Recent studies have established that cellular electrostatic interactions are more influential than assumed previously. Here, we use cryo-EM and perform steady-state kinetic studies to investigate electrostatic interactions between cytochrome (cyt.) c and the complex (C) III₂-IV supercomplex from Saccharomyces cerevisiae at low salinity. The kinetic studies show a sharp transition with a Hill coefficient ≥2, which together with the cryo-EM data at 2.4 Å resolution indicate multiple cyt. c molecules bound along the supercomplex surface. Negatively charged loops of CIII₂ subunits Qcr6 and Qcr9 become structured to interact with cyt. c. In addition, the higher resolution allows us to identify water molecules in proton pathways of CIV and, to the best of our knowledge, previously unresolved cardiolipin molecules. In conclusion, the lowered electrostatic screening renders engagement of multiple cyt. c molecules that are directed by electrostatically structured CIII₂ loops to conduct electron transfer between CIII₂ and CIV.

Fission yeast Schizosaccharomyces pombe serves as model organism for studying higher eukaryotes. We combined the use of cryo-EM and spectroscopy to investigate the structure and function of affinity purified respiratory complex IV (CIV) from S. pombe. The reaction sequence of the reduced enzyme with O-2 proceeds over a time scale of mu s-ms, similar to that of the mammalian CIV. The cryo-EM structure of CIV revealed eleven subunits as well as a bound hypoxia-induced gene 1 (Hig1) domain of respiratory supercomplex factor 2 (Rcf2). These results suggest that binding of Rcf2 does not require the presence of a CIII-CIV supercomplex, i.e. Rcf2 is a component of CIV. An AlphaFold-Multimer model suggests that the Hig1 domains of both Rcf1 and Rcf2 bind at the same site of CIV suggesting that their binding is mutually exclusive. Furthermore, the differential functional effect of Rcf1 or Rcf2 is presumably caused by interactions of CIV with their different non-Hig1 domain parts. Fission yeast Schizosaccharomyces pombe shares many characteristics with higher eukaryotes. Here, the authors investigate the structure and function of respiratory complex IV from S. pombe, reveal the subunit arrangements and the reaction sequence of O-2 reduction.

In the final step of cellular respiration, electrons are transferred through the respiratory chain to reduce molecular oxygen to water. The energy released in this chain is used to maintain a proton electrochemical gradient across the cell membrane, which is used, for example, by the ATP synthase to produce ATP. The enzyme complexes of the respiratory chain are known to organize in supramolecular assemblies, so-called respiratory supercomplexes.

In this work we investigated the functional significance of respiratory supercomplexes consisting of complexes III and IV in mitochondria. By combining structural and kinetic studies we showed that at the commonly assumed "physiological" ionic strength of 150 mM monovalent salt, the water-soluble cyt. c associates with the negatively charged surface of III₂-IV_1-2 supercomplexes in the yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. The data showed that one cyt. c diffuses in 2D, between complexes III and IV, indicating a kinetic advantage of forming supercomplexes. These studies also showed different relative orientation of the individual complexes in the supercomplexes from the two yeast species, indicating that 2D diffusion is a general mechanism, not limited to a specific relative orientation of complexes III and IV. 

More recent data in the literature indicate that a more realistic mimic of intracellular conditions is a monovalent salt concentration of 20 mM. We showed that under these conditions two cyt. c molecules bind simultaneously to the supercomplex. This result further supports a kinetic advantage of forming supercomplexes.

We also determined the cryo-EM structure of the obligate III₂-IV₂ supercomplex from the Gram-positive bacterium Corynebacterium glutamicum. The structure revealed an electronic connection between complexes III and IV by a di-heme cyt. cc subunit. The structure also showed that complexes III and IV are structurally intertwined and strongly connected with unique features conserved in the phylum actinobacteria. 

The respiratory chain in aerobic organisms is composed of a number of membrane-bound protein complexes that link electron transfer to proton translocation across the membrane. In mitochondria, the final electron acceptor, complex IV (CIV), receives electrons from dimeric complex III (CIII₂), via a mobile electron carrier, cytochrome c. In the present study, we isolated the CIII₂CIV supercomplex from the fission yeast Schizosaccharomyces pombe and determined its structure with bound cyt. c using single-particle electron cryomicroscopy. A respiratory supercomplex factor 2 was found to be bound at CIV distally positioned in the supercomplex. In addition to the redox-active metal sites, we found a metal ion, presumably Zn²⁺, coordinated in the CIII subunit Cor1, which is encoded by the same gene (qcr1) as the mitochondrial-processing peptidase subunit β. Our data show that the isolated CIII₂CIV supercomplex displays proteolytic activity suggesting a dual role of CIII₂ in S. pombe. As in the supercomplex from S. cerevisiae, subunit Cox5 of CIV faces towards one CIII monomer, but in S. pombe, the two complexes are rotated relative to each other by ~45°. This orientation yields equal distances between the cyt. c binding sites at CIV and at each of the two CIII monomers. The structure shows cyt. c bound at four positions, but only along one of the two symmetrical branches. Overall, this combined structural and functional study reveals the integration of peptidase activity with the CIII₂ respiratory system and indicates a two-dimensional cyt. c diffusion mechanism within the CIII₂–CIV supercomplex.

The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme’s catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.

Corynebacterium glutamicum is a preferentially aerobic gram-positive bacterium belonging to the phylum Actinobacteria, which also includes the pathogen Mycobacterium tuberculosis. In these bacteria, respiratory complexes III and IV form a CIII₂CIV₂ supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 Å resolution. The structure shows a central CIII₂ dimer flanked by a CIV on two sides. A menaquinone is bound in each of the Q_N and Q_P sites in each CIII and an additional menaquinone is positioned ∼14 Å from heme b_L. A di-heme cyt. cc subunit electronically connects each CIII with an adjacent CIV, with the Rieske iron-sulfur protein positioned with the iron near heme b_L. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a path for proton transfer into CIV.

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 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. 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 QH(2):O-2 oxidoreductase activity and suggests a possible regulatory role for supercomplex formation in the respiratory chain.

In the final steps of energy conservation in aerobic organisms, free energy from electron transfer through the respiratory chain is transduced into a proton electrochemical gradient across a membrane. In mitochondria and many bacteria, reduction of the dioxygen electron acceptor is catalyzed by cytochrome c oxidase (complex IV), which receives electrons from cytochrome bc(1) (complex III), via membrane-bound or watersoluble cytochrome c. These complexes function independently, but in many organisms they associate to form supercomplexes. Here, we review the structural features and the functional significance of the nonobligate III2IV1/2 Saccharomyces cerevisiae mitochondrial super-complex as well as the obligate III2IV2 supercomplex from actinobacteria. The analysis is centered around the Q-cycle of complex III, proton uptake by CytcO, as well as mechanistic and structural solutions to the electronic link between complexes III and IV.