Clustering and dynamics of cytochrome bd‐I complexes in the Escherichia coli plasma membrane in vivo

The cytochrome bd‐I complex of Escherichia coli is a respiratory terminal oxidase and an integral component of the cytoplasmic membrane. As with other respiratory components, the organization and dynamics of this complex in living membranes is unknown. We set out to visualize the distribution and dynamics of this complex in vivo. By exchanging cydB for cydB–gfpgcn4 on the E. coli chromosome, we produced a strain (YTL01) that expresses functional GFP‐tagged cytochrome bd‐I terminal oxidase complexes under wild‐type genetic control. We imaged live YTL01 cells using video‐rate epifluorescence and total internal reflection fluorescence (TIRF) microscopy in combination with fluorescence recovery after photobleaching (FRAP) and saw mobile spots of GFP fluorescence in plasma membranes. Numbers of GFP molecules per spot were quantified by step‐wise photobleaching giving a broad distribution with a mean of ～76, indicating that cytochrome bd‐I is concentrated in mobile patches in the E. coli plasma membrane. We hypothesize that respiration occurs in mobile membrane patches which we call ‘respirazones’.     

Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo′' to inhibition by the carbon monoxide-releasing molecule,CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration

Background: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO – a critical gasotransmitter – in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Methods: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli — cytochrome bd-I, cytochrome bd-II and cytochrome bo′, to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (K_m 0.24 μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. Results: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo′. Cytochromes bo′ and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. Conclusions: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. General significance: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Assembly of a chimeric respiratory chain from bovine heart submitochondrial particles and cytochrome bd terminal oxidase of Escherichia coli

Cytochrome bd is a terminal quinol oxidase in Escherichia coli. Mitochondrial respiration is inhibited at cytochrome bc₁ (complex III) by myxothiazol. Mixing purified cytochrome bd oxidase with myxothiazol-inhibited bovine heart submitochondrial particles (SMP) restores up to 50% of the original rotenone-sensitive NADH oxidase and succinate oxidase activities in the absence of exogenous ubiquinone analogs. Complex III bypassed respiration and is saturated at amounts of added cytochrome bd similar to that of other natural respiratory components in SMP. The cytochrome bd tightly binds to the mitochondrial membrane and operates as an intrinsic component of the chimeric respiratory chain.     

Crp‐dependent cytochrome bd oxidase confers nitrite resistance to Shewanella oneidensis

Shewanella oneidensis is able to respire on a variety of organic and inorganic substrates, including nitrate and nitrite. Conversion of nitrate to nitrite and nitrite to ammonium is catalysed by periplasmic nitrate and nitrite reductases (NAP and NRF) respectively. Global regulator Crp (c yclic AMP r eceptor p rotein) is essential for growth of S. oneidensis on both nitrate and nitrite. In this study, we discovered that crp mutants are not only severely deficient in nitrate or nitrite respiration, but are also hypersensitive to nitrite. This hypersusceptibility phenotype is independent of nitrite respiration. Using random transposon mutagenesis, we obtained 73 Δcrp suppressor strains resistant to nitrite. Transposon insertion sites in 24 suppressor strains were exclusively mapped in the region upstream of the cyd operon encoding a cytochrome bd oxidase, resulting in expression of the operon now driven by a Crp‐independent promoter. Further investigation indicated that the promoter in suppressor strains comes from the transposon. Mutational analysis of the cydB gene (encoding the essential subunit II of the bd oxidase) confirmed that the cytochrome bd oxidase confers nitrite resistance to S. oneidensis.     

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 K_i for the bo₃, bd-I and bd-II oxidases are 2.4 ± 0.3, 0.04 ± 0.01 and 0.2 ± 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.     

Peroxidase activity of cytochrome <Emphasis Type="Italic">bd</Emphasis> from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cytochrome bd from Escherichia coli is able to oxidize such substrates as guaiacol, ferrocene, benzohydroquinone, and potassium ferrocyanide through the peroxidase mechanism, while none of these donors is oxidized in the oxidase reaction (i.e. in the reaction that involves molecular oxygen as the electron acceptor). Peroxidation of guaiacol has been studied in detail. The dependence of the rate of the reaction on the concentration of the enzyme and substrates as well as the effect of various inhibitors of the oxidase reaction on the peroxidase activity have been tested. The dependence of the guaiacol-peroxidase activity on the H₂O₂ concentration is linear up to the concentration of 8 mM. At higher concentrations of H₂O₂, inactivation of the enzyme is observed. Guaiacol markedly protects the enzyme from inactivation induced by peroxide. The peroxidase activity of cytochrome bd increases with increasing guaiacol concentration, reaching saturation in the range from 0.5 to 2.5 mM, but then starts falling. Such inhibitors of the ubiquinol-oxidase activity of cytochrome bd as cyanide, pentachlorophenol, and 2-n-heptyl 4-hydroxyquinoline-N-oxide also suppress its guaiacol-peroxidase activity; in contrast, zinc ions have no influence on the enzyme-catalyzed peroxidation of guaiacol. These data suggest that guaiacol interacts with the enzyme in the center of ubiquinol binding and donates electrons into the di-heme center of oxygen reduction via heme b ₅₅₈, and H₂O₂ is reduced by heme d. Although the peroxidase activity of cytochrome bd from E. coli is low compared to peroxidases, it might be of physiological significance for the bacterium itself and plays a pathophysiological role for humans and animals.     

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T)

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181^T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181^T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa ₃ ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

     

Asymmetrical Evolution of Cytochrome bd Subunits

Functionally linked genes generally evolve at similar rates and the knowledge of this particular feature of genomic evolution has been used as the basis for the phylogenetic profiling method. We illustrate here an exception to this rule in the evolution of the cytochrome bd complex. This is a two-component oxidase complex, with the subunits I and II known to be widely present in bacteria. The subunits within the cytochrome bd complex are under the same evolutionary pressure and most likely behave in the same evolutionary manner. However, the sequence similarity of genes encoding subunit II varies considerably across species. Genes encoding subunit II evolve 1.2 times faster on most of the branches of their phylogeny than subunit I genes. Furthermore, the genes encoding subunit II in Oceanobacillus iheyensis, Bacillus halodurans, and Staphylococcus species do not have detectable homologues within E. coli due to their large divergence. Together, the two subunits of cytochrome bd reveal an interesting example of an asymmetric pattern of evolutionary change. [Reviewing Editor: Dr. Brian Morton]     

Role of a putative third subunit YhcB on the assembly and function of cytochrome bd-type ubiquinol oxidase from Escherichia coli

Recent proteome studies on the Escherichia coli membrane proteins suggested that YhcB is a putative third subunit of cytochrome bd-type ubiquinol oxidase (CydAB) (F. Stenberg, P. Chovanec, S.L. Maslen, C.V. Robinson, L.L. Ilag, G. von Heijne, D.O. Daley, Protein complexes of the Escherichia coli cell envelope. J. Biol. Chem. 280 (2005) 34409-34419). We isolated and characterized cytochrome bd from the ΔyhcB strain, and found that the formation of the CydAB heterodimer, the spectroscopic properties of bound hemes, and kinetic parameters for the ubiquinol-1 oxidation were identical to those of cytochrome bd from the wild-type strain. Anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis showed that YhcB was not associated with the cytochrome bd complex. We concluded that YhcB is dispensable for the assembly and function of cytochrome bd. YhcB, which is distributed only in γ-proteobacteria, may be a part of another membrane protein complex or may form a homo multimeric complex.     

Roles of respiratory oxidases in protecting Escherichia coli K12 from oxidative stress

Isogenic strains of Escherichia coli that were defective in either of the two major aerobic terminal respiratory oxidases (cytochromes bo and bd) or in the putative third oxidase (cytochrome bd-II) were studied to elucidate role(s) for oxidases in protecting cells from oxidative stress in the form of H₂O₂ and paraquat. Exponential phase cultures of all three oxidase mutants exhibited a greater decline in cell viability when exposed to H₂O₂ stress compared to the isogenic parent wild-type strain. Cytochrome bo mutants showed the greatest sensitivity to H₂O₂ under all conditions studied indicating that this oxidase was crucial for protection from H₂O₂ in E. coli. Cell killing of all oxidase mutants by H₂O₂ was by an uncharacterized mechanism (mode 2 killing) with cell growth rate affected. The expression of (katG-lacZ), an indicator of intracellular H₂O₂, was 2-fold higher in a cydAB::kan mutant compared to the wild-type strain at low H₂O₂ concentrations (< 100 M) suggesting that cytochrome bd mutants were experiencing higher intracellular levels of H₂O₂. Protein fusions to the three oxidase genes demonstrated that expression of genes encoding cytochrome bd, but not cytochrome bo or cytochrome bd-II was increased in the presence of external H₂O₂. This increase in expression of (cydA-lacZ) by H₂O₂ was further enhanced in a cyo::kan mutant. The level of cytochrome bd determined spectrally and (cydA-lacZ) expression was 5-fold and 2-fold higher respectively in an rpoS mutant compared to isogenic wild-type cells suggesting that RpoS was a negative regulator of cytochrome bd. Whether the effect of RpoS is direct or indirect remains to be determined.     

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.     

The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.     

Cytochrome bd biosynthesis in Escherichia coli: the sequences of the cydC and cydD genes suggest that they encode the components of an ABC membrane transporter

At least four genes are known to affect formation of the cytochrome bd-type terminal oxidase of Escherichia coli. In addition to the genes (cydA and cydB) encoding the two constituent subunits of this complex, a further two genes (cydC and cydD) map near 19 min on the E. coli chromosome. We report here the cloning of both genes on a 5.3 kb ClaI-HindIII restriction fragment, which, when used to transform either a cydC or cydD mutant, restored the ability of these mutants to grow on a selective medium containing azide and zinc ions and also restored the spectral signals associated with the cytochrome components of the oxidase complex. A subcloned 1.8 kb DdeI fragment similarly restored growth and cytochrome content of a cydD mutant, but not a cydC mutant. The complete nucleotide sequence of the ClaI-HindIII fragment reveals three open reading frames, one being trxB (19.3 min on the E. coli chromosome map, encoding thioredoxin reductase), confirming the mapping position of cydD previously established by P1-mediated transduction. Two ORFs identified by complementation experiments as cydD and cydC encode proteins with predicted molecular masses, respectively, of 65103 and 62 946 Da. The hydropathy profile of each protein reveals an N-terminal hydrophobic domain and a C-terminal hydrophilic domain containing a putative nucleotide-binding site. The gene products probably constitute an ABC (ATP-binding cassette) family membrane transporter, the function of which is necessary for the formation of the cytochrome bd quinol oxidase. The CydDC system appears to be the first prokaryotic example of a heterodimeric ABC transport system in which each polypeptide contains both hydrophobic and ATP-binding domains.     

Incorporation of specifically labeled cysteine into Escherichia coli protein

Protein obtained from several strains of Escherichia coli grown in the presence of [3,3′-¹⁴C]cystine contained the radiolabel in nearly all the other amino acids, suggesting catabolism of cysteine to pyruvic acid. Utilization in amino acid synthesis of the pyruvate thus generated can be blocked by growing the bacteria in a medium specifically enriched with most of the naturally occurring amino acids. Cysteine that is incorporated intact is diluted by de novo synthesis at low cystine concentrations; also, it was found that E. coli can use the sulfur of methionine for cysteine biosynthesis. Both of these latter two processes can be prevented by supplying an excess of exogenous cystine. This regiment leads to protein that is highly specifically labeled in the cysteine residues, with a minor amount (20–25%) of the label also appearing in alanine residues. Although this strategy was developed expressly for cysteine, it may be useful for incorporating other labeled amino acids that are also readily catabolized.     

The carboxy-terminal insert in the Q-loop is needed for functionality of Escherichia coli cytochrome bd-I

Cytochrome bd, a component of the prokaryotic respiratory chain, is important under physiological stress and during pathogenicity. Electrons from quinol substrates are passed on via heme groups in the CydA subunit and used to reduce molecular oxygen. Close to the quinol binding site, CydA displays a periplasmic hydrophilic loop called Q-loop that is essential for quinol oxidation. In the carboxy-terminal part of this loop, CydA from Escherichia coli and other proteobacteria harbors an insert of ~60 residues with unknown function. In the current work, we demonstrate that growth of the multiple-deletion strain E. coli MB43∆cydA (∆cydA∆cydB∆appB∆cyoB∆nuoB) can be enhanced by transformation with E. coli cytochrome bd-I and we utilize this system for assessment of Q-loop mutants. Deletion of the cytochrome bd-I Q-loop insert abolished MB43∆cydA growth recovery. Swapping the cytochrome bd-I Q-loop for the Q-loop from Geobacillus thermodenitrificans or Mycobacterium tuberculosis CydA, which lack the insert, did not enhance the growth of MB43∆cydA, whereas swapping for the Q-loop from E. coli cytochrome bd-II recovered growth. Alanine scanning experiments identified the cytochrome bd-I Q-loop insert regions Ile³¹⁸-Met³²², Gln³³⁸-Asp³⁴², Tyr³⁵³-Leu³⁵⁷, and Thr³⁶⁸-Ile³⁷² as important for enzyme functionality. Those mutants that completely failed to recover growth of MB43∆cydA also lacked oxygen consumption activity and heme absorption peaks. Moreover, we were not able to isolate cytochrome bd-I from these inactive mutants. The results indicate that the cytochrome bd Q-loop exhibits low plasticity and that the Q-loop insert in E. coli is needed for complete, stable, assembly of cytochrome bd-I.     

Respiratory Protection of Nitrogenase Activity in Azotobacter vinelandii—Roles of the Terminal Oxidases

Nitrogen fixation by aerobic prokaryotes appears paradoxical: the nitrogen-fixing enzymes—nitrogenases—are notoriously oxygen-labile, yet many bacteria fix nitrogen aerobically. This review summarises the evidence that cytochrome bd, a terminal oxidase unrelated to the mitochondrial and many other bacterial oxidases, plays a crucial role in aerotolerant nitrogen fixation in Azotobacter vinelandii and other bacteria by rapidly consuming oxygen during uncoupled respiration. We review the pertinent properties of this oxidase, particularly its complement of redox centres, the catalytic cycle of oxygen reduction, the affinity of the oxidase for oxygen, and the regulation of cytochrome bd gene expression. The roles of other oxidases and other mechanisms for limiting damage to nitrogenase are assessed.