Mechanistic and structural diversity between cytochrome bd isoforms of Escherichia coli

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.

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

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.

Aerobic Respiration and Its Regulation in the Metal Reducer Shewanella oneidensis

Shewanella oneidensis MR-1 is a facultative anaerobe known for its ability to reduce metal oxides. Anaerobic respiration, especially metal reduction, has been the subject of extensive research. In contrast, S. oneidensis aerobic respiration has received less attention. S. oneidensis expresses cbb3- and aa3-type cytochrome c oxidases and a bd-type quinol oxidase. The aa3-type oxidase, which in other bacteria is the major oxygen reductase under oxygen replete conditions, does not appear to contribute to aerobic respiration and growth in S. oneidensis. Our results indicated that although the aa3-type oxidase does not play a role in aerobic growth on lactate, the preferred carbon source for S. oneidensis, it is involved in growth on pyruvate or acetate. These results highlight the importance of testing multiple carbon and energy sources when attempting to identify enzyme activities and mutant phenotypes. Several regulatory proteins contribute to the regulation of aerobic growth in S. oneidensis including CRP and ArcA. The 3',5'-cAMP phosphodiesterase (CpdA) appears to play a more significant role in aerobic growth than either CRP or ArcA, yet the deficiency does not appear to be the result of reduced oxidase genes expression. Interestingly, the ∆cpdA mutant was more deficient in aerobic respiration with several carbon sources tested compared to ∆crp, which was moderately deficient only in the presence of lactate. To identify the reason for ∆cpdA aerobic growth deficiency, we isolated a suppressor mutant with transposon insertion in SO_3550. Inactivation of this gene, which encodes an anti-sigma factor, restored aerobic growth in the cpdA mutant to wild-type levels. Inactivation of SO_3550 in wild-type cells, however, did not affect aerobic growth. The S. oneidensis genome encodes two additional CRP-like proteins that we designated CrpB and CrpC. Mutants that lack crpB and crpC were deficient in aerobic growth, but this deficiency was not due to the loss of oxidase gene expression.

The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

Evolution of the cytochrome-bd type oxygen reductase superfamily and the function of cydAA’ in Archaea

Cytochrome bd-type oxygen reductases (cytbd) belong to one of three enzyme superfamilies that catalyze oxygen reduction to water. They are widely distributed in Bacteria and Archaea, but the full extent of their biochemical diversity is unknown. Here we used phylogenomics to identify 3 families and several subfamilies within the cytbd superfamily. The core architecture shared by all members of the superfamily consists of four transmembrane helices that bind two active site hemes, which are responsible for oxygen reduction. While previously characterized cytochrome bd-type oxygen reductases use quinol as an electron donor to reduce oxygen, sequence analysis shows that only one of the identified families has a conserved quinol binding site. The other families are missing this feature, suggesting that they use an alternative electron donor. Multiple gene duplication events were identified within the superfamily, resulting in significant evolutionary and structural diversity. The CydAA’ cytbd, found exclusively in Archaea, is formed by the co-association of two superfamily paralogs. We heterologously expressed CydAA’ from Caldivirga maquilingensis and demonstrated that it performs oxygen reduction with quinol as an electron donor. Strikingly, CydAA’ is the first isoform of cytbd containing only b-type hemes shown to be active when isolated, demonstrating that oxygen reductase activity in this superfamily is not dependent on heme d.

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

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.

Type 2 NADH Dehydrogenase Is the Only Point of Entry for Electrons into theStreptococcus agalactiaeRespiratory Chain and Is a Potential Drug Target

ABSTRACTThe opportunistic pathogenStreptococcus agalactiaeis the major cause of meningitis and sepsis in a newborn’s first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme and quinone are available in the environment, and a functional respiratory chain is required for full virulence. Remarkably, it is shown here that the entire respiratory chain ofS. agalactiaeconsists of only two enzymes, a type 2 NADH dehydrogenase (NDH-2) and a cytochromebdoxygen reductase. There are no respiratory dehydrogenases other than NDH-2 to feed electrons into the respiratory chain, and there is only one respiratory oxygen reductase to reduce oxygen to water. AlthoughS. agalactiaegrows well invitroby fermentative metabolism, it is shown here that the absence of NDH-2 results in attenuated virulence, as observed by reduced colonization in heart and kidney in a mouse model of systemic infection. The lack of NDH-2 in mammalian mitochondria and its important role for virulence suggest this enzyme may be a potential drug target. For this reason, in this study,S. agalactiaeNDH-2 was purified and biochemically characterized, and the isolated enzyme was used to screen for inhibitors from libraries of FDA-approved drugs. Zafirlukast was identified to successfully inhibit both NDH-2 activity and aerobic respiration in intact cells. This compound may be useful as a laboratory tool to inhibit respiration inS. agalactiaeand, since it has few side effects, it might be considered a lead compound for therapeutics development.IMPORTANCES. agalactiaeis part of the human intestinal microbiota and is present in the vagina of ~30% of healthy women. Although a commensal, it is also the leading cause of septicemia and meningitis in neonates and immunocompromised adults. This organism can aerobically respire, but only using external sources of heme and quinone, required to have a functional electron transport chain. Although bacteria usually have a branched respiratory chain with multiple dehydrogenases and terminal oxygen reductases, here we establish thatS. agalactiaeutilizes only one type 2 NADH dehydrogenase (NDH-2) and one cytochromebdoxygen reductase to perform respiration. NADH-dependent respiration plays a critical role in the pathogen in maintaining NADH/NAD+redox balance in the cell, optimizing ATP production, and tolerating oxygen. In summary, we demonstrate the essential role of NDH-2 in respiration and its contribution toS. agalactiaevirulence and propose it as a potential drug target.

The monoheme cytochrome c subunit of Alternative Complex III is a direct electron donor to caa3 oxygen reductase in Rhodothermus marinus

Alternative Complex III (ACIII) is an example of the robustness and flexibility of prokaryotic respiratory chains. It performs quinol:cytochrome c oxidoreductase activity, being functionally equivalent to the bc1 complex but structurally unrelated. In this work we further explored ACIII investigating the role of its monoheme cytochrome c subunit (ActE). We expressed and characterized the individually isolated ActE, which allowed us to suggest that ActE is a lipoprotein and to show its function as a direct electron donor to the caa3 oxygen reductase.