Cytochrome Bd Research Articles

Requirement of Bacillus subtilis succinate: Menaquinone oxidoreductase activity for membrane energization depends on the direction of catalysis

Succinate:quinone oxidoreductases (SQR) from Bacilli catalyze reduction of menaquinone by succinate, as well as the reverse reaction. The direct activity is energetically unfavorable and lost upon ΔμН+ dissipation, thus suggesting ΔμН+ to be consumed during catalysis. Paradoxically, the generation of ΔμН+ upon fumarate reduction was never confirmed. Thus, the exact role of ΔμН+ in the operation of bacillary-type SQRs remained questionable. The purpose of this work was to clarify this issue.We have described the different operating modes of the membrane-bound SQR from Bacillus subtilis. Tightly coupled membrane vesicles from both wild-type cells and the mutant containing cytochrome bd as the only terminal oxidase were studied. This made it possible to compare the respiratory chains with 2 versus 1H+/e− stoichiometry of ΔμН+ generation. Direct and reverse activities of SQR were determined under either energized or deenergized conditions.The wild-type membranes demonstrated high succinate oxidase activity very sensitive to uncoupling. On the contrary, the mutant showed extremely low succinate oxidase activity resistant to uncoupling. ΔμН+ generation at the cost of ATP hydrolysis restored the uncoupling sensitive succinate respiration in the mutant. Membranes of the both types effectively reduced fumarate by menaquinol. This activity was not affected by energization or uncoupling, neither it was followed by ΔμН+ generation.Thus, B. subtilis SQR demonstrates two regimes: ΔμН+-coupled and not coupled. This behavior can be explained by assuming the presence of two menaquinone binding sites which drastically differ in affinity for the oxidized and reduced substrate.

Mechanisms of nano zero-valent iron in enhancing dibenzofuran degradation by a Rhodococcus sp.: Trade-offs between ATP production and protection against reactive oxygen species.

Nano zero-valent iron (nZVI) can enhance pollutants biodegradation, but it displays toxicity towards microorganisms. Gram-positive (G+) bacteria exhibit greater resistance to nZVI than Gram-negative bacteria. However, mechanisms of nZVI accelerating pollutants degradation by G+ bacteria remain unclear. Herein, we explored effects of nZVI on a G+ bacterium, Rhodococcus sp. strain p52, and mechanisms by which nZVI accelerates biodegradation of dibenzofuran, a typical polycyclic aromatic compound. Electron microscopy and energy dispersive spectroscopy analysis revealed that nZVI could penetrate cell membranes, which caused damage and growth inhibition. nZVI promoted dibenzofuran biodegradation at certain concentrations, while higher concentration functioned later due to the delayed reactive oxygen species (ROS) mitigation. Transcriptomic analysis revealed that cells adopted response mechanisms to handle the elevated ROS induced by nZVI. ATP production was enhanced by accelerated dibenzofuran degradation, providing energy for protein synthesis related to antioxidant stress and damage repair. Meanwhile, electron transport chain (ETC) was adjusted to mitigate ROS accumulation, which involved downregulating expression of ETC complex I-related genes, as well as upregulating expression of the genes for the ROS-scavenging cytochrome bd complex and ETC complex II. These findings revealed the mechanisms underlying nZVI-enhanced biodegradation by G+ bacteria, offering insights into optimizing bioremediation strategies involving nZVI.

Biosensor-based growth-coupling as an evolutionary strategy to improve heme export in Corynebacterium glutamicum

The iron-containing porphyrin heme is of high interest for the food industry for the production of artificial meat as well as for medical applications. Recently, the biotechnological platform strain Corynebacterium glutamicum has emerged as a promising host for animal-free heme production. Beyond engineering of complex heme biosynthetic pathways, improving heme export offers significant yet untapped potential for enhancing production strains. In this study, a growth-coupled biosensor was designed to impose a selection pressure on the increased expression of the hrtBA operon encoding an ABC-type heme exporter in C. glutamicum. For this purpose, the promoter region of the growth-regulating genes pfkA (phosphofructokinase) and aceE (pyruvate dehydrogenase) was replaced with that of PhrtB, creating biosensor strains with a selection pressure for hrtBA activation. Resulting sensor strains were used for plate-based selections and for a repetitive batch f(luorescent)ALE using a fully automated laboratory platform. Genome sequencing of isolated clones featuring increased hrtBA expression revealed three distinct mutational hotspots: (i) chrS, (ii) chrA, and (iii) cydD. Mutations in the genes of the ChrSA two-component system, which regulates hrtBA in response to heme levels, were identified as a promising target to enhance export activity. Furthermore, causal mutations within cydD, encoding an ABC-transporter essential for cytochrome bd oxidase assembly, were confirmed by the construction of a deletion mutant. Reversely engineered strains showed strongly increased hrtBA expression as well as increased cellular heme levels. These results further support the proposed role of CydDC as a heme transporter in bacteria. Mutations identified in this study therefore underline the potential of biosensor-based growth coupling and provide promising engineering targets to improve microbial heme production.

Proton uptake in cytochrome bd -I from E. coli: electrocatalytic and spectroscopic investigations on D58 variants

Proton uptake in cytochrome bd -I from E. coli: electrocatalytic and spectroscopic investigations on D58 variants

Menaquinone-specific oxidation by M. tuberculosis cytochrome bd is redox regulated by the Q-loop disulfide bond

Menaquinone-specific oxidation by M. tuberculosis cytochrome bd is redox regulated by the Q-loop disulfide bond

A cytochrome bd repressed by a MarR family regulator confers resistance to metals, nitric oxide, sulfide, and cyanide in Chromobacterium violaceum.

The terminal oxidases of bacterial respiratory chains rely on heme-copper (heme-copper oxidases) or heme (cytochrome bd ) to catalyze reduction of molecular oxygen to water. Chromobacterium violaceum is a facultative anaerobic bacterium that uses oxygen and other electron acceptors for respiration under conditions of varying oxygen availability. The C. violaceum genome encodes multiple respiratory terminal oxidases, but their role and regulation remain unexplored. Here, we demonstrate that CioAB, the single cytochrome bd from C. violaceum , protects this bacterium against multiple stressors that are inhibitors of heme-copper oxidases, including nitric oxide, sulfide, and cyanide. CioAB also confers C. violaceum resistance to iron, zinc, and hydrogen peroxide. This cytochrome bd is encoded by the cioRAB operon, which is under direct repression by the MarR-type regulator CioR. In addition, the cioRAB operon responds to quorum sensing and to cyanide, suggesting a protective mechanism of increasing CioAB in the setting of high endogenous cyanide production.

Genome reduction in novel, obligately methyl-reducing Methanosarcinales isolated from arthropod guts (Methanolapillus gen. nov. and Methanimicrococcus).

Recent metagenomic studies have identified numerous lineages of hydrogen-dependent, obligately methyl-reducing methanogens. Yet, only a few representatives have been isolated in pure culture. Here, we describe six new species with this capability in the family Methanosarcinaceae (order Methanosarcinales), which makes up a substantial fraction of the methanogenic community in arthropod guts. Phylogenomic analysis placed the isolates from cockroach hindguts into the genus Methanimicrococcus (M. hacksteinii, M. hongohii, and M. stummii) and the isolates from millipede hindguts into a new genus, Methanolapillus (M. africanus, M. millepedarum, and M. ohkumae). Members of this intestinal clade, which includes also uncultured representatives from termites and vertebrates, have substantially smaller genomes (1.6-2.2 Mbp) than other Methanosarcinales. Genome reduction was accompanied by the loss of the upper part of the Wood-Ljungdahl pathway, several energy-converting membrane complexes (Fpo, Ech, and Rnf), and various biosynthetic pathways. However, genes involved in the protection against reactive oxygen species (catalase and superoxide reductase) were conserved in all genomes, including cytochrome bd (CydAB), a high-affinity terminal oxidase that may confer the capacity for microaerobic respiration. Since host-associated Methanosarcinales are nested within omnivorous lineages, we conclude that the specialization on methyl groups is an adaptation to the intestinal environment.

The role of Listeria monocytogenes PstA in β-lactam resistance requires the cytochrome bd oxidase activity.

PstA is a structurally conserved c-di-AMP-binding protein that is broadly present among Firmicutes bacteria. Furthermore, PstA binds c-di-AMP at high affinity and specificity, indicating an important role in the c-di-AMP signaling network. However, the molecular function of PstA remains elusive. Our findings reveal contrasting roles of PstA in β-lactam resistance depending on c-di-AMP-binding status. We also define physiological conditions for PstA function during aerobic growth. Future efforts can exploit these conditions to identify PstA interaction partners under β-lactam stress.

Targeting Tuberculosis: Novel Scaffolds for Inhibiting Cytochrome bd Oxidase.

Discovered in the 1920s, cytochrome bd is a terminal oxidase that has received renewed attention as a drug target since its atomic structure was first determined in 2016. Only found in prokaryotes, we study it here as a drug target for Mycobacterium tuberculosis (Mtb). Most previous drug discovery efforts toward cytochrome bd have involved analogues of the canonical substrate quinone, known as Aurachin D. Here, we report six new cytochrome bd inhibitor scaffolds determined from a computational screen and confirmed on target activity through in vitro testing. These scaffolds provide new avenues for lead optimization toward Mtb therapeutics.

Probiotic Characteristics and Anti-Inflammatory Effects of Limosilactobacillus fermentum 664 Isolated from Chinese Fermented Pickles.

Limosilactobacillus fermentum (L. fermentum) is widely used in industrial food fermentations, and its probiotic and health-promoting roles attracted much attention in the past decades. In this work, the probiotic potential of L. fermentum 664 isolated from Chinese fermented pickles was assessed. In addition, the anti-inflammatory properties and mechanisms were investigated using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Results indicated that L. fermentum 664 demonstrated excellent acid and bile salt tolerance, adhesion capability, antimicrobial activity, and safety profile. L. fermentum 664 downregulated the release of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2) stimulated with LPS. Moreover, L fermentum 664 inhibited the nuclear translocation of the nuclear factor κB (NF-κB) and the activation of mitogen-activated protein kinases (MAPKs) induced by LPS. This action was associated with a reduction in reactive oxygen species (ROS) levels and an enhanced expression of heme oxygenase-1 (HO-1) protein. Additionally, whole genome sequencing indicated that L. fermentum 664 contained genes that encode proteins with antioxidant and anti-inflammatory functions, including Cytochrome bd ubiquinol oxidase subunit I (CydA), Cytochrome bd ubiquinol oxidase subunit II (CydB), and NAD(P)H dehydrogenase quinone 1 (NQO1). In conclusion, our study suggested that L. fermentum 664 has the potential to become a probiotic and might be a promising strategy for the prevention of inflammation.

Steroid Drugs Inhibit Bacterial Respiratory Oxidases and Are Lethal Toward Methicillin-Resistant Staphylococcus aureus.

Cytochrome bd complexes are respiratory oxidases found exclusively in prokaryotes that are important during infection for numerous bacterial pathogens. In silico docking was employed to screen approved drugs for their ability to bind to the quinol site of Escherichia coli cytochrome bd-I. Respiratory inhibition was assessed with oxygen electrodes using membranes isolated from E. coli and methicillin-resistant Staphylococcus aureus strains expressing single respiratory oxidases (ie, cytochromes bd, bo', or aa3). Growth/viability assays were used to measure bacteriostatic and bactericidal effects. The steroid drugs ethinylestradiol and quinestrol inhibited E. coli bd-I activity with median inhibitory concentration (IC50) values of 47 ± 28.9 µg/mL (158 ± 97.2 µM) and 0.2 ± 0.04 µg/mL (0.5 ± 0.1 µM), respectively. Quinestrol inhibited growth of an E. coli "bd-I only" strain with an IC50 of 0.06 ± 0.02 µg/mL (0.2 ± 0.07 µM). Growth of an S. aureus "bd only" strain was inhibited by quinestrol with an IC50 of 2.2 ± 0.43 µg/mL (6.0 ± 1.2 µM). Quinestrol exhibited potent bactericidal effects against S. aureus but not E. coli. Quinestrol inhibits cytochrome bd in E. coli and S. aureus membranes and inhibits the growth of both species, yet is only bactericidal toward S. aureus.

Unraveling response mechanism of electron transport mechanism to uranium-exposure in Bacillus sp. X02

With the development of the uranium mining industry, the remediation of uranium pollution is an urgent environmental problem to be solved. Bioreduction is currently recognized as a U(VI) bioremediation method with good application prospects. However, inadequate reporting of uranium-reducing bacteria and low bioreduction efficiency are the main reasons limiting the application of the bioreduction method. In this paper, we isolated an efficient uranium-reducing bacterium, Bacillus sp. X02, explored the effect of U(VI) on bioreduction efficiency through electron transport mechanisms. The results showed that the maximum removal and reduction rate were 98.35 % and 62.43 %, respectively. Further studies revealed that the efficiency of extracellular electron transport of Bacillus sp. X02 was enhanced under U(VI) exposure. But uranium stress down-regulated the expression of F-type ATPase, Cytochrome c reductase ISP, Cytochrome c oxidase, and Cytochrome bd complex, inhibited the processes of complex III, complex IV, and ATP synthesis in the oxidative phosphorylation electron transport pathway, decreasing the electron transfer and U(VI) reduction capacity of Bacillus sp. X02 ultimately. This work proposed a new electron transfer pathway for U(VI) bioreduction and provided new insights into the effect of uranium on bioreduction efficiency.

Cytochrome bd oxidase: an emerging anti-tubercular drug target.

Cytochrome bd (cyt-bd) oxidase, one of the two terminal oxidases in the Mycobacterium tuberculosis (Mtb) oxidative phosphorylation pathway, plays an indispensable role in maintaining the functionality of the metabolic pathway under stressful conditions. However, the absence of this oxidase in eukaryotic cells allows researchers to select it as a potential drug target for the synthesis of anti-tubercular (anti-TB) molecules. Cyt-bd inhibitors have often been combined with cytochrome bcc/aa3 super-complex inhibitors in anti-TB drug regimens to achieve a desired bactericidal response. The functional redundancy between both the terminal oxidases is responsible for this. The cryo-EM structure of cyt-bd oxidase from Mtb (PDB ID: 7NKZ) further accelerated the research to identify its inhibitor. Herein, we have summarized the reported anti-TB cyt-bd inhibitors, insight into the rationale behind targeting cyt-bd oxidase, and an outline of the architecture of Mtb cyt-bd oxidase.

Strategies of chemolithoautotrophs adapting to high temperature and extremely acidic conditions in a shallow hydrothermal ecosystem

BackgroundActive hydrothermal vents create extreme conditions characterized by high temperatures, low pH levels, and elevated concentrations of heavy metals and other trace elements. These conditions support unique ecosystems where chemolithoautotrophs serve as primary producers. The steep temperature and pH gradients from the vent mouth to its periphery provide a wide range of microhabitats for these specialized microorganisms. However, their metabolic functions, adaptations in response to these gradients, and coping mechanisms under extreme conditions remain areas of limited knowledge. In this study, we conducted temperature gradient incubations of hydrothermal fluids from moderate (pH = 5.6) and extremely (pH = 2.2) acidic vents. Combining the DNA-stable isotope probing technique and subsequent metagenomics, we identified active chemolithoautotrophs under different temperature and pH conditions and analyzed their specific metabolic mechanisms.ResultsWe found that the carbon fixation activities of Nautiliales in vent fluids were significantly increased from 45 to 65 °C under moderately acidic condition, while their heat tolerance was reduced under extremely acidic conditions. In contrast, Campylobacterales actively fixed carbon under both moderately and extremely acidic conditions under 30 − 45 °C. Compared to Campylobacterales, Nautiliales were found to lack the Sox sulfur oxidation system and instead use NAD(H)-linked glutamate dehydrogenase to boost the reverse tricarboxylic acid (rTCA) cycle. Additionally, they exhibit a high genetic potential for high activity of cytochrome bd ubiquinol oxidase in oxygen respiration and hydrogen oxidation at high temperatures. In terms of high-temperature adaption, the rgy gene plays a critical role in Nautiliales by maintaining DNA stability at high temperature. Genes encoding proteins involved in proton export, including the membrane arm subunits of proton-pumping NADH: ubiquinone oxidoreductase, K+ accumulation, selective transport of charged molecules, permease regulation, and formation of the permeability barrier of bacterial outer membranes, play essential roles in enabling Campylobacterales to adapt to extremely acidic conditions.ConclusionsOur study provides in-depth insights into how high temperature and low pH impact the metabolic processes of energy and main elements in chemolithoautotrophs living in hydrothermal ecosystems, as well as the mechanisms they use to adapt to the extreme hydrothermal conditions.4ap83QbxwyKb6GHBwDJLyuVideo

Cytochrome <i>bd</i> as Antioxidant Redox Enzyme

One of the main functions of enzyme complexes that constitute electron transport (respiratory) chains of organisms is to maintain cellular redox homeostasis by oxidizing reducing equivalents, NADH and quinol. Cytochrome bd is a unique terminal oxidase of the chains of many bacteria including pathogenic species. This redox enzyme couples the oxidation of ubiquinol or menaquinol by molecular oxygen to the generation of proton motive force, a universal energy currency. The latter is used by the organism to produce ATP, another cellular energy currency, via oxidative phosphorylation. Escherichia coli contains two bd–type oxidases, bd-I and bd-II, encoded by the cydAB and appCB operons, respectively. Surprisingly, both bd enzymes make a further contribution to molecular mechanisms of maintaining the appropriate redox balance in the bacterial cell by means of elimination of reactive oxygen species, such as hydrogen peroxide. This review summarizes recent data on the redox-modulated H2O2-scavenging activities of cytochromes bd-I and bd-II from E. coli. The possibility of such antioxidant properties in cytochromes bd from other bacteria is also discussed.

Generation of Membrane Potential by Cytochrome bd.

An overview of current notions on the mechanism of generation of a transmembrane electric potential difference (Δψ) during the catalytic cycle of a bd-type triheme terminal quinol oxidase is presented in this work. It is suggested that the main contribution to Δψ formation is made by the movement of H+ across the membrane along the intra-protein hydrophilic proton-conducting pathway from the cytoplasm to the active site for oxygen reduction of this bacterial enzyme.

Cytochrome bd as Antioxidant Redox Enzyme

One of the main functions of enzyme complexes that constitute electron transport (respiratory) chains of organisms is to maintain cellular redox homeostasis by oxidizing reducing equivalents, NADH and quinol. Cytochrome bd is a unique terminal oxidase of the chains of many bacteria including pathogenic species. This redox enzyme couples the oxidation of ubiquinol or menaquinol by molecular oxygen to the generation of proton motive force, a universal energy currency. The latter is used by the organism to produce ATP, another cellular energy currency, via oxidative phosphorylation. Escherichia coli contains two bd-type oxidases, bd-I and bd-II, encoded by the cydAB and appCB operons, respectively. Surprisingly, both bd enzymes make a further contribution to molecular mechanisms of maintaining the appropriate redox balance in the bacterial cell by means of elimination of reactive oxygen species, such as hydrogen peroxide. This review summarizes recent data on the redox-modulated H2O2-scavenging activities of cytochromes bd-I and bd-II from E. coli. The possibility of such antioxidant properties in cytochromes bd from other bacteria is also discussed.

Electrocatalytic and Spectroscopic Studies on Cytochrome Bd Oxidase, a Highly Diverse Bacterial Defense Factor

The selective reduction of oxygen to water is crucial to life and a central process in aerobic organisms. It is catalyzed by several different enzymes, including cytochrome bd oxidases that are solely present in prokaryotes, including several pathogens. In addition, these enzymes play a crucial role in protection against oxidative stress, in virulence, adaptability and antibiotics resistance. The reduction of O2 occurs at the high spin D-type heme in all cytochrome bd oxidases, that is also the binding site for several ligands from signaling processes, including NO, H2S and CO.Here we present the electrocatalytic study of the cytochrome bd I and bd II oxidases from Escherichia coli (1,2) as well on other related bd oxidases. Structural parameters that are crucial for the reactivity towards oxygen are analyzed. The pH dependency of the binding and release of NO, an important signaling factor is presented. The influence of mutants in the proton channel on the NO release is discussed.(1) Grauel, A. Kägi, J. Rasmussen, T. Makarchuk, I. Oppermann, S. Moumbock, A., Wohlwend, D., Müller, R., Melin, F., Günther, S., Hellwig, P., Böttcher, B., and Friedrich, T., ‘Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D’ (2021) Nat. Commun., 12:6498.(2) Nikolaev, A., Safarian, S., Thesseling, A., Wohlwend, D., Friedrich, T., Michel, H., Kusumoto, T., Sakamoto, J., Melin, F., Hellwig P. ‘Electrocatalytic evidence of the diversity of the oxygen reaction in the bacterial bd oxidase from different organisms’ (2021) Biochim. Biophys. Acta 1862, 148436.

Evaluation of Nanaerobic Digestion as a Mechanism to Explain Surplus Methane Production in Animal Rumina and Engineered Digesters.

Nanaerobes are a newly described class of microorganisms that use a unique cytochrome bd oxidase to achieve nanaerobic respiration at <2 μM dissolved oxygen (∼1% of atmospheric oxygen) but are not viable above this value due to the lack of other terminal oxidases. Although sharing an overlapping ecological niche with methanogenic archaea, the role of nanaerobes in methanogenic systems has not been studied so far. To explore their occurrence and significance, we re-analyzed published meta-omic datasets from animal rumina and waste-to-energy digesters, including conventional anaerobic digesters and anaerobic digesters with ultra-low oxygenation. Results show that animal rumina share broad similarities in the microbial community and system performance with oxygenated digesters, rather than with conventional anaerobic digesters, implying that trace levels of oxygen drive the efficient digestion in ruminants. The rumen system serves as an ideal model for the newly named nanaerobic digestion, as it relies on the synergistic co-occurrence of nanaerobes and methanogens for methane yield enhancement. The most abundant ruminal bacterial family Prevotellaceae contains many nanaerobes, which perform not only anaerobic fermentation but also nanaerobic respiration using cytochrome bd oxidase. These nanaerobes generally accompany hydrogenotrophic methanogens to constitute a thermodynamically and physiologically consistent framework for efficient methane generation. Our findings provide new insights into ruminal methane emissions and strategies to enhance methane generation from biomass.

Complete genome sequence of Halomonas alkaliantarctica MSP3 isolated from marine sediment, Jeju Island.

As a moderate halophilic-heterotrophic bacterium, Halomonas alkaliantarctica MSP3 was isolated from marine sediment located in Jeju island, South Korea. The complete genome of strain MSP3 was sequenced and analyzed to reveal its genetic features and metabolic potential. The genome size of MSP3 was about 4.23 Mbp with 54.7% G+C content, and it contained 3811 protein-coding sequences and 79 RNA genes (61 tRNA and 18 rRNA). According to the genome annotation, it was revealed that the strain MSP3 harbors genes encoding for urease and urea transporters, which play a crucial role in the process of urea degradation and utilization. In addition, it is noteworthy that the MSP3 strain possesses genes encoding for both cytochrome c oxidase and cytochrome bd oxidase, thereby conferring upon it the ability to adapt to various levels of oxygen (oxic to microoxic) and to execute denitrification processes in the absence of oxygen. Moreover, it was observed that strain MSP3 had genes for the glyoxylate cycle, which is an alternative pathway to the TCA cycle. Furthermore, it was observed that the MSP3 strain exhibited the ability to thrive across a diverse spectrum of NaCl concentrations, ranging from 2% to 10% (w/v). Collectively, strain MSP3 may possess an advantage over competitors within the marine ecosystem, particularly in conditions where carbon substrates are restricted. The genomic-based assumption could potentially be substantiated by the presence of a multitude of transporter genes within the genome.