Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b558, heme b595 and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O2 reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer. Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b558 is pH dependent, with a maximum rate constant (~104 s−1) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pKa ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58B➔Asn and Asp105B➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105B➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105B in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pKa proton donor, and the conservation pattern of the Asp-105B in the cyt. bd superfamily.

 Cytochrome bd is a terminal oxidase expressed under low oxygen conditions that is central for the survival of many pathogens and hence a potential drug target. The bd oxidases can be grouped into three evolutionary classes, with most structural data originating from the qOR-1 clades, while the other families remain poorly understood. Here we combine biochemical studies with cryo-electron microscopy (cryo-EM) and multiscale simulations to functionally characterise the first qOR-2 type oxidase from Mycobacterium smegmatis. By over-expressing the appCB gene in its native host, we produce an active bd-II protein with a coupled menaquinol-oxidoreductase activity of 30 e^-s^-1. We determine the cryo-EM structure of the mycobacterial bd-II at 2.8 Å that together with multiscale simulations reveal unique putative proton pathways and oxygen channels, and a molecular basis for its function. We identify a putative pH-dependent coordination change of the redox-active heme d that couples to conformational switching of a bulky Phe114 and regulate substrate access into the active site. Taken together, our findings provide molecular insights into the mechanistic principles and evolution of the qOR-2 typebdoxidases and basis for understanding their mechanism of action.

Aerobic organisms obtain energy by linking electron transfer from NADH to O₂, through the respiratory chain, to transmembrane proton translocation. In mycobacteria the respiratory chain is branched; the membrane-bound electron carrier menaquinol (MQH₂) donates electrons either to the O₂-reducing cytochrome bd or a supercomplex that is composed of a complex (C) III₂ dimer flanked two CIVs. Here, we measured the MQH₂ oxidation:O₂ reduction activities of the CIII₂CIV₂ supercomplex and cytochrome bd in the presence of the mammalian electron carrier, ubiquinol (UQH₂). The data show that UQH₂ inhibits both the CIII₂CIV₂ and cytochrome bd activities, suggesting that UQ/UQH₂ interferes with both branches of the respiratory chain. Cryo-EM data of the M.smegmatis supercomplex shows that oxidized UQ binds in the electron donor site (Q_o) of CIII₂. Accordingly, growth of M.smegmatis cells was impaired in the presence of UQ. Remarkably, UQ also impairs intracellular growth of virulent M.tuberculosis cells in human primary macrophages suggesting that the compound could potentially be used as an adjuvant during tuberculosis disease treatment.