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Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D

ABSTRACT: Cytochrome bd quinol:O2 oxidoreductases are respiratory terminal oxidases so far only identified in prokaryotes, including several pathogenic bacteria. Escherichia coli contains two bd oxidases of which only the bd-I type is structurally characterized. Here, we report the structure of the Escherichia coli cytochrome bd-II type oxidase with the bound inhibitor aurachin D as obtained by electron cryo-microscopy at 3 Å resolution. The oxidase consists of subunits AppB, C and X that show an architecture similar to that of bd-I. The three heme cofactors are found in AppC, while AppB is stabilized by a structural ubiquinone-8 at the homologous positions. A fourth subunit present in bd-I is lacking in bd-II. Accordingly, heme b595 is exposed to the membrane but heme d embedded within the protein and showing an unexpectedly high redox potential is the catalytically active centre. The structure of the Q-loop is fully resolved, revealing the specific aurachin binding. Terminal bd oxidases endow bacterial pathogens with resistance to cellular stressors. The authors report the structure of E. coli bd-II type oxidase with the bound inhibitor aurachin D, providing a structural basis for the design of specifically binding antibiotics.

SUBMITTER: Grauel A

PROVIDER: S-EPMC8585947 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Short-chain aurachin D derivatives are selective inhibitors of E. coli cytochrome bd-I and bd-II oxidases.

Project description:Cytochrome bd-type oxidases play a crucial role for survival of pathogenic bacteria during infection and proliferation. This role and the fact that there are no homologues in the mitochondrial respiratory chain qualify cytochrome bd as a potential antimicrobial target. However, few bd oxidase selective inhibitors have been described so far. In this report, inhibitory effects of Aurachin C (AurC-type) and new Aurachin D (AurD-type) derivatives on oxygen reductase activity of isolated terminal bd-I, bd-II and bo3 oxidases from Escherichia coli were potentiometrically measured using a Clark-type electrode. We synthesized long- (C10, decyl or longer) and short-chain (C4, butyl to C8, octyl) AurD-type compounds and tested this set of molecules towards their selectivity and potency. We confirmed strong inhibition of all three terminal oxidases for AurC-type compounds, whereas the 4(1H)-quinolone scaffold of AurD-type compounds mainly inhibits bd-type oxidases. We assessed a direct effect of chain length on inhibition activity with highest potency and selectivity observed for heptyl AurD-type derivatives. While Aurachin C and Aurachin D are widely considered as selective inhibitors for terminal oxidases, their structure-activity relationship is incompletely understood. This work fills this gap and illustrates how structural differences of Aurachin derivatives determine inhibitory potency and selectivity for bd-type oxidases of E. coli.

| S-EPMC8668966 | biostudies-literature

Respiration of Escherichia coli can be fully uncoupled via the nonelectrogenic terminal cytochrome bd-II oxidase.

Project description:The respiratory chain of Escherichia coli is usually considered a device to conserve energy via the generation of a proton motive force, which subsequently may drive ATP synthesis by the ATP synthetase. It is known that in this system a fixed amount of ATP per oxygen molecule reduced (P/O ratio) is not synthesized due to alternative NADH dehydrogenases and terminal oxidases with different proton pumping stoichiometries. Here we show that P/O ratios can vary much more than previously thought. First, we show that in wild-type E. coli cytochrome bo, cytochrome bd-I, and cytochrome bd-II are the major terminal oxidases; deletion of all of the genes encoding these enzymes results in a fermentative phenotype in the presence of oxygen. Second, we provide evidence that the electron flux through cytochrome bd-II oxidase is significant but does not contribute to the generation of a proton motive force. The kinetics support the view that this system is as an energy-independent system gives the cell metabolic flexibility by uncoupling catabolism from ATP synthesis under non-steady-state conditions. The nonelectrogenic nature of cytochrome bd-II oxidase implies that the respiratory chain can function in a fully uncoupled mode such that ATP synthesis occurs solely by substrate level phosphorylation. As a consequence, the yield with a carbon and energy source can vary five- to sevenfold depending on the electron flux distribution in the respiratory chain. A full understanding and control of this distribution open new avenues for optimization of biotechnological processes.

| S-EPMC2725625 | biostudies-literature

Compensations for diminished terminal oxidase activity in Escherichia coli: cytochrome bd-II-mediated respiration and glutamate metabolism.

Project description:Escherichia coli possesses cytochrome bo' (CyoABCDE), cytochrome bd-I (CydAB), and cytochrome bd-II (AppBC) quinol oxidases, all of which can catalyze the terminal step in the aerobic respiratory chain, the reduction of oxygen by ubiquinol. Although CydAB has a role in the generation of DeltapH, AppBC has been proposed to alleviate the accumulation of electrons in the quinone pool during respiratory stress via electroneutral ubiquinol oxidation. A cydB mutant strain exhibited lower respiration rates while maintaining a wild type growth rate. Transcriptomic analysis revealed a dramatic up-regulation of AppBC in the cydB strain, accompanied by the induction of genes involved in glutamate/gamma-aminobutyric acid (GABA) antiport, the GABA shunt, the glyoxylate shunt, respiration (including appBC), motility, and osmotic stress. Transcription factor modeling suggests that the underpinning regulation is largely controlled by H-NS, GadX, FlhDC, and AppY. The transcriptional adaptations imply that cydB cells contribute to the proton motive force via consumption of intracellular protons and glutamate/GABA antiport. Indeed, supplementation of culture medium with l-glutamate stimulates growth in a cydB strain. Phenotype analyses of the cydB strain confirm decreased motility and elevated acid resistance and also an elevated cytochrome d spectroscopic signal in cells grown at low pH. We propose a mechanism via which E. coli can compensate for the loss of cytochrome bd-I activity; cytochrome bd-II-mediated quinol oxidation prevents the accumulation of NADH, whereas GABA synthesis/antiport maintains the proton motive force for ATP production.

| S-EPMC2881772 | biostudies-literature

The Escherichia coli CydX protein is a member of the CydAB cytochrome bd oxidase complex and is required for cytochrome bd oxidase activity.

Project description:Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ?30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ?cydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.

| S-EPMC3754557 | biostudies-literature

The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection.

Project description:Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract.

| S-EPMC5073308 | biostudies-literature

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration

Project description: Not available

| S-EPMC10815991 | biostudies-literature

The cytochrome bd oxidase of Escherichia coli prevents respiratory inhibition by endogenous and exogenous hydrogen sulfide.

Project description:When sulfur compounds are scarce or difficult to process, Escherichia coli adapts by inducing the high-level expression of sulfur-compound importers. If cystine then becomes available, the cystine is rapidly overimported and reduced, leading to a burgeoning pool of intracellular cysteine. Most of the excess cysteine is exported, but some is adventitiously degraded, with the consequent release of sulfide. Sulfide is a potent ligand of copper and heme moieties, raising the prospect that it interferes with enzymes. We observed that when cystine was provided and sulfide levels rose, E. coli became strictly dependent upon cytochrome bd oxidase for continued respiration. Inspection revealed that low-micromolar levels of sulfide inhibited the proton-pumping cytochrome bo oxidase that is regarded as the primary respiratory oxidase. In the absence of the back-up cytochrome bd oxidase, growth failed. Exogenous sulfide elicited the same effect. The potency of sulfide was enhanced when oxygen concentrations were low. Natural oxic-anoxic interfaces are often sulfidic, including the intestinal environment where E. coli dwells. We propose that the sulfide resistance of the cytochrome bd oxidase is a key trait that permits respiration in such habitats.

| S-EPMC4925259 | biostudies-literature

In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I.

Project description:Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

| S-EPMC7824442 | biostudies-literature

Transcriptional response of Mycobacterium tuberculosis to inhibition of Cytochrome bd oxidase and Cytochrome bcc:aa3 oxidase

Project description:Comparative analysis of the transcriptional response, as quantified by RNA-Seq, of Mycobacterium tuberculosis (Mtb) during inhibition of the terminal cytochrome oxidases, Cytochrome bd oxidase and Cytochrome bcc:aa3. This study was designed to evaluate the efficacy if a novel small molecule inhibitor of the Cytochrome bd oxidase. Specifically, we compared the transcriptional response of Mtb during inhibition by Q203 with a Cytochrome bd deletion mutant to the transcriptional response of Mtb treated with a combination of Q203 and the purported Cytorchomre bd small molecule inhibitor (ND-011992).

2020-10-07 | GSE159080 | GEO

Mechanistic and structural diversity between cytochrome bd isoforms of Escherichia coli.

Project description: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.

| S-EPMC8685929 | biostudies-literature

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