the slow-relaxing SQ(i) (SQ(iS)) reflects the form present alongside b(H) reduced (and diamagnetic) heme b(H). This so far unreported SQ(iF) calls for a re-investigation of the thermodynamic properties of SQ(i) and the Q(i) site. The existence of SQ(iF) in the native enzyme reveals a possibility of an extended electron equilibration within the dimer, involving all four hemes b and both Q(i) sites. This substantiates the predicted earlier electron transfer acting to sweep the b-chain of reduced hemes b to diminish generation of reactive oxygen species by cytochrome bc(1). In analogy to the Q(i) site, we anticipate that the quinone binding sites in other enzymes may contain yet undetected semiquinones which interact magnetically with oxidized hemes upon progress of catalytic reactions.