Cryo-EM structure and kinetics reveal electron transfer by 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex

Significance In the last steps of food oxidation in living organisms, electrons are transferred to oxygen through the membrane-bound respiratory chain. This electron transfer is mediated by mobile carriers, such as membrane-bound quinone and water-soluble cytochrome c. The latter transfers electrons from respiratory complex III to complex IV. In yeast, these complexes assemble into III2IV1/2 supercomplexes, but its role has remained enigmatic. This study establishes a functional role for this supramolecular assembly in the mitochondrial membrane. We used cryo-EM and kinetic studies to show that cytochrome c shuttles electrons by two-dimensional diffusion, sliding along the surface of III2IV1/2. The structural arrangement of III2IV1/2 supercomplexes suggests a mechanism to regulate cellular respiration. Energy conversion in aerobic organisms involves an electron current from low-potential donors, such as NADH and succinate, to dioxygen through the membrane-bound respiratory chain. Electron transfer is coupled to transmembrane proton transport, which maintains the electrochemical proton gradient used to produce ATP and drive other cellular processes. Electrons are transferred from respiratory complexes III to IV (CIII and CIV) by water-soluble cytochrome (cyt.) c. In Saccharomyces cerevisiae and some other organisms, these complexes assemble into larger CIII2CIV1/2 supercomplexes, the functional significance of which has remained enigmatic. In this work, we measured the kinetics of the S. cerevisiae supercomplex cyt. c-mediated QH2:O2 oxidoreductase activity under various conditions. The data indicate that the electronic link between CIII and CIV is confined to the surface of the supercomplex. Single-particle electron cryomicroscopy (cryo-EM) structures of the supercomplex with cyt. c show the positively charged cyt. c bound to either CIII or CIV or along a continuum of intermediate positions. Collectively, the structural and kinetic data indicate that cyt. c travels along a negatively charged patch on the supercomplex surface. Thus, rather than enhancing electron transfer rates by decreasing the distance that cyt. c must diffuse in three dimensions, formation of the CIII2CIV1/2 supercomplex facilitates electron transfer by two-dimensional (2D) diffusion of cyt. c. This mechanism enables the CIII2CIV1/2 supercomplex to increase QH2:O2 oxidoreductase activity and suggests a possible regulatory role for supercomplex formation in the respiratory chain.