Aerobic cellular respiration relies on the efficient and safe conversion of energy. This process is driven largely by the cytochrome bc complexes and cytochrome aa3 oxidases, which establish the proton motive force (PMF) crucial for ATP synthesis. Respiratory complexes often associate into supercomplexes, a higher-order organization hypothesized to enhance energy conversion efficiency and suppress the generation of harmful reactive oxygen species (ROS). However, the precise mechanisms by which these supercomplexes control electron and proton transfer to achieve both efficiency and safety remain poorly understood. This research aimed to address this gap by determining the high-resolution structure of a respiratory supercomplex and analyzing its structural features related to electron and proton transfer pathways. Understanding these mechanisms is not only fundamentally important for understanding energy metabolism but also has significant implications for the development of novel therapeutics targeting pathogenic actinobacteria responsible for diseases like diphtheria and tuberculosis, as these bacteria rely on efficient respiratory chains for survival.

Previous studies have suggested that respiratory supercomplexes play a vital role in optimizing energy conversion and ROS production. Studies on mitochondrial respiratory supercomplexes have provided some insights, but the mechanisms at play in other organisms, particularly actinobacteria, are less clear. Prior structural studies of *M. smegmatis* supercomplexes have offered conflicting information regarding the role of the di-haem subunit QcrC – whether it acts as a direct electron wire or a conformational switch regulating electron transfer. The current study aims to provide a more detailed picture of the supercomplex structure and its function, resolving these inconsistencies and providing a basis for a more complete understanding of respiratory chain organization and function in actinobacteria.

The research utilized cryo-electron microscopy (cryo-EM) to determine the high-resolution structure of the cytochrome bcc-aa3 (III2-IV2) supercomplex from *Corynebacterium glutamicum*. The supercomplex was purified, and cryo-EM data were collected and processed using advanced computational methods. Density maps were generated, and atomic models were built and refined using available software and restraints derived from previously solved structures of related protein complexes. Analysis of the resulting structure focused on the identification of redox-active cofactors, quinone binding sites, proton transfer pathways, and conformational states of key residues. Electron transfer rates were calculated based on edge-to-edge distances of cofactors. Bioinformatic tools were used to analyze protein interaction surface areas, protein channels and cavities, multiple sequence alignments, and ligand interactions. Mass spectrometry was used to analyze protein composition and quantify menaquinone content.

The 2.8 Å resolution cryo-EM structure revealed several key features. First, the structure resolved three menaquinone molecules: one at the Qi site (menaquinone reduction), one at the unexpected Qe site, and one at the Qo site (menaquinone oxidation). The structure of the Qo site included a transition-state analog (stigmatellin) bound to the site, along with identified proton release pathways (Ex1 and Ex2). The Qi site was found to have an uptake pathway (En1) connected to a cardiolipin molecule. The Qe site, a novel finding, appeared to serve as a transient electron storage site, preventing ROS production. The di-haem subunit QcrC was found to be stably integrated into the supercomplex, acting as a defined electron wire rather than a conformational switch, contradicting some previous reports. Two proton transfer pathways (K-channel and D-channel) were identified in the aa3 oxidase, facilitating rapid proton uptake for oxygen reduction and proton pumping. The structure provides evidence for bifurcated electron and proton transfer, optimizing energy conversion efficiency. The detailed analysis of the identified residues, including the highly conserved ones in actinobacteria and the less conserved residues relevant for species-specific targeting, revealed a sophisticated mechanism for efficient and controlled electron and proton movement within the supercomplex.

The high-resolution structure of the *C. glutamicum* cytochrome bcc-aa3 supercomplex provides critical insights into how safe and efficient energy conversion is achieved in a respiratory supercomplex. The findings show how controlled electron and proton transfer, facilitated by specific interactions between subunits and cofactors, minimizes ROS production and maximizes ATP synthesis. The identification of the novel Qe site suggests a mechanism for preventing electron overreduction and limiting wasteful bypass reactions. The clarified role of QcrC as a static electron wire clarifies previous conflicting reports and provides a more complete understanding of electron transfer dynamics. The identification of the proton transfer pathways in the aa3 oxidase highlights the mechanisms for efficient proton delivery required for oxygen reduction and proton pumping. The structural information revealed in this study is highly relevant for understanding bacterial respiration, energy conversion, and ROS generation and for the rational design of anti-bacterial drugs that target the respiratory chain.

This study presents a high-resolution structure of a bacterial respiratory supercomplex, revealing the structural basis for efficient and safe energy conversion. The findings highlight the roles of controlled electron and proton transfer, a novel Qe site for electron storage, and the defined role of QcrC. This research provides critical insights into bacterial energy metabolism and opens avenues for the development of novel antimicrobial therapies targeting the respiratory chain of pathogenic actinobacteria. Future studies could investigate the dynamic interactions within the supercomplex and the effects of mutations on function and efficiency.

The study focuses on a single species of actinobacteria (*C. glutamicum*). The generalizability of these findings to other species within this large and diverse phylum requires further investigation. The study relies on a static structure determined by cryo-EM; thus, it may not capture all the dynamic aspects of the respiratory chain’s function. Further studies using other biophysical techniques would complement this research to further explore the dynamic aspects of the supercomplex function and its response to environmental changes.