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

The terminal oxidases of bacterial aerobic respiratory chains are redox-active electrogenic enzymes that catalyze the four-electron reduction of O2 to 2H2O taking out electrons from quinol or cytochrome c. Living bacteria often deal with carbon monoxide (CO) which can act as both a signaling molecule and a poison. Bacterial terminal oxidases contain hemes; therefore, they are potential targets for CO. However, our knowledge of this issue is limited and contradictory. Here, we investigated the effect of CO on the cell growth and aerobic respiration of three different Escherichia coli mutants, each expressing only one terminal quinol oxidase: cytochrome bd-I, cytochrome bd-II, or cytochrome bo3. We found that following the addition of CO to bd-I-only cells, a minimal effect on growth was observed, whereas the growth of both bd-II-only and bo3-only strains was severely impaired. Consistently, the degree of resistance of aerobic respiration of bd-I-only cells to CO is high, as opposed to high CO sensitivity displayed by bd-II-only and bo3-only cells consuming O2. Such a difference between the oxidases in sensitivity to CO was also observed with isolated membranes of the mutants. Accordingly, O2 consumption of wild-type cells showed relatively low CO sensitivity under conditions favoring the expression of a bd-type oxidase.

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.

Six Functions of Respiration: Isn't It Time to Take Control over ROS Production in Mitochondria, and Aging Along with It?

Cellular respiration is associated with at least six distinct but intertwined biological functions. (1) biosynthesis of ATP from ADP and inorganic phosphate, (2) consumption of respiratory substrates, (3) support of membrane transport, (4) conversion of respiratory energy to heat, (5) removal of oxygen to prevent oxidative damage, and (6) generation of reactive oxygen species (ROS) as signaling molecules. Here we focus on function #6, which helps the organism control its mitochondria. The ROS bursts typically occur when the mitochondrial membrane potential (MMP) becomes too high, e.g., due to mitochondrial malfunction, leading to cardiolipin (CL) oxidation. Depending on the intensity of CL damage, specific programs for the elimination of damaged mitochondria (mitophagy), whole cells (apoptosis), or organisms (phenoptosis) can be activated. In particular, we consider those mechanisms that suppress ROS generation by enabling ATP synthesis at low MMP levels. We discuss evidence that the mild depolarization mechanism of direct ATP/ADP exchange across mammalian inner and outer mitochondrial membranes weakens with age. We review recent data showing that by protecting CL from oxidation, mitochondria-targeted antioxidants decrease lethality in response to many potentially deadly shock insults. Thus, targeting ROS- and CL-dependent pathways may prevent acute mortality and, hopefully, slow aging.

The terminal oxidase cytochrome bd-I confers carbon monoxide resistance to Escherichia coli cells.

Carbon monoxide (CO) plays a multifaceted role in the physiology of organisms, from poison to signaling molecule. Heme proteins, including terminal oxidases, are plausible CO targets. Three quinol oxidases terminate the branched aerobic respiratory chain of Escherichia coli. These are the heme­copper cytochrome bo3 and two copper-lacking bd-type cytochromes, bd-I and bd-II. All three enzymes generate a proton motive force during the four-electron oxygen reduction reaction that is used for ATP production. The bd-type oxidases also contribute to mechanisms of bacterial defense against various types of stresses. Here we report that in E. coli cells, at the enzyme concentrations tested, cytochrome bd-I is much more resistant to inhibition by CO than cytochrome bd-II and cytochrome bo3. The apparent half-maximal inhibitory concentration values, IC50, for inhibition of O2 consumption of the membrane-bound bd-II and bo3 oxidases by CO at ~150 µM O2 were estimated to be 187.1 ± 11.1 and 183.3 ± 13.5 µM CO, respectively. Under the same conditions, the maximum inhibition observed with the membrane-bound cytochrome bd-I was 20 ± 10% at ~200 µM CO.

Interaction of Terminal Oxidases with Amphipathic Molecules.

The review focuses on recent advances regarding the effects of natural and artificial amphipathic compounds on terminal oxidases. Terminal oxidases are fascinating biomolecular devices which couple the oxidation of respiratory substrates with generation of a proton motive force used by the cell for ATP production and other needs. The role of endogenous lipids in the enzyme structure and function is highlighted. The main regularities of the interaction between the most popular detergents and terminal oxidases of various types are described. A hypothesis about the physiological regulation of mitochondrial-type enzymes by lipid-soluble ligands is considered.

F1·Fo ATP Synthase/ATPase: Contemporary View on Unidirectional Catalysis.

F1·Fo-ATP synthases/ATPases (F1·Fo) are molecular machines that couple either ATP synthesis from ADP and phosphate or ATP hydrolysis to the consumption or production of a transmembrane electrochemical gradient of protons. Currently, in view of the spread of drug-resistant disease-causing strains, there is an increasing interest in F1·Fo as new targets for antimicrobial drugs, in particular, anti-tuberculosis drugs, and inhibitors of these membrane proteins are being considered in this capacity. However, the specific drug search is hampered by the complex mechanism of regulation of F1·Fo in bacteria, in particular, in mycobacteria: the enzyme efficiently synthesizes ATP, but is not capable of ATP hydrolysis. In this review, we consider the current state of the problem of "unidirectional" F1·Fo catalysis found in a wide range of bacterial F1·Fo and enzymes from other organisms, the understanding of which will be useful for developing a strategy for the search for new drugs that selectively disrupt the energy production of bacterial cells.

Preparations of Terminal Oxidase Cytochrome bd-II Isolated from Escherichia coli Reveal Significant Hydrogen Peroxide Scavenging Activity.

Cytochrome bd-II is one of the three terminal quinol oxidases of the aerobic respiratory chain of Escherichia coli. Preparations of the detergent-solubilized untagged bd-II oxidase isolated from the bacterium were shown to scavenge hydrogen peroxide (H2O2) with high rate producing molecular oxygen (O2). Addition of H2O2 to the same buffer that does not contain enzyme or contains thermally denatured cytochrome bd-II does not lead to any O2 production. The latter observation rules out involvement of adventitious transition metals bound to the protein. The H2O2-induced O2 production is not susceptible to inhibition by N-ethylmaleimide (the sulfhydryl binding compound), antimycin A (the compound that binds specifically to a quinol binding site), and CO (diatomic gas that binds specifically to the reduced heme d). However, O2 formation is inhibited by cyanide (IC50 = 4.5 ± 0.5 µM) and azide. Addition of H2O2 in the presence of dithiothreitol and ubiquinone-1 does not inactivate cytochrome bd-II and apparently does not affect the O2 reductase activity of the enzyme. The ability of cytochrome bd-II to detoxify H2O2 could play a role in bacterial physiology by conferring resistance to the peroxide-mediated stress.

Bioenergetics and Reactive Nitrogen Species in Bacteria.

The production of reactive nitrogen species (RNS) by the innate immune system is part of the host's defense against invading pathogenic bacteria. In this review, we summarize recent studies on the molecular basis of the effects of nitric oxide and peroxynitrite on microbial respiration and energy conservation. We discuss possible molecular mechanisms underlying RNS resistance in bacteria mediated by unique respiratory oxygen reductases, the mycobacterial bcc-aa3 supercomplex, and bd-type cytochromes. A complete picture of the impact of RNS on microbial bioenergetics is not yet available. However, this research area is developing very rapidly, and the knowledge gained should help us develop new methods of treating infectious diseases.

Recent Advances in Structural Studies of Cytochrome bd and Its Potential Application as a Drug Target.

Cytochrome bd is a triheme copper-free terminal oxidase in membrane respiratory chains of prokaryotes. This unique molecular machine couples electron transfer from quinol to O2 with the generation of a proton motive force without proton pumping. Apart from energy conservation, the bd enzyme plays an additional key role in the microbial cell, being involved in the response to different environmental stressors. Cytochrome bd promotes virulence in a number of pathogenic species that makes it a suitable molecular drug target candidate. This review focuses on recent advances in understanding the structure of cytochrome bd and the development of its selective inhibitors.

Impact of Hydrogen Sulfide on Mitochondrial and Bacterial Bioenergetics.

This review focuses on the effects of hydrogen sulfide (H2S) on the unique bioenergetic molecular machines in mitochondria and bacteria-the protein complexes of electron transport chains and associated enzymes. H2S, along with nitric oxide and carbon monoxide, belongs to the class of endogenous gaseous signaling molecules. This compound plays critical roles in physiology and pathophysiology. Enzymes implicated in H2S metabolism and physiological actions are promising targets for novel pharmaceutical agents. The biological effects of H2S are biphasic, changing from cytoprotection to cytotoxicity through increasing the compound concentration. In mammals, H2S enhances the activity of FoF1-ATP (adenosine triphosphate) synthase and lactate dehydrogenase via their S-sulfhydration, thereby stimulating mitochondrial electron transport. H2S serves as an electron donor for the mitochondrial respiratory chain via sulfide quinone oxidoreductase and cytochrome c oxidase at low H2S levels. The latter enzyme is inhibited by high H2S concentrations, resulting in the reversible inhibition of electron transport and ATP production in mitochondria. In the branched respiratory chain of Escherichia coli, H2S inhibits the bo3 terminal oxidase but does not affect the alternative bd-type oxidases. Thus, in E. coli and presumably other bacteria, cytochrome bd permits respiration and cell growth in H2S-rich environments. A complete picture of the impact of H2S on bioenergetics is lacking, but this field is fast-moving, and active ongoing research on this topic will likely shed light on additional, yet unknown biological effects.

Proton Pumping and Non-Pumping Terminal Respiratory Oxidases: Active Sites Intermediates of These Molecular Machines and Their Derivatives.

Terminal respiratory oxidases are highly efficient molecular machines. These most important bioenergetic membrane enzymes transform the energy of chemical bonds released during the transfer of electrons along the respiratory chains of eukaryotes and prokaryotes from cytochromes or quinols to molecular oxygen into a transmembrane proton gradient. They participate in regulatory cascades and physiological anti-stress reactions in multicellular organisms. They also allow microorganisms to adapt to low-oxygen conditions, survive in chemically aggressive environments and acquire antibiotic resistance. To date, three-dimensional structures with atomic resolution of members of all major groups of terminal respiratory oxidases, heme-copper oxidases, and bd-type cytochromes, have been obtained. These groups of enzymes have different origins and a wide range of functional significance in cells. At the same time, all of them are united by a catalytic reaction of four-electron reduction in oxygen into water which proceeds without the formation and release of potentially dangerous ROS from active sites. The review analyzes recent structural and functional studies of oxygen reduction intermediates in the active sites of terminal respiratory oxidases, the features of catalytic cycles, and the properties of the active sites of these enzymes.

ROS Defense Systems and Terminal Oxidases in Bacteria.

Reactive oxygen species (ROS) comprise the superoxide anion (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen (1O2). ROS can damage a variety of macromolecules, including DNA, RNA, proteins, and lipids, and compromise cell viability. To prevent or reduce ROS-induced oxidative stress, bacteria utilize different ROS defense mechanisms, of which ROS scavenging enzymes, such as superoxide dismutases, catalases, and peroxidases, are the best characterized. Recently, evidence has been accumulating that some of the terminal oxidases in bacterial respiratory chains may also play a protective role against ROS. The present review covers this role of terminal oxidases in light of recent findings.

Terminal Oxidase Cytochrome bd Protects Bacteria Against Hydrogen Sulfide Toxicity.

Hydrogen sulfide (H2S) is often called the third gasotransmitter (after nitric oxide and carbon monoxide), or endogenous gaseous signaling molecule. This compound plays important roles in organisms from different taxonomic groups, from bacteria to animals and humans. In mammalian cells, H2S has a cytoprotective effect at nanomolar concentrations, but becomes cytotoxic at higher concentrations. The primary target of H2S is mitochondria. At submicromolar concentrations, H2S inhibits mitochondrial heme-copper cytochrome c oxidase, thereby blocking aerobic respiration and oxidative phosphorylation and eventually leading to cell death. Since the concentration of H2S in the gut is extremely high, the question arises - how can gut bacteria maintain the functioning of their oxygen-dependent respiratory electron transport chains under such conditions? This review provides an answer to this question and discusses the key role of non-canonical bd-type terminal oxidases of the enterobacterium Escherichia coli, a component of the gut microbiota, in maintaining aerobic respiration and growth in the presence of toxic concentrations of H2S in the light of recent experimental data.

Bacterial Oxidases of the Cytochrome bd Family: Redox Enzymes of Unique Structure, Function, and Utility As Drug Targets.

Significance: Cytochrome bd is a ubiquinol:oxygen oxidoreductase of many prokaryotic respiratory chains with a unique structure and functional characteristics. Its primary role is to couple the reduction of molecular oxygen, even at submicromolar concentrations, to water with the generation of a proton motive force used for adenosine triphosphate production. Cytochrome bd is found in many bacterial pathogens and, surprisingly, in bacteria formally denoted as anaerobes. It endows bacteria with resistance to various stressors and is a potential drug target. Recent Advances: We summarize recent advances in the biochemistry, structure, and physiological functions of cytochrome bd in the light of exciting new three-dimensional structures of the oxidase. The newly discovered roles of cytochrome bd in contributing to bacterial protection against hydrogen peroxide, nitric oxide, peroxynitrite, and hydrogen sulfide are assessed. Critical Issues: Fundamental questions remain regarding the precise delineation of electron flow within this multihaem oxidase and how the extraordinarily high affinity for oxygen is accomplished, while endowing bacteria with resistance to other small ligands. Future Directions: It is clear that cytochrome bd is unique in its ability to confer resistance to toxic small molecules, a property that is significant for understanding the propensity of pathogens to possess this oxidase. Since cytochrome bd is a uniquely bacterial enzyme, future research should focus on harnessing fundamental knowledge of its structure and function to the development of novel and effective antibacterial agents.

His57 controls the efficiency of ESR, a light-driven proton pump from Exiguobacterium sibiricum at low and high pH.

ESR, a light-driven proton pump from Exiguobacterium sibiricum, contains a lysine residue (Lys96) in the proton donor site. Substitution of Lys96 with a nonionizable residue greatly slows reprotonation of the retinal Schiff base. The recent study of electrogenicity of the K96A mutant revealed that overall efficiency of proton transport is decreased in the mutant due to back reactions (Siletsky et al., BBA, 2019). Similar to members of the proteorhodopsin and xanthorhodopsin families, in ESR the primary proton acceptor from the Schiff base, Asp85, closely interacts with His57. To examine the role of His57 in the efficiency of proton translocation by ESR, we studied the effects of H57N and H57N/K96A mutations on the pH dependence of light-induced pH changes in suspensions of Escherichia coli cells, kinetics of absorption changes and electrogenic proton transfer reactions during the photocycle. We found that at low pH (<5) the proton pumping efficiency of the H57N mutant in E. coli cells and its electrogenic efficiency in proteoliposomes is substantially higher than in the WT, suggesting that interaction of His57 with Asp85 sets the low pH limit for H+ pumping in ESR. The electrogenic components that correspond to proton uptake were strongly accelerated at low pH in the mutant indicating that Lys96 functions as a very efficient proton donor at low pH. In the H57N/K96A mutant, a higher H+ pumping efficiency compared with K96A was observed especially at high pH, apparently from eliminating back reactions between Asp85 and the Schiff base by the H57N mutation.

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.

Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa3 Supercomplex.

Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa3 supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatisaa3-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O2 and reductants is resistant to inhibition by the ligand, being able to metabolize NO at 25 °C with an apparent turnover number as high as ≈303 mol NO (mol enzyme)-1 min-1 at 30 µM NO. The rate of NO consumption proved to be proportional to that of O2 consumption, with 2.65 ± 0.19 molecules of NO being consumed per O2 molecule by the mycobacterial bcc-aa3. The enzyme was found to metabolize the ligand even under anaerobic reducing conditions with a turnover number of 2.8 ± 0.5 mol NO (mol enzyme)-1 min-1 at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial bcc-aa3 supercomplexes against NO stress.

In the respiratory chain of Escherichia coli cytochromes bd-I and bd-II are more sensitive to carbon monoxide inhibition than cytochrome bo3.

Bacteria can not only encounter carbon monoxide (CO) in their habitats but also produce the gas endogenously. Bacterial respiratory oxidases, thus, represent possible targets for CO. Accordingly, host macrophages were proposed to produce CO and release it into the surrounding microenvironment to sense viable bacteria through a mechanism that in Escherichia (E.) coli was suggested to involve the targeting of a bd-type respiratory oxidase by CO. The aerobic respiratory chain of E. coli possesses three terminal quinol:O2-oxidoreductases: the heme-copper oxidase bo3 and two copper-lacking bd-type oxidases, bd-I and bd-II. Heme-copper and bd-type oxidases differ in the mechanism and efficiency of proton motive force generation and in resistance to oxidative and nitrosative stress, cyanide and hydrogen sulfide. Here, we investigated at varied O2 concentrations the effect of CO gas on the O2 reductase activity of the purified cytochromes bo3, bd-I and bd-II of E. coli. We found that CO, in competition with O2, reversibly inhibits the three enzymes. The inhibition constants Ki for the bo3, bd-I and bd-II oxidases are 2.4 ±â€¯0.3, 0.04 ±â€¯0.01 and 0.2 ±â€¯0.1 µM CO, respectively. Thus, in E. coli, bd-type oxidases are more sensitive to CO inhibition than the heme-copper cytochrome bo3. The possible physiological consequences of this finding are discussed.

Cytochrome bd and Gaseous Ligands in Bacterial Physiology.

Cytochrome bd is a unique prokaryotic respiratory terminal oxidase that does not belong to the extensively investigated family of haem-copper oxidases (HCOs). The enzyme catalyses the four-electron reduction of O2 to 2H2O, using quinols as physiological reducing substrates. The reaction is electrogenic and cytochrome bd therefore sustains bacterial energy metabolism by contributing to maintain the transmembrane proton motive force required for ATP synthesis. As compared to HCOs, cytochrome bd displays several distinctive features in terms of (i) metal composition (it lacks Cu and harbours a d-type haem in addition to two haems b), (ii) overall three-dimensional structure, that only recently has been solved, and arrangement of the redox cofactors, (iii) lesser energetic efficiency (it is not a proton pump), (iv) higher O2 affinity, (v) higher resistance to inhibitors such as cyanide, nitric oxide (NO) and hydrogen sulphide (H2S) and (vi) ability to efficiently metabolize potentially toxic reactive oxygen and nitrogen species like hydrogen peroxide (H2O2) and peroxynitrite (ONOO-). Compelling evidence suggests that, beyond its bioenergetic role, cytochrome bd plays multiple functions in bacterial physiology and affords protection against oxidative and nitrosative stress. Relevant to human pathophysiology, thanks to its peculiar properties, the enzyme has been shown to promote virulence in several bacterial pathogens, being currently recognized as a target for the development of new antibiotics. This review aims to give an update on our current understanding of bd-type oxidases with a focus on their reactivity with gaseous ligands and its potential impact on bacterial physiology and human pathophysiology.

Photosystem II and terminal respiratory oxidases: molecular machines operating in opposite directions.

In the thylakoid membrane of green plants, cyanobacteria and algae, photosystem II (PSII) uses light energy to split water and generate molecular oxygen. In the opposite process of the biochemical transformation of dioxygen, in heterotrophs, the terminal respiratory oxidases (TRO) are at the end of the respiratory chain in mitochondria and in plasma membrane of many aerobic bacteria reducing dioxygen back to water. Despite the different sources of free energy (light or oxidation of the substrates), energy conversion by these enzymes is based on the spatial organization of enzymatic reactions in which the conversion of water to dioxygen (and vice versa) involves the transfer of protons and electrons in opposite directions across the membrane, which is accompanied by generation of proton-motive force. Similar and distinctive features in structure and function of these important energy-converting molecular machines are described. Information about many fascinating parallels between the mechanisms of TRO and PSII could be used in the artificial light-driven water-splitting process and elucidation of energy conversion mechanism in protein pumps.