Structural basis for safe and efficient energy conversion in a respiratory supercomplex

The study investigates how respiratory supercomplexes achieve safe and efficient energy conversion while minimizing wasteful electron and proton transfer that can lead to reactive oxygen species. Cytochrome bc complexes and cytochrome aa3 oxidases generate the proton motive force, yet the mechanisms controlling electron/proton transfer efficiency and safety within supercomplexes remained unclear. The authors address this by determining a 2.8 Å cryo-EM structure of the cytochrome bcc-aa3 supercomplex from Corynebacterium glutamicum, enabling visualization of quinones, ligands, dioxygen, proton pathways, and key protonable residues involved in catalysis.

Background work established roles of respiratory supercomplexes in electron transport chain organization and efficiency in mitochondria and bacteria, and canonical features of cytochrome bc1 and A-type cytochrome c oxidases (including K and D proton channels). Prior structures of Mycobacterium smegmatis III-IV supercomplexes provided conflicting models for QcrC behavior (static electron wire versus conformational switch). Mitochondrial bc1 structures and biochemical studies described Qo/Qi site architecture, Rieske domain mobility, and cardiolipin interactions at quinone sites. This study builds on these insights to analyze actinobacterial-specific features, including a distinct Qo motif and unique quinone-binding architecture.

- Determined a 2.8 Å resolution cryo-EM structure of the cytochrome bcc-aa3 (III2-IV2) supercomplex from Corynebacterium glutamicum, resolving endogenous ligands (menaquinones at multiple sites), substrate mimics (stigmatellin at Qo), lycopene, dioxygen, ordered waters, and key side-chain conformations. - Mapped electron and proton transfer pathways from the cryo-EM density, including identifying ordered water networks and hydrogen-bonded protonable residues. - Calculated electron transfer rates from edge-to-edge cofactor distances using methods of Dutton and colleagues; distances for haems measured from the edge of the conjugated ring. - Performed structural/bioinformatic analyses: signal peptide prediction (SignalP 5.0), interaction surface calculations (PISA), channel/cavity analysis and visualization (HOLLOW; defined van der Waals radii for oxygen tunnel), multiple sequence alignment (Clustal Omega; visualized with JalView), and ligand interaction analysis (LIGPLOT). - Data resources: cryo-EM maps deposited (EMD-13976, EMD-13977); atomic models deposited (PDB 7qhm, 7qho). Mass spectrometry datasets deposited (MassIVE MSV000083873; ProteomeXchange PXD014069).

- Two proton-release pathways at the Qo site (Ex1 and Ex2) were identified. Ex1 runs from the FeS-cluster ligand His355QcrA via Asp302QcrB, Arg306QcrB, Asp387QcrB and ordered waters to the protein surface. Ex2 uses Asp295QcrB (the protonable residue of the actinobacterial Qo motif) and a chain of ordered waters toward the QcrB:QcrC interface where PE lipids are located. This supports bifurcated proton release concomitant with bifurcated electron transfer during menaquinol oxidation, contributing to proton motive force generation. - The Rieske-homologous QcrA extrinsic domain is locked by extensive interfaces (~5000 Å2), occluding direct exposure of the Qo site and necessitating defined proton-release pathways rather than simple solvent exposure. - Qi site: An endogenous menaquinone (MKQi) is bound in a cavity formed by QcrB helices A, E, and D, 4.6 Å from haem b for rapid electron transfer. Binding is stabilized by non-polar interactions and a single H-bond from Glu38QcrB to one quinone carbonyl. A proton uptake route (En1) connects a cardiolipin molecule via ordered waters to Lys253QcrB and Glu38QcrB, enabling proton delivery for quinone reduction. Unlike mitochondrial bc1, only one carbonyl of MKQi forms an H-bond, highlighting a distinct Qi architecture. - Discovery of a previously unknown central quinone site (Qe): a co-purified menaquinone (MKQe) binds in a hydrophobic pocket formed by QcrB, QcrA, and QcrA' (from the opposite protomer), with the ring 12.6 Å from haem b1 and the tail extending into the quinone exchange cavity. The pocket lacks ionizable residues and solvent access, suggesting stabilization of a semiquinone anion and a role as a single-electron mediator/electron buffer. Quinone extraction showed ~4.2 menaquinone molecules per supercomplex monomer, consistent with occupancy of three resolved sites and additional cavity density. The Qe site likely stores surplus electrons, keeping haem b1 oxidized to favor electron bifurcation and limit ROS-producing bypass reactions. - QcrC (di-haem c subunit) is statically integrated: both c-type haem domains are deeply embedded with interface areas >2600 Å2 each, forming a static electron busbar via FeS → haem → haem → CuA with edge-to-edge distances of approximately 16.5, 12.7, and 14.4 Å, supporting rapid, defined inter-complex electron transfer. - In the aa3 oxidase, two proton uptake pathways corresponding to canonical K and D channels were resolved. The K-channel features entry at Glu110CtaC leading to Lys341CtaD. The D-channel ends at Glu267CtaD; the typical entry Asp116CtaD is shielded but connected to the surface via an ordered water and conserved residues His529CtaD and Glu453QcrB (part of a C-terminal extension unique to actinobacterial QcrB), providing a structural basis for efficient proton delivery for oxygen reduction and pumping. - Additional ligands and features include stigmatellin bound at the Qo site and lycopene associated with the complex. Overall architecture and mapped cofactor distances support efficient and controlled electron transfer.

The structure reveals how the actinobacterial III2–IV2 supercomplex achieves efficient and safe energy conversion. Locking of the QcrA extrinsic domain restricts solvent access to the Qo site, enforcing controlled, bifurcated proton release through defined Ex1 and Ex2 pathways that are structurally supported by hydrogen-bonded residues and ordered waters. Identification of a central Qe menaquinone site provides a mechanistic basis for transient electron storage as a stabilized semiquinone, maintaining haem b1 oxidized to promote electron bifurcation at Qo and suppress ROS-generating bypass reactions. The static, deeply integrated QcrC forms a robust electron conduit between complexes III and IV with optimized distances for rapid transfer, while the resolved K and D proton channels in aa3 oxidase, including a QcrB-facilitated connection to the D-channel entry, ensure rapid proton uptake for catalysis and pumping. Together, these features explain how the supercomplex coordinates electron and proton flows to maximize proton motive force generation while minimizing reactive oxygen species formation, addressing the core question of efficiency and safety in respiratory supercomplexes.

This work provides a 2.8 Å cryo-EM structure of the Corynebacterium glutamicum cytochrome bcc–aa3 (III2–IV2) supercomplex, mapping cofactors, ligands, and water networks that underlie controlled electron and proton transfer. Key advances include discovery of a central Qe site for semiquinone stabilization/electron buffering, delineation of bifurcated proton-release pathways at Qo, structural resolution of a static QcrC electron wire, and visualization of canonical K and D proton channels in aa3 oxidase with an actinobacteria-specific connection. These insights explain mechanisms enabling safe and efficient energy conversion and provide a structural framework to guide rational drug design against pathogenic actinobacteria (e.g., diphtheria and tuberculosis). Future research may exploit species-specific features (e.g., Qo motif environment, quinone-binding pockets, QcrB extensions) for targeted inhibitor development and probe dynamic aspects of electron/proton transfer under varying physiological conditions.