The cytochrome bd oxidases are terminal oxidases that are present in bacteria and archaea. They reduce molecular oxygen (dioxygen) to water, avoiding the production of reactive oxygen species. In addition to their contribution to the proton motive force, they mediate viability under oxygen-related stress conditions and confer tolerance to nitric oxide, thus contributing to the virulence of pathogenic bacteria. Here we present the atomic structure of the bd oxidase from Geobacillus thermodenitrificans, revealing a pseudosymmetrical subunit fold. The arrangement and order of the heme cofactors support the conclusions from spectroscopic measurements that the cleavage of the dioxygen bond may be mechanistically similar to that in the heme-copper–containing oxidases, even though the structures are completely different.

The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refined using X-ray cryodata to 2.25 A resolution in order to gain further insights into its mechanism of action. The refined structural model shows a number of new features including many additional solvent and detergent molecules. The electron density bridging the heme a(3) iron and Cu(B) of the active site is fitted best by a peroxo-group or a chloride ion. Two waters or OH(-) groups do not fit, one water (or OH(-)) does not provide sufficient electron density. The analysis of crystals of cytochrome c oxidase isolated in the presence of bromide instead of chloride appears to exclude chloride as the bridging ligand. In the D-pathway a hydrogen bonded chain of six water molecules connects Asn131 and Glu278, but the access for protons to this water chain is blocked by Asn113, Asn131 and Asn199. The K-pathway contains two firmly bound water molecules, an additional water chain seems to form its entrance. Above the hemes a cluster of 13 water molecules is observed which potentially form multiple exit pathways for pumped protons. The hydrogen bond pattern excludes that the Cu(B) ligand His326 is present in the imidazolate form.

No abstract

Using a combination of stopped-flow spectrophotometric proton pumping measurements and time-resolved potential measurements on black lipid membranes, we have investigated the effect of Zn 2+ ions on the proton transfer properties of Paracoccus denitrificans cytochrome c oxidase. When zinc was enclosed in the interior of cytochrome c oxidase containing liposomes, the H/e stoichiometry was found to gradually decrease with increasing Zn 2+ concentration. Halfinhibition of proton pumping was observed at [Zn 2+ ] i = 75 W WM corresponding to about 5^6 Zn 2+ ions per oxidase molecule. In addition, there was a significant increase in the respiratory control ratio of the proteoliposomes upon incorporation of Zn 2+ . Time-resolved potential measurements on a black lipid membrane showed that the electrogenic phases slowed down in the presence of Zn 2+ correspond to phases that have been attributed to proton uptake from the cytoplasmic side and to proton pumping. We conclude that Zn 2+ ions bind close to or within the two proton transfer pathways of the bacterial cytochrome c oxidase. ß

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I. Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.