Gene fusions with beta-lactamase show that subunit I of the cytochrome bd quinol oxidase from E. coli has nine transmembrane helices with the O2 reactive site near the periplasmic surface

The cytochrome bd quinol oxidase is a component of the respiratory chain of many prokaryotes. The enzyme contains two subunits, CydA and CydB, which were initially predicted based on the sequence of the Escherichia coli oxidase to have seven and eight transmembrane spans, respectively. More recently, the topological model of CydA was revised to predict nine transmembrane helices, based on additional sequence information from other organisms. In the current work, the topology of the E. coli oxidase was experimentally examined using beta-lactamase gene fusions. The results confirm the revised topology, which places the oxygen reactive site near the periplasmic surface.     

Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42 degrees C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.     

A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168.

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.     

Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection

Brucellosis is characterized by abortion in ruminants and a protracted undulant fever in humans, which often results in severe pathological manifestations. Scant information exists about the molecular mechanisms employed by Brucella abortus to combat host defenses or to persist and replicate within host cells. Transposon (Tn5) mutagenesis of B. abortus and the subsequent screening of mutants for sensitivity to killing in murine macrophages and in the mouse model led to the identification of mutants which were severely attenuated for intracellular survival. One group of mutants was interrupted in cydB, a gene that is part of the cydAB operon encoding cytochrome bd oxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration. The elevated affinity for molecular oxygen of this enzyme in Escherichia coli has suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage. B. abortus cydB::Tn5 strains exhibited heightened sensitivity to the respiratory inhibitors zinc and azide, highly reactive oxygen species such as hydrogen peroxide, low pH, and attenuated virulence in the mouse model of infection. Virulence was restored by an intact copy of cydAB or by B. abortus genes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase. These results suggest a bifunctional role for the products of the cydAB operon, both in preventing the buildup of oxidative free radicals and in detoxifying the intracellular compartment, thus indicating the importance of these products in preventing intracellular destruction. Intracellular conditions that favor expression of the cydAB operon are under investigation and may be linked to the acid sensitivity also observed in this strain.     

Cloning and mutagenesis of genes encoding the cytochrome bd terminal oxidase complex in Azotobacter vinelandii: mutants deficient in the cytochrome d complex are unable to fix nitrogen in air.

The genome of Azotobacter vinelandii contains DNA sequences homologous to the structural genes for the Escherichia coli cytochrome bd terminal oxidase complex. Two recombinant clones bearing cydA- and cydB-like sequence were isolated from an A. vinelandii gene library and subcloned into the plasmid vector pACYC184. Physical mapping demonstrated that the cydA- and cydB-like regions in A. vinelandii are contiguous. The cydAB and flanking DNA was mutagenized by the insertion of Tn5-B20. Mutations in the cydB-hybridizing region resulted in the loss of spectral features associated with cytochromes b595 and d. A new locus, cydB, encoding cytochromes b595 and d in A. vinelandii is proposed. A second region adjacent to cydB was also involved in expression of the cytochrome bd complex in A. vinelandii, since mutations in this region resulted in an increase in the levels of both cytochrome b595 and cytochrome d. The regions involved in expression of the cytochrome bd complex and cydB are transcribed in the same direction. Mutants deficient in cytochromes b595 and d were unable to grow on N-deficient medium when incubated in air but could fix nitrogen when the environmental O2 concentration was reduced to 1.5% (vol/vol). It is proposed that the branch of the respiratory chain terminated by the cytochrome bd complex supports the high respiration rates required for the respiratory protection of nitrogenase.     

Terminal oxidases of Bacillus subtilis strain 168: one quinol oxidase, cytochrome aa(3) or cytochrome bd, is required for aerobic growth

The gram-positive endospore-forming bacterium Bacillus subtilis has, under aerobic conditions, a branched respiratory system comprising one quinol oxidase branch and one cytochrome oxidase branch. The system terminates in one of four alternative terminal oxidases. Cytochrome caa(3) is a cytochrome c oxidase, whereas cytochrome bd and cytochrome aa(3) are quinol oxidases. A fourth terminal oxidase, YthAB, is a putative quinol oxidase predicted from DNA sequence analysis. None of the terminal oxidases are, by themselves, essential for growth. However, one quinol oxidase (cytochrome aa(3) or cytochrome bd) is required for aerobic growth of B. subtilis strain 168. Data indicating that cytochrome aa(3) is the major oxidase used by exponentially growing cells in minimal and rich medium are presented. We show that one of the two heme-copper oxidases, cytochrome caa(3) or cytochrome aa(3), is required for efficient sporulation of B. subtilis strain 168 and that deletion of YthAB in a strain lacking cytochrome aa(3) makes the strain sporulation deficient.     

Cytochrome bd oxidase and nitric oxide: from reaction mechanisms to bacterial physiology

Experimental evidence suggests that the prokaryotic respiratory cytochrome bd quinol oxidase is responsible for both bioenergetic functions and bacterial adaptation to different stress conditions. The enzyme, phylogenetically unrelated to the extensively studied heme-copper terminal oxidases, is found in many commensal and pathogenic bacteria. Here, we review current knowledge on the catalytic intermediates of cytochrome bd and their reactivity towards nitric oxide (NO). Available information is discussed in the light of the hypothesis that, owing to its high NO dissociation rate, cytochrome bd confers resistance to NO-stress, thereby providing a strategy for bacterial pathogens to evade the NO-mediated host immune attack.     

Small-Colony Variant Selection as a Survival Strategy for Staphylococcus aureus in the Presence of Pseudomonas aeruginosa

Previously it has been demonstrated that Staphylococcus aureus is sensitive toward Pseudomonas-secreted exotoxins, which preferentially target the electron transport chain in staphylococci. Here it is shown that a subpopulation of S. aureus survives these respiratory toxins of Pseudomonas aeruginosa by selection of the small-colony variant (SCV) phenotype. Purified pyocyanin alone causes the same effect. A hemB mutant of S. aureus survives cocultivation with P. aeruginosa without a decrease in CFU.Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and frequently coinfect the lungs of patients with cystic fibrosis (CF). P. aeruginosa excretes an arsenal of small respiratory inhibitors, like pyocyanin (5), hydrogen cyanide (2), or quinoline N-oxides (9), that may act against the commensal microbiota as well as host cells. Previously it has been demonstrated that S. aureus is sensitive toward the toxic products generated by P. aeruginosa and that these exotoxins preferentially target the electron transport chain (17).Staphylococcal species can be divided into two groups: the sensitive group, comprising pathogenic species such as S. aureus and S. epidermidis, and the resistant group, represented by nonpathogenic species such as S. carnosus, S. piscifermentans, and S. gallinarum. The resistance in the latter group was due to cydAB genes, which encode a pyocyanin- and cyanide-resistant cytochrome bd quinol oxidase (17). It has also been shown that the resistant or sensitive phenotype is determined by the CydB subunit, which is part of the cytochrome bd quinol oxidase of S. aureus. Despite its sensitivity to these exotoxins, S. aureus has frequently been coisolated with P. aeruginosa from the skin, eyes, and catheter infections and from the lungs of patients with CF. The aim of this study is to elucidate the escape mechanism of S. aureus by cocultivating S. aureus and P. aeruginosa. The findings indicate that a subpopulation of the staphylococcal community can survive in the presence of P. aeruginosa by the selection of small-colony variants (SCVs), which usually are defective in the electron transport chain. SCVs grow as tiny, nonpigmented colonies and are auxotrophic to hemin, menadione, or thymidine (14). Here we show that both a culture supernatant of P. aeruginosa and purified pyocyanin select for the SCV phenotype in S. aureus.     

The Content of Alternative Oxidase of Euglena Mitochondria is Increased by Growth in the Presence of Cyanide and is not Cytochrome o.

ABSTRACT A study of the effect of respiratory inhibitors on O₂ uptake of Euglena gracilis mitochondria, isolated from cells grown in the presence of cyanide or with ethanol as carbon source, was undertaken. The contents of cytochrome c oxidase and alternative oxidase were also determined. Inhibition of respiration by antimycin and cyanide was only partial and it was dependent on the oxidizable substrate used. Succinate oxidation was the most sensitive to cyanide whereas lactate oxidation was the most resistant. Cell growth in the presence of cyanide or with ethanol as carbon source brought about an enhanced content of alternative oxidase without a concomitant increase in cytochrome aa₃ content. However, a correlation between cyanide-resistant respiration and alternative oxidase content was not found. Analysis of heme types in mitochondrial membranes revealed the absence of heme O. The data suggest the presence of an inducible alternative oxidase in Euglena mitochondria which has high resistance to cyanide and contains heme B. A close relationship between Euglena alternative oxidase and bacterial quinol oxidases containing B-type heme is proposed.     

Kinetic mechanism of quinol oxidation by cytochrome bd studied with ubiquinone-2 analogs

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase of Escherichia coli under microaerophilic growth conditions. The oxidase activity shows sigmoidal concentration-dependence with low concentrations of ubiquinols, and a marked substrate inhibition with high concentrations of ubiquinol-2 analogs [Sakamoto, K., Miyoshi, H., Takegami, K., Mogi, T., Anraku, Y., and Iwamura H. (1996) J. Biol. Chem. 271, 29897-29902]. Kinetic analysis of the oxidation of the ubiquinol-2 analogs, where the 2- or 3-methoxy group has been substituted with an azido or ethoxy group, suggested that its peculiar enzyme kinetics can be explained by a modified ping-pong bi-bi mechanism with the formation of inactive binary complex FS in the one-electron reduced oxygenated state and inactive ternary complex (E2S)S(n) on the oxidation of the second quinol molecule. Structure-function studies on the ubiquinol-2 analogs suggested that the 6-diprenyl group and the 3-methoxy group on the quinone ring are involved in the substrate inhibition. We also found that oxidized forms of ubiquinone-2 analogs served as weak noncompetitive inhibitors. These results indicate that the mechanism for the substrate oxidation by cytochrome bd is different from that of the heme-copper terminal quinol oxidase and is tightly coupled to dioxygen reduction chemistry.     

Antibiotics LL-Z1272 identified as novel inhibitors discriminating bacterial and mitochondrial quinol oxidases

To counter antibiotic-resistant bacteria, we screened the Kitasato Institute for Life Sciences Chemical Library with bacterial quinol oxidase, which does not exist in the mitochondrial respiratory chain. We identified five prenylphenols, LL-Z1272β, γ, δ, ? and ζ, as new inhibitors for the Escherichia coli cytochrome bd. We found that these compounds also inhibited the E. coli bo-type ubiquinol oxidase and trypanosome alternative oxidase, although these three oxidases are structurally unrelated. LL-Z1272β and ? (dechlorinated derivatives) were more active against cytochrome bd while LL-Z1272γ, δ, and ζ (chlorinated derivatives) were potent inhibitors of cytochrome bo and trypanosome alternative oxidase. Thus prenylphenols are useful for the selective inhibition of quinol oxidases and for understanding the molecular mechanisms of respiratory quinol oxidases as a probe for the quinol oxidation site. Since quinol oxidases are absent from mammalian mitochondria, LL-Z1272β and δ, which are less toxic to human cells, could be used as lead compounds for development of novel chemotherapeutic agents against pathogenic bacteria and African trypanosomiasis.     

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

Gram-positive thermophilic Bacillus species contain cytochrome caa3-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 caa3-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 alpha-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 50195 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 38992 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.     

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.     

Mutagenesis of a gene encoding a cytochrome o-like terminal oxidase of Azotobacter vinelandii: A cytochrome o mutant is aero-tolerant during nitrogen fixation

Abstract The amino acid sequence obtained by translating the nucleotide sequence of a 0.55 kb fragment, amplified from Azotobacter vinelandii chromosomal DNA by PCR, was 57% identical to part of the Escherichia coli cyoB gene, encoding subunit I of the cytochrome bo -type quinol oxidase. This fragment was mutated in vitro by insertion of a kanamycin-resistance cassette and introduced into the chromosome of A. vinelandii by homologous recombination. The mutant contained no spectrally detectable cytochrome o . However, in the stationary phase of growth, the level of the alternative oxidase (cytochrome bd ) was 11-fold higher than in the wild-type strain. Respiration of the mutant was insensitive to chlorpromazine, an inhibitor thought to act specifically on cytochrome o . Cytochrome o -deficient mutants fixed nitrogen in air, clearly distinguishing the role of this oxidase from that of cytochrome bd , which is required for respiratory protection of oxygen-labile nitrogenase.     

Electron transport routes in whole cells of Synechocystis sp. strain PCC 6803: the role of the cytochrome bd-type oxidase

The plastoquinone pool is the central switching point of both respiratory and photosynthetic electron transport in cyanobacteria. Its redox state can be monitored noninvasively in whole cells using chlorophyll fluorescence induction, avoiding possible artifacts associated with thylakoid membrane preparations. This method was applied to cells of Synechocystis sp. PCC 6803 to study respiratory reactions involving the plastoquinone pool. The role of the respiratory oxidases known from the genomic sequence of Synechocystis sp. PCC 6803 was investigated by a combined strategy using inhibitors and deletion strains that lack one or more of these oxidases. The putative quinol oxidase of the cytochrome bd-type was shown to participate in electron transport in thylakoid membranes. The activity of this enzyme in thylakoids was strongly dependent on culture conditions; it was increased under conditions where the activity of the cytochrome b(6)f complex alone may be insufficient for preventing over-reduction of the PQ pool. In contrast, no indication of quinol oxidase activity in thylakoids was found for a second alternative oxidase encoded by the ctaII genes.     

Glutamate 107 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli is protonated and near the heme d/heme b595 binuclear center

Cytochrome bd is a quinol oxidase from Escherichia coli, which is optimally expressed under microaerophilic growth conditions. The enzyme catalyzes the two-electron oxidation of either ubiquinol or menaquinol in the membrane and scavenges O2 at low concentrations, reducing it to water. Previous work has shown that, although cytochrome bd does not pump protons, turnover is coupled to the generation of a proton motive force. The generation of a proton electrochemical gradient results from the release of protons from the oxidation of quinol to the periplasm and the uptake of protons used to form H2O from the cytoplasm. Because the active site has been shown to be located near the periplasmic side of the membrane, a proton channel must facilitate the delivery of protons from the cytoplasm to the site of water formation. Two conserved glutamic acid residues, E107 and E99, are located in transmembrane helix III in subunit I and have been proposed to form part of this putative proton channel. In the current work, it is shown that mutations in either of these residues results in the loss of quinol oxidase activity and can result in the loss of the two hemes at the active site, hemes d and b595. One mutant, E107Q, while being totally inactive, retains the hemes. Fourier transform infrared (FTIR) redox difference spectroscopy has identified absorption bands from the COOH group of E107. The data show that E107 is protonated at pH 7.6 and that it is perturbed by the reduction of the heme d/heme b595 binuclear center at the active site. In contrast, mutation of an acidic residue known to be at or near the quinol-binding site (E257A) also inactivates the enzyme but has no substantial influence on the FTIR redox difference spectrum. Mutagenesis shows that there are several acidic residues, including E99 and E107 as well as D29 (in CydB), which are important for the assembly or stability of the heme d/heme b595 active site.     

A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase

Abstract Using transposon mutagenesis and screening for the loss of the ability to oxidise the artificial electron donor N , N , N ', N '-tetrarnethyl- p -phenylenediarnine, we have isolated a mutant of Pseudomonas aeruginosa that lacks all c -type cytochromes. This mutant is unable to grow anaerobically with nitrate as a terminal electron acceptor. Analysis of its respiratory function indicates that the mutant has lost its cytochrome c oxidase-terminated respiratory pathway but the cyanide-insensitive oxidase-terminated branch remains functional. Complementation of the mutant by in vivo cloning led to recovery of the wild-type characteristics. These data are consistent with the idea that the cyanide-insensitive respiratory pathway does not contain haem c and that the pathway's terminal oxidase is a quinol oxidase.