The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca²⁺ dissipates the H⁺ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca²⁺ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg²⁺ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca²⁺ can transform the energy-conserving F-ATP synthase into an energy-dissipating device.

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H2O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately −240 mV) and low-spin (approximately −110, −190 and −265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (Em=−80 mV) in the presence of NADH (Em=−320 mV) and an NADH–menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.

The biochemical and molecular mechanisms used by alkaliphilic bacterial communities to reduce metals in the environment are currently unknown. We demonstrate that an alkaliphilic (pH > 9) consortium dominated by Tissierella , Clostridium , and Alkaliphilus spp. is capable of using iron (Fe ³⁺ ) as a final electron acceptor under anaerobic conditions. Iron reduction is associated with the production of a freely diffusible species that, upon rudimentary purification and subsequent spectroscopic, high-performance liquid chromatography, and electrochemical analysis, has been identified as a flavin species displaying properties indistinguishable from those of riboflavin. Due to the link between iron reduction and the onset of flavin production, it is likely that riboflavin has an import role in extracellular metal reduction by this alkaliphilic community.

Little is known about enzymatic quinone-quinol interconversions in the lipid membrane when compared with our knowledge of substrate transformations by globular enzymes. Here, the smallest example of a quinol dehydrogenase in nature, CymA, has been studied. CymA is a monotopic membrane tetraheme c-type cytochrome belonging to the NapC/NirT family and central to anaerobic respiration in Shewanella sp. Using protein-film electrochemistry, it is shown that vesicle-bound menaquinone-7 is not only a substrate for this enzyme but is also required as a cofactor when converting other quinones. Here, we propose that the high concentration of quinones in the membrane negates the evolutionary pressure to create a high affinity active site. However, the instability and reactivity of reaction intermediate, semiquinone, might require a cofactor that functions to minimize damaging side reactions.

Significance: Antibiotics generally target one of five essential cellular functions in bacteria, but many of these targets are now compromised through rapidly spreading antibiotic resistance. Bedaquiline (BDQ), a new FDA-approved antitubercular drug, targets energy metabolism: defining cellular energetics as a new target space for antibiotics. This is a relatively unexplored area, as BDQ was only FDA approved in 2012. Several studies have recently found that BDQ stimulates mycobacterial respiration, in addition to inhibiting its molecular target, the F ₁ F _o -ATP synthase. We show that BDQ is an ionophore, which shuttles H ⁺ and K ⁺ ions across membranes, and propose that this activity may contribute to killing of mycobacteria by BDQ. Combining ionophoric activity with high-affinity membrane protein inhibition may enhance the specificity and potency of antibiotics.

Protein–protein interactions are well-known to regulate enzyme activity in cell signaling and metabolism. Here, we show that protein–protein interactions regulate the activity of a respiratory-chain enzyme, CymA, by changing the direction or bias of catalysis. CymA, a member of the widespread NapC/NirT superfamily, is a menaquinol-7 (MQ-7) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of Shewanella oneidensis. We report the incorporation of CymA within solid-supported membranes that mimic the inner membrane architecture of S. oneidensis. Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c₃ (Fcc₃) fumarate reductase. Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc₃ complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7. We propose that MQ-7 oxidation in CymA is limited by electron transfer to the hemes and that complex formation with Fcc₃ facilitates the electron-transfer rate along the heme redox chain. These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein–protein interactions.