Cytochrome Bd-type Quinol Oxidase Research Articles

Hemes switch spots in a terminal oxidase

Respiratory Enzymes Reduction of molecular oxygen to water is the driving force for respiration in aerobic organisms and is catalyzed by several distinct integral membrane complexes. These include an exclusively prokaryotic enzyme, cytochrome bd–type quinol oxidase, which is a potential antimicrobial target. Safarian et al. determined a high-resolution cryo–electron microscopy structure of this enzyme from the enteric bacterium Escherichia coli . Comparison to a homolog reveals a complete relocation of the site of oxygen binding and reduction caused by a change in the arrangement of heme cofactors and channels in the protein scaffold. This switch illustrates the diversity of structure and function in this family of enzymes and might reflect different biochemical roles of these homologs. Science , this issue p. [100][1] [1]: /lookup/doi/10.1126/science.aay0967

Genomes of ubiquitous marine and hypersaline Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira spp. encode a diversity of mechanisms to sustain chemolithoautotrophy in heterogeneous environments.

Chemolithoautotrophic bacteria from the genera Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira are common, sometimes dominant, isolates from sulfidic habitats including hydrothermal vents, soda and salt lakes and marine sediments. Their genome sequences confirm their membership in a deeply branching clade of the Gammaproteobacteria. Several adaptations to heterogeneous habitats are apparent. Their genomes include large numbers of genes for sensing and responding to their environment (EAL- and GGDEF-domain proteins and methyl-accepting chemotaxis proteins) despite their small sizes (2.1-3.1 Mbp). An array of sulfur-oxidizing complexes are encoded, likely to facilitate these organisms' use of multiple forms of reduced sulfur as electron donors. Hydrogenase genes are present in some taxa, including group 1d and 2b hydrogenases in Hydrogenovibrio marinus and H. thermophilus MA2-6, acquired via horizontal gene transfer. In addition to high-affinity cbb3 cytochrome c oxidase, some also encode cytochrome bd-type quinol oxidase or ba3 -type cytochrome c oxidase, which could facilitate growth under different oxygen tensions, or maintain redox balance. Carboxysome operons are present in most, with genes downstream encoding transporters from four evolutionarily distinct families, which may act with the carboxysomes to form CO2 concentrating mechanisms. These adaptations to habitat variability likely contribute to the cosmopolitan distribution of these organisms.

Cytochrome bd as survival factor in mycobacteria

Targeting respiration and ATP synthesis has received strong interest as a new strategy for combating drug-resistant Mycobacterium tuberculosis [1,2]. Mycobacteria employ a respiratory chain terminating with two branches. One of the branches includes a cytochrome bc1 complex and an aa3-type cytochrome c oxidase while the other branch terminates with a cytochrome bd-type quinol oxidase. We have shown that genetic inactivation of cytochrome bd, but not of cytochrome bc1, enhances the susceptibility of Mycobacterium smegmatis to hydrogen peroxide and antibiotic-induced stress [3]. The type-II NADH dehydrogenase effector clofazimine and the ATP synthase inhibitor bedaquiline were bacteriostatic against wild-type M. smegmatis, but strongly bactericidal against a cytochrome bd knock-out mutant [3,4]. These results identify cytochrome bd as a key survival factor in mycobacteria. The quinone-analog aurachin D inhibited mycobacterial cytochrome bd at sub-micromolar concentrations. Progress towards the characterization of cytochrome bd and its inhibitors will be discussed. Targeting the cytochrome bd respiratory branch appears to be a promising strategy that may enhance the bactericidal activity of existing tuberculosis drugs.

The extracytoplasmic function σ factor σ(C) regulates expression of a branched quinol oxidation pathway in Corynebacterium glutamicum.

Bacteria modify their expression of different terminal oxidases in response to oxygen availability. Corynebacterium glutamicum, a facultative anaerobic bacterium of the phylum Actinobacteria, possesses aa3 -type cytochrome c oxidase and cytochrome bd-type quinol oxidase, the latter of which is induced by oxygen limitation. We report that an extracytoplasmic function σ factor, σ(C) , is responsible for the regulation of this process. Chromatin immunoprecipitation with microarray analysis detected eight σ(C) -binding regions in the genome, facilitating the identification of a consensus promoter sequence for σ(C) recognition. The promoter sequences were found upstream of genes for cytochrome bd, heme a synthesis enzymes and uncharacterized membrane proteins, all of which were upregulated by sigC overexpression. However, one consensus promoter sequence found on the antisense strand upstream of an operon encoding the cytochrome bc1 complex conferred a σ(C) -dependent negative effect on expression of the operon. The σ(C) regulon was induced by cytochrome aa3 deficiency without modifying sigC expression, but not by bc1 complex deficiency. These findings suggest that σ(C) is activated in response to impaired electron transfer via cytochrome aa3 and not directly to a shift in oxygen levels. Our results reveal a new paradigm for transcriptional regulation of the aerobic respiratory system in bacteria.

The cytochrome bd-type quinol oxidase is important for survival of Mycobacterium smegmatis under peroxide and antibiotic-induced stress.

Targeting respiration and ATP synthesis has received strong interest as a new strategy for combatting drug-resistant Mycobacterium tuberculosis. Mycobacteria employ a respiratory chain terminating with two branches. One of the branches includes a cytochrome bc1 complex and an aa3-type cytochrome c oxidase while the other branch terminates with a cytochrome bd-type quinol oxidase. In this communication we show that genetic inactivation of cytochrome bd, but not of cytochrome bc1, enhances the susceptibility of Mycobacterium smegmatis to hydrogen peroxide and antibiotic-induced stress. The type-II NADH dehydrogenase effector clofazimine and the ATP synthase inhibitor bedaquiline were bacteriostatic against wild-type M. smegmatis, but strongly bactericidal against a cytochrome bd mutant. We also demonstrated that the quinone-analog aurachin D inhibited mycobacterial cytochrome bd at sub-micromolar concentrations. Our results identify cytochrome bd as a key survival factor in M. smegmatis during antibiotic stress. Targeting the cytochrome bd respiratory branch therefore appears to be a promising strategy that may enhance the bactericidal activity of existing tuberculosis drugs.

Purification and characterization of malate:quinone oxidoreductase from thermophilic Bacillus sp. PS3

Several bacteria possess membrane-bound dehydrogenases other than cytosolic dehydrogenases in their respiratory chains. In many cases, the membrane-bound malate:quinone oxidoreductases (MQOs) are essential for growth. However, these MQOs are absent in mammalian mitochondria, and therefore may be a potential drug target for pathogenic bacteria. To characterize the kinetic properties of MQOs, we purified MQO from Bacillus sp. PS3, which is a gram-positive and thermophilic bacterium, and cloned the gene encoding MQO based on the obtained partial N-terminus sequence. Purified MQOs showed a molecular mass of ~90 kDa, which was estimated using gel filtration, and it consists of two subunits with a molecular mass of ~50 kDa. Phylogenetic analysis showed a high similarity to the MQO of the Geobacillus group rather than the Bacillus group. Additionally, the purified enzyme was thermostable and it retained menaquinol reduction activity at high temperatures. Although it is difficult to conduct experiments using menaquinol because of its instability, we were able to measure the oxidase activity of cytochrome bd-type quinol oxidase by using menaquinol-1 by coupling this molecule with the menaquinol reduction reaction using purified MQOs.

Global transcriptome analysis of the tetrachloroethene-dechlorinating bacterium Desulfitobacterium hafniense Y51 in the presence of various electron donors and terminal electron acceptors

Desulfitobacterium hafniense Y51 is a dechlorinating bacterium that encodes an unusually large set of O-demethylase paralogs and specialized respiratory systems including specialized electron donors and acceptors. To use this organism in bioremediation of tetrachloroethene (PCE) or trichloroethene (TCE) pollution, expression patterns of its 5,060 genes were determined under different conditions using 60-mer probes in DNA microarrays. PCE, TCE, fumarate, nitrate, and dimethyl sulfoxide (DMSO) respiration all sustain the growth of strain Y51. Global transcriptome analyses were thus performed using various electron donor and acceptor couples (respectively, pyruvate and either fumarate, TCE, nitrate, or DMSO, and vanillate/fumarate). When TCE is used as terminal electron acceptor, resulting in its detoxification, a series of electron carriers comprising a cytochrome bd-type quinol oxidase (DSY4055-4056), a ferredoxin (DSY1451), and four Fe-S proteins (DSY1626, DSY1629, DSY0733, DSY3309) are upregulated, suggesting that the products of these genes are involved in PCE oxidoreduction. Interestingly, the PCE dehalogenase cluster (pceABCT) is constitutively expressed in the media tested, with pceT being upregulated and pceC downregulated in pyruvate/TCE-containing medium. In addition, another dehalogenation enzyme (DSY1155 coding for a putative chlorophenol reductive dehalogenase), is induced 225-fold in that medium, despite not being involved in PCE respiration. Remarkably since the reducing equivalents formed during pyruvate conversion to acetyl-CoA are channeled to electron acceptors including halogenated compounds, pyruvate induces expression of a pyruvate:ferredoxin oxidoreductase. This study paves the way to understanding the physiology of D. hafniense, optimizing this microbe as a bioremediation agent, and designing bioarray sensors to monitor the presence of dechlorinating organisms in the environment.

The genome of Polaromonas naphthalenivorans strain CJ2, isolated from coal tar‐contaminated sediment, reveals physiological and metabolic versatility and evolution through extensive horizontal gene transfer

We analysed the genome of the aromatic hydrocarbon-degrading, facultatively chemolithotrophic betaproteobacterium, Polaromonas naphthalenivorans strain CJ2. Recent work has increasingly shown that Polaromonas species are prevalent in a variety of pristine oligotrophic environments, as well as polluted habitats. Besides a circular chromosome of 4.4 Mb, strain CJ2 carries eight plasmids ranging from 353 to 6.4 kb in size. Overall, the genome is predicted to encode 4929 proteins. Comparisons of DNA sequences at the individual gene, gene cluster and whole-genome scales revealed strong trends in shared heredity between strain CJ2 and other members of the Comamonadaceae and Burkholderiaceae. blastp analyses of protein coding sequences across strain CJ2's genome showed that genetic commonalities with other betaproteobacteria diminished significantly in strain CJ2's plasmids compared with the chromosome, especially for the smallest ones. Broad trends in nucleotide characteristics (GC content, GC skew, Karlin signature difference) showed at least six anomalous regions in the chromosome, indicating alteration of genome architecture via horizontal gene transfer. Detailed analysis of one of these anomalous regions (96 kb in size, containing the nag-like naphthalene catabolic operon) indicates that the fragment's insertion site was within a putative MiaB-like tRNA-modifying enzyme coding sequence. The mosaic nature of strain CJ2's genome was further emphasized by the presence of 309 mobile genetic elements scattered throughout the genome, including 131 predicted transposase genes, 178 phage-related genes, and representatives of 12 families of insertion elements. A total of three different terminal oxidase genes were found (putative cytochrome aa(3)-type oxidase, cytochrome cbb(3)-type oxidase and cytochrome bd-type quinol oxidase), suggesting adaptation by strain CJ2 to variable aerobic and microaerobic conditions. Sequence-suggested abilities of strain CJ2 to carry out nitrogen fixation and grow on the aromatic compounds, biphenyl and benzoate, were experimentally verified. These new phenotypes and genotypes set the stage for gaining additional insights into the physiology and biochemistry contributing to strain CJ2's fitness in its native habitat, contaminated sediment.

Gramicidin S identified as a potent inhibitor for cytochrome bd-type quinol oxidase

Gramicidin S, a cationic cyclic decapeptide, exhibits the potent antibiotic activity through perturbation of lipid bilayers of the bacterial membrane. From the screening of natural antibiotics, we identified gramicidin S as a potent inhibitor for cytochrome bd-type quinol oxidase from Escherichia coli. We found that gramicidin S inhibited the oxidase with IC 50 of 3.5 μM by decreasing V max and the affinity for substrates but showed the stimulatory effect at low concentrations. Our findings would provide a new insight into the development of gramicidin S analogs, which do not share the target and mechanism with conventional antibiotics.

Menaquinol oxidase activity and primary structure of cytochrome bd from the amino-acid fermenting bacterium Corynebacterium glutamicum.

Cytochrome d was spectroscopically detected in membrane fractions of the amino-acid-fermenting, high-G+C gram-positive bacterium Corynebacterium glutamicum. Inhibition of NADH oxidase activity in the membranes by cyanide suggested that the main terminal respiratory oxidase during the stationary phase was a type of cytochrome bd. Cytochrome bd-type quinol oxidase, purified from the membranes, was composed of two subunits. Its reduced form showed absorption peaks at 627, 595, and 560 nm, which were due to haem d, high-spin protohaem, and low-spin protohaem, respectively. The air-oxidised form showed a peak at 645 nm, which might be due to oxygenated ferrous haem d. The spectral features and the size of subunit I are more similar to the properties of cytochromes bd from Proteobacteria, such as Escherichia coli, than to those of cytochrome bd from low-G+C gram-positive bacteria, such as Bacillus stearothermophilus. The menaquinol oxidase activity of the purified cytochrome bd was low, but was enhanced about fivefold by pre-incubating the enzyme with menaquinones. The order of effectiveness of quinols as oxidase substrates was clearly different from that of quinones as the activators of enzyme activity. Furthermore, activation was destroyed by ultraviolet irradiation of the pre-incubated enzyme and then restored by a second incubation with menaquinone. These results indicate that the enzymatic properties of this new oxidase are more similar to the properties of cytochromes bd from low-G+C gram-positive bacterial than to those of proteobacterial counterparts. They also suggest that the enzyme has a second quinone-binding site essential for full activity, in addition to the active centre for substrate oxidation. By using probes based on partial peptide sequences of the subunits, the genes for the two subunits of C. glutamicum cytochrome bd were cloned. The deduced amino acid sequence demonstrated that subunit I lacks the C-terminal half of the Q loop and that the primary structure of C. glutamicum cytochrome bd is more similar to that of other gram-positive bacteria than to proteobacterial cytochromes bd.

Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus

Gram-positive thermophilic Bacillus species contain cytochrome caa 3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. We previously identified and purified an alternative oxidase, cytochrome bd-type quinol oxidase, from a mutant of Bacillus stearothermophilus defective in the caa 3-type oxidase activity (J. Sakamoto et al., FEMS Microbiol. Lett. 143 (1996) 151–158). Compared with proteobacterial counterparts, B. stearothermophilus cytochrome bd showed lower molecular weights of the two subunits, shorter wavelength of α-band absorption maximum due to heme D, and lower quinol oxidase activity. Preincubation with menaquinone-2 enhanced the enzyme activity up to 40 times, suggesting that, besides the catalytic site, there is another quinone-binding site which largely affects the enzyme activity. In order to clarify the molecular basis of the differences of cytochromes bd between B. stearothermophilus and proteobacteria, the genes encoding for the B. stearothermophilus bd was cloned based on its partial peptide sequences. The gene for subunit I ( cbdA) encodes 448 amino acid residues with a molecular weight of 50 195 Da, which is 14 and 17% shorter than those of Escherichia coli and Azotobacter vinelandii, respectively, and CbdA lacks the C-terminal half of the long hydrophilic loop between the putative transmembrane segments V and VI (Q loop), which has been suggested to include the substrate quinone-binding site for the E. coli enzyme. The gene for subunit II ( cbdB) encodes 342 residues with a molecular weight of 38 992 Da. Homology search indicated that the B. stearothermophilus cbdAB has the highest sequence similarity to ythAB in B. subtilis genome rather than to cydAB, the first set of cytochrome bd genes identified in the genome. Sequence comparison of cytochromes bd and their homologs from various organisms demonstrates that the proteins can be classified into two subfamilies, a proteobacterial type including E. coli bd and a more widely distributed type including the B. stearothermophilus enzyme, suggesting that the latter type is evolutionarily older.

Direct correlationship between proton translocation and growth yield: An analysis of the respiratory chain of Bacillus stearothermophilus

Thermophilic bacilli contain cytochrome caa 3-type cytochrome c oxidase as the main terminal oxidase in the respiratory chain. A mutant strain, named K-17, lacking cytochrome caa 3 and exhibiting very low N,N,N′,N′-tetramethyl- p-phenylene diamine oxidase activity, was isolated by random mutation from Bacillus stearothermophilus K1041 (Sakamoto, J. et al., FEMS Microbiol. Lett., 143, 151–158, 1996). Comparing this mutant with the parent strain K1041, we obserbed the following differences in energy-yielding properties. (i) K-17 gave an cell yield less than one half of that of the wild type, although the doubling time of K-17 was only a little slower than that of the parent strain. (ii) In cellular respiration, the H +/O ratio of K-17 was 2.9–3.1, while that of the wild type was 6.1–6.5. (iii) A low concentration of cyanide inhibited endogenous respiration of the wild-type cells partly with a concomitant reduction of the H +/O ratio to around 3, while it did not significantly affect the respiration rate and the H +/O ratio of the K-17 cells. (iv) Cytochrome bd-type quinol oxidase seemed to operate in the wild-type cells when a low concentration (below 0.5 mM) of cyanide was added, while this enzyme is the main terminal oxidase in K-17. The K-17 cells also contained cytochrome b(o/a) 3-type cytochrome c-551 oxidase. These results demonstrated that the combination of the enzymes involved in the respiratory chain determines the H +/O ratios of the cell and consequently the growth yield of the bacteria.

A factor produced by Escherichia coli K-12 inhibits the growth of E. coli mutants defective in the cytochrome bd quinol oxidase complex: enterochelin rediscovered.

Escherichia coli produces an extracellular factor that inhibits the aerobic growth of Cyd- mutants, defective in the synthesis or assembly of the cytochrome bd-type quinol oxidase. This paper shows that such a factor is the iron-chelating siderophore enterochelin. Mutants in entA or aroB, defective in the production of enterochelin, did not produce the factor that inhibits the growth of cydAB and cydDC mutants; purified enterochelin inhibited the growth of Cyd- mutants, but not that of wild-type cells. Other iron-chelating agents, particularly ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA), whose complex with Fe(III) has a large stability constant (log K = 33.9), also inhibited the growth of Cyd- mutants at micromolar concentrations, but not that of wild-type cells. Supplementation of agar plates with Fe(III) or boiled catalase prevented the inhibition of Cyd- mutants by the extracellular factor. Spontaneous mutants isolated by being able to grow in the presence of the extracellular factor on plates also showed increased resistance to iron chelators. The reducing agent ascorbate, ascorbate plus In(III), ascorbate plus Ga(III), or Ga(III) alone, also alleviated inhibition by the extracellular factor, presumably by reducing iron to Fe(II) and complexing of the siderophore with alternative trivalent metal cations. The preferential inhibition of Cyd- mutants by the extracellular factor and other iron chelators is not due to decrease in expression, activity or assembly of cytochrome bo', the major alternative oxidase mediating quinol oxidation. Cyd- mutants overproduce siderophores, presumably reflecting intracellular iron deprivation.

Cytochrome bd-typQ quinol oxidase in a mutant of Bacillus stearothermophilus deficient in caa 3-type cytochrome c oxidase

Gram-positive thermophilic Bacillus species contain cytochrome ozas-type cytochrome c oxidase as a terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated B. stearothermophilus mutants defective in the caa 3-type oxidase activity. One mutant contained little cytochrome a and had low cytochrome c oxidase activity. However, growth and the respiratory activity of membranes in the presence of NADH were close to normal, suggesting that the mutant contains an alternative electron transfer pathway. A novel oxidase was isolated from the membrane fraction of the mutant. The enzyme is a cytochrome bd-type quinol oxidase composed of two subunits of 52 and 40 kDa, whose N-terminal regions show sequence similarity to polypeptides of the bd-type oxidase from Escherichia coli and Azotobacter vinelandii. This is the first report of a bd-type terminal oxidase purified from a Gram-positive bacterium.

Cytochrome bd-type quinol oxidase in a mutant of Bacillus stearothermophilus deficient in caa3-type cytochrome c oxidase.

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as a terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated B. stearothermophilus mutants defective in the caa3-type oxidase activity. One mutant contained little cytochrome a and had low cytochrome c oxidase activity. However, growth and the respiratory activity of membranes in the presence of NADH were close to normal, suggesting that the mutant contains an alternative electron transfer pathway. A novel oxidase was isolated from the membrane fraction of the mutant. The enzyme is a cytochrome bd-type quinol oxidase composed of two subunits of 52 and 40 kDa, whose N-terminal regions show sequence similarity to polypeptides of the bd-type oxidase from Escherichia coli and Azotobacter vinelandii. This is the first report of a bd-type terminal oxidase purified from a Gram-positive bacterium.