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

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 that maintains the electrochemical proton gradient used to produce ATP and drive other cellular processes. Electrons are transferred between respiratory complexes III and IV (CIII and CIV) by water-soluble cyt. c . In S. cerevisiae and some other organisms, these complexes assemble into larger CIII 2 CIV 1/2 supercomplexes, the functional significance of which has remained enigmatic. In this work, we measured the kinetics of the S. cerevisiae supercomplex’s cyt. c -mediated QH 2 :O 2 oxidoreductase activity under various conditions. The data indicate that the electronic link between CIII and CIV is confined to the surface of the supercomplex. Cryo-EM structures of the supercomplex with cyt. c reveal distinct states where the positively-charged cyt. c is bound either to CIII or CIV, or resides at intermediate positions. Collectively, the structural and kinetic data indicate that cyt. c travels along a negatively-charged surface patch of the supercomplex. Thus, rather than enhancing electron-transfer rates by decreasing the distance cyt. c must diffuse in 3D, formation of the CIII 2 CIV 1/2 supercomplex facilitates electron transfer by 2D diffusion of cyt. c . This mechanism enables the CIII 2 CIV 1/2 supercomplex to increase QH 2 :O 2 oxidoreductase activity and suggests a possible regulatory role for supercomplex formation in the respiratory chain. Significance Statement 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 cyt. c . The latter transfers electrons from respiratory complex III to IV. In yeast these complexes assemble into III 2 IV 1/2 supercomplexes, but their 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 cyt. c shuttles electrons by sliding along the surface of III 2 IV 1/2 (2D diffusion). The structural arrangement into III 2 IV 1/2 supercomplexes suggests a mechanism to regulate cellular respiration.