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Cell physiology relies on metalloenzymes and can be easily disrupted by imbalances in metal ion pools. Bacillus subtilis requires manganese for growth and has highly regulated mechanisms for import and efflux that help maintain homeostasis. Cells defective for manganese (Mn) efflux are highly sensitive to intoxication, but the processes impaired by Mn excess are often unknown. Here, we employed a forward genetics approach to identify pathways affected by manganese intoxication. Our results highlight a central role for the membrane-localized electron transport chain (ETC) in metal intoxication during aerobic growth. In the presence of elevated manganese there is an increased generation of reactive radical species associated with dysfunction of the major terminal oxidase, the cytochrome aa(3) heme-copper menaquinol oxidase (QoxABCD). Intoxication is suppressed by diversion of menaquinol to alternative oxidases or by a mutation affecting heme A synthesis that is known to convert QoxABCD from an aa(3) to a bo(3)-type oxidase. Manganese sensitivity is also reduced by derepression of the MhqR regulon, which protects cells against reactive quinones. These results suggest that dysfunction of the cytochrome aa(3)-type quinol oxidase contributes to metal-induced intoxication.

In order to reduce nitrate in vivo, the spore-specific respiratory nitrate reductase, Nar1, of Streptomyces coelicolor relies on an active cytochrome bcc-aa3 oxidase supercomplex (bcc-aa3 supercomplex). This suggests that membrane-associated Nar1, comprising NarG1, NarH1, and NarI1 subunits, might not act as a classical menaquinol oxidase but could either receive electrons from the bcc-aa3 supercomplex, or require the supercomplex to stabilize the reductase in the membrane to allow it to function. To address the biochemical basis for this dependence on the bcc-aa3 supercomplex, we purified two different Strep-tagged variants of Nar1 and enriched the native enzyme complex from spore extracts using different chromatographic and electrophoretic procedures. Polypeptides associated with the isolated Nar1 complexes were identified using mass spectrometry and included components of the bcc-aa3 supercomplex, along with an alternative, spore-specific cytochrome b component, QcrB3. Surprisingly, we also co-enriched the Nar3 enzyme with Nar1 from the wild-type strain of S. coelicolor. Two differentially migrating active Nar1 complexes could be identified after clear native polyacrylamide gel electrophoresis; these had masses of approximately 450 and 250 kDa. The distribution of active Nar1 in these complexes was influenced by the presence of cytochrome bd oxidase and by QcrB3; the presence of the latter shifted Nar1 into the larger complex. Together, these data suggest that several respiratory complexes can associate in the spore membrane, including Nar1, Nar3, and the bcc-aa3 supercomplex. Moreover, these findings provide initial support for the hypothesis that Nar1 and the bcc-aa3 supercomplex physically associate.

Mycobacteria have shown enormous resilience to survive and persist by remodeling and altering metabolic requirements. Under stringent conditions or exposure to drugs, mycobacteria have adapted to rescue themselves by shutting down their major metabolic activity and elevate certain survival factor levels and efflux pathways to survive and evade the effects of drug treatments. A fundamental feature in this adaptation is the ability of mycobacteria to vary the enzyme composition of the electron transport chain (ETC), which generates the proton motive force for the synthesis of adenosine triphosphate via oxidative phosphorylation. Mycobacteria harbor dehydrogenases to fuel the ETC, and two terminal respiratory oxidases, an aa3-type cytochrome c oxidase (cyt-bcc-aa3) and a bacterial specific cytochrome bd-type menaquinol oxidase (cyt-bd). In this study, we employed homology modeling and structure-based virtual screening studies to target mycobacteria-specific residues anchoring the b558 menaquinol binding region of Mycobacterium tuberculosis cyt-bd oxidase to obtain a focused library. Furthermore, ATP synthesis inhibition assays were carried out. One of the ligands MQL-H2 inhibited both NADH2- and succinate-driven ATP synthesis inhibition of Mycobacterium smegmatis inside-out vesicles in micromolar potency. Similarly, MQL-H2 also inhibited NADH2-driven ATP synthesis in inside-out vesicles of the cytochrome-bcc oxidase deficient M. smegmatis strain. Since neither varying the electron donor substrates nor deletion of the cyt-bcc oxidase, a major source of protons, hindered the inhibitory effects of the MQL-H2, reflecting that MQL-H2 targets the terminal oxidase cytochrome bd oxidase, which was consistent with molecular docking studies. Characterization of novel cytochrome bd oxidase Menaquinol binding domain inhibitor (MQL-H2) using virtual screening and ATP synthesis inhibition assays.

Cytochrome c oxidase in the respiratory chain of bacteria and mitochondria couples the reduction of molecular oxygen to form water with the generation of a transmembrane proton gradient. Bacillus subtilis has two heme A-containing heme–copper oxidases: the menaquinol oxidase cytochrome aa3 and the cytochrome c oxidase cytochrome caa3. By screening three collections of mutants for defective cytochrome c oxidase, we found the genes for two, new membrane-bound assembly factors in B. subtilis: ytkA and yozB (renamed ctaK and ctaM, respectively). CtaK is a lipoprotein without sequence similarity to any protein of known function. We show that CtaK functions together with Sco1 (YpmQ) in a pathway, leading to the assembly of the CuA center in cytochrome caa3 and seems to be a functional analogue to proteins of the periplasmic CuA chaperone family (PCuAC). CtaM is required for the activity of both cytochrome caa3 and cytochrome aa3 and dispensable for the insertion of heme A into these oxidases. The orthologous Bacillus anthracis protein and the distantly related Staphylococcus aureus CtaM complemented CtaM deficiency in B. subtilis, establishing a common function of CtaM in these bacteria. As the overall result of our work, 12 different proteins are known to function in the biosynthesis of cytochrome c oxidase in B. subtilis. (Less)

Causing approximately 10 million incident cases and 1.3–1.5 million deaths every year, Mycobacterium tuberculosis remains a global health problem. The risk is further exacerbated with latent tuberculosis (TB) infection, the HIV pandemic, and increasing anti-TB drug resistance. Therefore, unexplored chemical scaffolds directed towards new molecular targets are increasingly desired. In this context, mycobacterial energy metabolism, particularly the oxidative phosphorylation (OP) pathway, is gaining importance. Mycobacteria possess primary dehydrogenases to fuel electron transport; aa3-type cytochrome c oxidase and bd-type menaquinol oxidase to generate a protonmotive force; and ATP synthase, which is essential for both growing mycobacteria as well as dormant mycobacteria because ATP is produced under both aerobic and hypoxic conditions. Small organic molecules targeting OP are active against latent TB as well as resistant TB strains. FDA approval of the ATP synthase inhibitor bedaquiline and the discovery of clinical candidate Q203, which both interfere with the cytochrome bc1 complex, have already confirmed mycobacterial energy metabolism to be a valuable anti-TB drug target. This review highlights both preferable molecular targets within mycobacterial OP and promising small organic molecules targeting OP. Progressive research in the area of mycobacterial OP revealed several highly potent anti-TB compounds with nanomolar-range MICs as low as 0.004 μM against Mtb H37Rv. Therefore, we are convinced that targeting the OP pathway can combat resistant TB and latent TB, leading to more efficient anti-TB chemotherapy.

Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa 3 -600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO ( N -oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.

Geobacter sulfurreducenswas originally considered a strict anaerobe. However, this bacterium was later shown to not only tolerate exposure to oxygen but also to use it as terminal electron acceptor. Research performed has so far only revealed the general ability ofG. sulfurreducensto reduce oxygen, but the oxygen uptake rate has not been quantified yet, nor has evidence been provided as to how the bacterium achieves oxygen reduction. Therefore, microaerobic growth ofG. sulfurreducenswas investigated here with better defined operating conditions as previously performed and a transcriptome analysis was performed to elucidate possible metabolic mechanisms important for oxygen reduction inG. sulfurreducens. The investigations revealed that cell growth with oxygen is possible to the same extent as with fumarate if the maximum specific oxygen uptake rate (sOUR) of 95 mgO2gCDW-1h-1is not surpassed. Hereby, the entire amount of introduced oxygen is reduced. When oxygen concentrations are too high, cell growth is completely inhibited and there is no partial oxygen consumption. Transcriptome analysis suggests a menaquinol oxidase to be the enzyme responsible for oxygen reduction. Transcriptome analysis has further revealed three different survival strategies, depending on the oxygen concentration present. When prompted with small amounts of oxygen,G. sulfurreducenswill try to escape the microaerobic area; if oxygen concentrations are higher, cells will focus on rapid and complete oxygen reduction coupled to cell growth; and ultimately cells will form protective layers if a complete reduction becomes impossible. The results presented here have important implications for understanding howG. sulfurreducenssurvives exposure to oxygen.

Cytochrome aa3-600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses menaquinol as its electron donor instead of cytochrome c when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made the R70H mutant and have characterized the menasemiquinone radical by advanced X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (a(14)N ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e(2)qQ/h = 1.44 MHz, typical for protonated imidazole nitrogens. In the wild-type cyt aa3-600, the SQ is instead hydrogen bonded with Nε from the Arg70 side chain. Analysis of the (1)H 2D electron spin echo envelope modulation (ESEEM) spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ and the surrounding protein. Despite the alterations in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo3 ubiquinol oxidase from Escherichia coli, where the R71H mutation eliminates function.

We deleted subunits I ( cydA ) and II ( cydB ) of the Mycobacterium tuberculosis cytochrome bd menaquinol oxidase. The resulting Δ cydA and Δ cydAB mutants were hypersusceptible to compounds targeting the mycobacterial bc 1 menaquinol-cytochrome c oxidoreductase and exhibited bioenergetic profiles indistinguishable from strains deficient in the ABC-type transporter, CydDC, predicted to be essential for cytochrome bd assembly. These results confirm CydAB and CydDC as potential targets for drugs aimed at inhibiting a terminal respiratory oxidase implicated in pathogenesis.

Staphylococcus aureus is the leading cause of skin and soft tissue infections, bacteremia, osteomyelitis, and endocarditis in the developed world. The ability of S. aureus to cause substantial disease in distinct host environments is supported by a flexible metabolism that allows this pathogen to overcome challenges unique to each host organ. One feature of staphylococcal metabolic flexibility is a branched aerobic respiratory chain composed of multiple terminal oxidases. Whereas previous biochemical and spectroscopic studies reported the presence of three different respiratory oxygen reductases (o type, bd type, and aa3 type), the genome contains genes encoding only two respiratory oxygen reductases, cydAB and qoxABCD. Previous investigation showed that cydAB and qoxABCD are required to colonize specific host organs, the murine heart and liver, respectively. This work seeks to clarify the relationship between the genetic studies showing the unique roles of the cydAB and qoxABCD in virulence and the respiratory reductases reported in the literature. We establish that QoxABCD is an aa3-type menaquinol oxidase but that this enzyme is promiscuous in that it can assemble as a bo3-type menaquinol oxidase. However, the bo3 form of QoxABCD restricts the carbon sources that can support the growth of S. aureus. In addition, QoxABCD function is supported by a previously uncharacterized protein, which we have named CtaM, that is conserved in aerobically respiring Firmicutes. In total, these studies establish the heme A biosynthesis pathway in S. aureus, determine that QoxABCD is a type aa3 menaquinol oxidase, and reveal CtaM as a new protein required for type aa3 menaquinol oxidase function in multiple bacterial genera., IMPORTANCE Staphylococcus aureus relies upon the function of two terminal oxidases, CydAB and QoxABCD, to aerobically respire and colonize distinct host tissues. Previous biochemical studies support the conclusion that a third terminal oxidase is also present. We establish the components of the S. aureus electron transport chain by determining the heme cofactors that interact with QoxABCD. This insight explains previous observations by revealing that QoxABCD can utilize different heme cofactors and confirms that the electron transport chain of S. aureus is comprised of two terminal menaquinol oxidases. In addition, a newly identified protein, CtaM, is found to be required for the function of QoxABCD. These results provide a more complete assessment of the molecular mechanisms that support staphylococcal respiration.

Cytochrome aa(3)-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O(2) to 2H(2)O. Cytochrome aa(3)-600 is of interest because it is a very close homologue of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo(3) has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa(3)-600. Cytochrome aa(3)-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa(3)-600 in comparison with the ubi-semiquinone in cytochrome bo(3). Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa(3)-600 in comparison with ubiquinone in cytochrome bo(3). In addition, the electronic structure of the semiquinone cyt aa(3)-600 is more shifted toward the anionic form from the neutral state in cyt bo(3).

Cytochrome aa3-600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme c...

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron–sulfur subunit which contains three distinct iron–sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b556 is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.

Corynebacterium glutamicum has a branched respiratory chain: one of the branches is cytochrome bcc complex and cytochrome aa3-type cytochrome c oxidase, and the other is cytochrome bd-type menaquinol oxidase. The factors that influence the expression patterns of these respiratory enzymes remain unclear. To investigate the expressional control mechanism of the enzymes, we have previously constructed a promoter assay system utilizing enhanced green fluorescence protein. Here, we monitored respiratory enzymes’ expression by using this system during growth in various culture media, with and without Cu2+ ion supplementation. The promoter activities of cytochrome aa3 oxidase in the early stationary phase in the media supplemented with Cu2+ ion at 40 or 400 μM were significantly increased 1.49-fold or 1.99-fold, respectively, as compared to the control. Moreover, the H+/O ratio, or the proton-pumping activity of the cells, increased about 1.6 times by the Cu2+ supplementation. These facts indicate that copper ions can switch the branches.

Mycobacteria inhabit a wide range of intracellular and extracellular environments. Many of these environments are highly dynamic, and therefore mycobacteria are faced with the constant challenge of redirecting their metabolic activity to be commensurate with either replicative growth or a nonreplicative quiescence. A fundamental feature in this adaptation is the ability of mycobacteria to respire, regenerate reducing equivalents, and generate ATP via oxidative phosphorylation. Mycobacteria harbor multiple primary dehydrogenases to fuel the electron transport chain, and two terminal respiratory oxidases, an aa 3 -type cytochrome c oxidase and a cytochrome bd -type menaquinol oxidase, are present for dioxygen reduction coupled to the generation of a proton motive force (PMF). Hypoxia leads to the downregulation of key respiratory complexes, but the molecular mechanisms regulating this expression are unknown. Despite being obligate aerobes, mycobacteria have the ability to metabolize in the absence of oxygen, and a number of reductases are present to facilitate the turnover of reducing equivalents under these conditions (e.g., nitrate reductase, succinate dehydrogenase/fumarate reductase). Hydrogenases and ferredoxins are also present in the genomes of mycobacteria, suggesting the ability of these bacteria to adapt to an anaerobic type of metabolism in the absence of oxygen. ATP synthesis by the membrane-bound F 1 F 0 -ATP synthase is essential for growing and nongrowing mycobacteria, and the enzyme is able to function over a wide range of PMF values (aerobic to hypoxic). The discovery of lead compounds that target respiration and oxidative phosphorylation in Mycobacterium tuberculosis highlights the importance of this area for the generation of new frontline drugs to combat tuberculosis.

The genus Mycobacterium comprises a group of obligately aerobic bacteria that have adapted to inhabit a wide range of intracellular and extracellular environments. A fundamental feature in this adaptation is the ability of mycobacteria to respire and generate energy for growth or to sustain latency. Mycobacteria harbor multiple primary dehydrogenases to fuel the electron transport chain and two terminal respiratory oxidases, an aa 3 -type cytochrome c oxidase and cytochrome bd-type menaquinol oxidase, are present for dioxygen reduction coupled to the generation of a protonmotive force. In mycobacteria, Type II NADH dehydrogenases are favoured over complex I for NADH oxidation and menaquinone acts as the primary conduit between electron-donating and electron-accepting reactions. The molecular mechanisms regulating the expression of the electron transport chain components in mycobacteria remains unknown. Despite being obligate aerobes, mycobacteria have the ability to metabolize in the absence of oxygen and a number of reductases are present to facilitate the turnover of reducing equivalents under these conditions (e.g., nitrate reductase, fumarate reductase). Hydrogenases and ferredoxins are also present in the genomes of mycobacteria suggesting the ability of these bacteria to adapt to an anaerobic-type of metabolism in the absence of oxygen. The exact roles of reductases and hydrogenases is poorly understood. ATP synthesis by the membrane-bound F1FO-ATP synthase (see Chap. 6) is essential for growing and non-growing mycobacteria and the enzyme is able to function over a wide range of proton-motive force values (aerobic to hypoxic). Research into mycobacterial respiration and oxidative phosphorylation have been energized by the discovery of a new drug (TMC207) that targets the ATP synthase of mycobacteria, suggesting that inhibitors of respiration and ATP synthesis will provide the next generation of front line drugs to combat tuberculosis and nontuberculous mycobacterial disease.

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 acitivity 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.

The present studies provide new data on the involvement of menaquinol oxidases in substrate oxidation and energy conservation in aerobically grown, resting cells of Staphylococcus aureus 17810R, starved of endogenous energy reserves and supplemented with glutamate or L-lactate. These cells were energetically competent, since they oxidized both substrates, generated an electrochemical proton gradient (deltamuH+) and synthesized ATP via oxidative phosphorylation. Studies with KCN showed that: (i) L-lactate oxidation occurred via two terminal menaquinol oxidases - the ba3-type sensitive to low KCN and the bo-type insensitive to cyanide, (ii) glutamate oxidation proceeded via the bo-type oxidase, and (iii) ATP synthesis with glutamate or L-lactate was coupled only to the bo-type oxidase. Also in glucose-grown cells oxidizing L-lactate, ATP synthesis was coupled to the highly repressed bo-type oxidase. It is suggested that in the respiratory chain of strain 17810R two energy coupling sites may be present: in the complex of NADH-menaquinone oxidoreductase and in the complex of the bo-type menaquinol oxidase. The rate of ATP synthesis was similar with both substrates, but the rate of their oxidation differed significantly: the P/O ratios were 1.5 and 0.03 with glutamate and L-lactate, respectively. CCCP accelerated glutamate oxidation by 50% but was without effect on L-lactate oxidation. In cell lysates, the rates of NADH and L-lactate oxidation were equal. It is concluded that in whole cells of S. aureus 17810R oxidation of NADH derived from glutamate breakdown is tightly coupled to phosphorylation, while L-lactate oxidation seems to be rather loosely coupled.

Menaquinol oxidase isolated from the membrane of Bacillus subtilis W23 was found to consist of four polypeptides (QoxA, B, C, and D) that were predicted by the sequence of the qox operon of B. subtilis 168 (Santana et al. 1992). The preparation contained 7 mol cytochrome aa3 per g protein, which corresponds to 2 mol heme A per mol enzyme of 144 kDa molecular mass. Respiration with dimethylnaphthoquinol catalyzed by the enzyme was ten times faster than that with menadiol. Activities with more electropositive quinols were negligible. The activity of the enzyme was inhibited by equimolar amounts of HQNO, while antimycin, myxothiazol, and stigmatellin were more than tenfold less effective. When cells of both strains of B. subtilis (W23 and 168) were grown with glucose, quinol respiration was an order of magnitude more active than respiration with N,N,N',N'-tetramethyl-1,4-phenylenediamine plus ascorbate. Surprisingly, the same result was obtained with mutant strains lacking qoxB. As cytochromes a and d were virtually absent, a second quinol oxidase, possibly of the cytochrome o-type, was apparently formed by the mutants.

The quinol oxidase appears to be mainly responsible for the oxidation of the bacterial MKH2 in Bacillus subtilis W23 growing with either glucose or succinate. The activity of the enzyme was maximum with dimethylnaphthoquinol, a water-soluble analogue of the bacterial menaquinol. Menadiol or duroquinol were less actively respired, and naphthoquinol was not oxidized at all. After fourtyfold purification the isolated enzyme contained 5.3 mumol cytochrome aa3 per gram of protein and negligible amounts of cytochrome b and c. The turnover number based on cytochrome aa3 was about 10(3) electrons.s-1 at pH 7 and 37 degrees C. The preparation consisted mainly of a M(r) 57,000 and a M(r) 36,000 polypeptide. The N-terminal amino acid sequence of the latter polypeptide differed from that predicted by the qoxA gene of B. subtilis strain 168 (Santana et al. 1992), in that asp-14 predicted by qoxA was missing in the M(r) 36,000 polypeptide.

Escherichia coli fumarate reductase (FRD) is a four-subunit enzyme that catalyzes the terminal step in anaerobic respiration to fumarate. The hydrophobic FrdC and FrdD subunits anchor the FrdA and FrdB catalytic subunits to the inner surface of the cytoplasmic membrane and are required for the enzyme to interact with quinones. Thirty-five single-site mutations were constructed in the FrdC and FrdD polypeptides by site-directed mutagenesis. Each mutant enzyme was characterized for its ability to catalyze quinone oxidation and reduction and to support growth of E. coli DW35 (delta frdABCD sdhC::kan) under selective conditions requiring functional enzyme. Replacement of FrdCE29 with Asp, Leu, Lys, or Phe had a deleterious effect both on quinol oxidase and quinone reductase activities. Substitution of FrdCH82 with Arg, Leu, Tyr, or Glu also decreased menaquinol oxidase activity, but had variable effects on the reverse reaction, the reduction of ubiquinone. Data are presented to support the hypothesis that the positive charge at FrdCH82 is required for stabilization of the quinone radical intermediate and the negative charge at FrdCE29 for deprotonation of menaquinol. Other critical amino acids identified in FrdC included Ala-32, Phe-38, Trp-86, Phe-87, and in FrdD residues Phe-57, Gln-59, Ser-60, and His-80. The established roles of such residues in the QA and QB sites of the photosynthetic reaction center would suggest a similar type of structure operative in the FRD complex. In such a model, Glu-29, Ala-32, His-82, Trp-86 of FrdC and His-80 of FrdD are considered participants in a QB-type site, and FrdD Phe-57, Gln-59, and Ser-60 components in an apolar QA-type site.

Corynebacterium glutamicum contains at least two terminal oxidases in the respiratory chain; cytochrome aa(3)-type cytochrome c oxidase and bd-type menaquinol oxidase. Thus, the chain has two branches of electron flow. The bcc-aa(3) branch translocates three protons per electron transferred, while the bd branch translocates only one. In this study, we constructed two mutant strains, lacking of either subunit I of the cytochrome c oxidase (DeltactaD) or subunits I and II of the quinol oxidase (DeltacydAB), and also plasmids to complement the deficient genes to investigate their effects on energy conservation and cell growth. We measured H(+)/O ratios of C. glutamicum wild-type and mutant cells grown aerobically. The H(+)/O ratio of the wild-type cells grown in the semi-synthetic medium was 3.94 +/- 0.30, while the value was 2.76 +/- 0.25 for the DeltactaD mutant. In contrast, the value was 5.23 +/- 0.36 for the DeltacydAB mutant. The cells grown in the LB medium showed higher value compared to that of cells grown in the semi-synthetic medium. The DeltactaD mutant grew less than the wild-type in LB medium, while they grew about equally in semi-synthetic medium. Correlation between bioenergetics and growth of C. glutamicum was significantly affected by the growth nutrients.

Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration. Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features. Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e. NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, D-lactate dehydrogenase, L-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and L-proline dehydrogenase. All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell. From menaquinol, the electrons are passed either via the cytochrome bc(1) complex to the aa(3)-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity. The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c(1) to the Cu(A) center in subunit II of cytochrome aa(3). Rather, cytochrome c(1) contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c. The bc(1) complex and cytochrome aa(3) oxidase form a supercomplex in C. glutamicum. The phenotype of defined mutants revealed that the bc(1)-aa(3) branch, but not the bd branch, is of major importance for aerobic growth in minimal medium. Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F(1)F(0)-ATP synthase were found to have strong effects on metabolism and amino acid production. Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering.

Cytochrome aa3-600 or menaquinol oxidase, from Bacillus subtilis, is a member of the heme-copper oxidase family. Cytochrome aa3-600 contains cytochrome a, cytochrome a3, and CuB, and each is coordinated via histidine residues to subunit I. Subunit II of cytochrome aa3-600 lacks CuA, which is a common feature of the cytochrome c oxidase family members. Anaerobic reduction of cytochrome aa3-600 by the substrate analogue 2,3-dimethyl-1,4-naphthoquinone (DMN) resolves two distinct kinetic phases by stopped-flow, single-wavelength spectrometry. Global analysis of time-resolved, multiwavelength spectra shows that during these distinct phases cytochromes a and a3 are both reduced. Cyanide binding to cytochrome a3 enhances the fast phase rate, which in the presence of cyanide can be assigned to cytochrome a reduction, whereas cytochrome a3-cyanide reduction is slow. The steady-state activity of cytochrome aa3-600 exhibits saturation kinetics as a function of DMN concentration with a Km of 300 microM and a maximal turnover of 63.5 s(-1). Global kinetic analysis of steady-state spectra reveals a species that is characteristic of a partially reduced oxygen adduct of cytochrome a3-CuB, whereas cytochrome a remains oxidized. Electron paramagnetic resonance (EPR) spectroscopy of the oxidase in the steady state shows the expected signal from ferricytochrome a, and a new EPR signal at g = 2.01. A model of the catalytic cycle for cytochrome aa3-600 proposes initial electron delivery from DMN to cytochrome a, followed by rapid heme to heme electron transfer, and suggests possible origins of the radical signal in the steady-state form of the enzyme.

A search of the Bacillus subtilis genome identifies a potential homolog, ypmQ, of the inner mitochondrial membrane protein Sco1 from yeast. Sco1 has been found to aid the delivery of copper to cytochrome c oxidase. B. subtilis expresses two members of the cytochrome oxidase family, a cytochrome c oxidase that has two copper centers, Cu(A) and Cu(B), and a menaquinol oxidase that has only Cu(B). Deletion of ypmQ in B. subtilis depresses expression of cytochrome c oxidase but not menaquinol oxidase. Levels of cytochrome c oxidase recover when copper is added to the growth medium of the DeltaypmQ strain or when ypmQ is expressed from a plasmid. Neither treatment affects the amount or activity of menaquinol oxidase. YpmQ in which two conserved cysteines are replaced by serines and a conserved histidine is replaced by alanine do not complement the deletion of ypmQ even though these mutant forms are found in the membrane extract at a level similar to the wild type protein. We propose that the two cysteines and the histidine are critical for the function of YpmQ and suggest they are involved in copper exchange between YpmQ and the Cu(A) site of cytochrome c oxidase.

The cytochrome aa 3 (600 nm) complex, or menaquinol oxidase, from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory membrane protein complexes. We have characterized some spectral properties of this enzyme and its reaction with cyanide. The magnetic circular dichroism (MCD) spectrum of the oxidized enzyme has a single band at 1560 nm in the near-infrared region assigned to bis-histidine-ligated, low-spin ferricytochrome a. The other heme, cytochrome a 3 , is presumably high-spin in the oxidized enzyme, as isolated. The absence of a trough in the MCD spectrum at 790 nm, observed previously with mammalian cytochrome c oxidase and assigned to Cu A (Greenwood et al., Biochem. J. 215, 303–316, 1983), is consistent with the absence of this center from the menaquinol oxidase. When the heme ligand cyanide is added to oxidized menaquinol oxidase, a new MCD band appears at 2010 nm, while the band at 1560 nm is unperturbed. The new band is assigned to low-spin ferricytochrome a 3 bound with cyanide. The long-wavelength position of this cyanide-induced band is proposed to arise from the close interaction of cytochrome a 3 with the copper atom, Cu B . The kinetics of cyanide binding to oxidized cytochrome aa 3 (600 nm) reveal a spectrally simple, yet kinetically complex process. The reaction is biphasic with second-order rate constants of 45 and 0.61 M −1 s −1 at 1 mM KCN, with each phase constituting about 50% of the overall reaction. When the enzyme is subjected to a cycle of anaerobic reduction and air oxidation, the subsequent reaction with cyanide occurs in a single phase at the faster rate. This behavior is ascribed to different conformations of the binuclear center exhibiting different reactivities with cyanide, and is in keeping with that previously established for the structurally more complex mitochondrial cytochrome c oxidase. However, the electronic spectral characteristics of some of the species involved in these reactions are different in the present bacterial case from those of reported eukaryotic systems.

Cytochrome aa3-600 or menaquinol oxidase from Bacillus subtilis is a member of the heme-copper oxidase family that includes mitochondrial cytochrome c oxidase. A distinguishing feature of cytochrome aa3-600 is that it does not oxidize cytochrome c and does not contain a CuA center, but instead uses menaquinol as reducing substrate to convert O2 to water. A radical signal is observed when cytochrome aa3-600 is frozen during the course of steady-state catalysis. The nature of this radical is not fully characterized and we aim to understand it further by using the radical traps 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 2-methyl-2-nitrosopropane (MNP) and N-tert-butyl-phenylnitrone (BPN). TEMPO appears to inhibit menaquinol oxidase's steady state activity, whereas MNP and BPN are without effect. Heme-copper oxidases form a series of intermediates when exposed to H2O2 that are related to the intermediates formed in the much faster reaction with oxygen. Addition of H2O2 to oxidized cytochrome aa3-600 leads to formation of the “P-state” (606 nm), which is followed by progression to the “F-state” (580 nm). The progress of this reaction is halted at the P-state when performed in the presence of TEMPO (50 μM- 5mM). In addition if TEMPO is added at the end of the H2O2 reaction the F-state is converted back to the P-state. We propose that the inhibition of cytochrome aa3-600 by TEMPO is mediated by its ability to trap the P-state of the enzyme and slow its progress through the catalytic cycle.

Geobacter sulfurreducens was originally considered a strict anaerobe. However, this bacterium was later shown to not only tolerate exposure to oxygen but also to use it as terminal electron acceptor. Research performed has so far only revealed the general ability of G. sulfurreducens to reduce oxygen, but the oxygen uptake rate has not been quantified yet, nor has evidence been provided as to how the bacterium achieves oxygen reduction. Therefore, microaerobic growth of G. sulfurreducens was investigated here with better defined operating conditions as previously performed and a transcriptome analysis was performed to elucidate possible metabolic mechanisms important for oxygen reduction in G. sulfurreducens. The investigations revealed that cell growth with oxygen is possible to the same extent as with fumarate if the maximum specific oxygen uptake rate (sOUR) of 95 mgO2 gCDW-1 h-1 is not surpassed. Hereby, the entire amount of introduced oxygen is reduced. When oxygen concentrations are too high, cell growth is completely inhibited and there is no partial oxygen consumption. Transcriptome analysis suggests a menaquinol oxidase to be the enzyme responsible for oxygen reduction. Transcriptome analysis has further revealed three different survival strategies, depending on the oxygen concentration present. When prompted with small amounts of oxygen, G. sulfurreducens will try to escape the microaerobic area; if oxygen concentrations are higher, cells will focus on rapid and complete oxygen reduction coupled to cell growth; and ultimately cells will form protective layers if a complete reduction becomes impossible. The results presented here have important implications for understanding how G. sulfurreducens survives exposure to oxygen.

Corynebacterium glutamicum is not only industrially important but also useful as a model organism of pathogenic Gram-positive bacteria, such as C. diphtheria and Mycobacterium tuberculosis. This actinobacterium contains at least two terminal oxidases in the respiratory chain; cytochrome aa3-type cytochrome c oxidase [1] and bd-type menaquinol oxidase [2]. Thus, the chain has two branches of electron flow. The bcc-aa3 branch translocates three protons per electron transferred, while the bd branch translocates only one. Here, we constructed two mutant strains, lacking of either the cytochrome aa3 (ΔctaD) or cytochrome bd oxidase (ΔcydAB), and also plasmids for complementing the deficient genes to investigate their effects on energy conservation and cell growth [3]. The amount of cytochrome bd oxidase was very low even in the ΔctaD mutant, because the expression of the oxidase may be tightly limited with a regulation system. Therefore, we also constructed the mutant overexpressing cytochrome bd to investigate the cytochrome bd branch in more detail. First, we measured H/O ratios of wild-type and mutant cells to evaluate the efficiency of the respiratory chain. The H/O ratio of the wild-type cells grown in the semi-synthetic medium was 3.94±0.30, while the value was 2.76±0.25 for the ΔctaD mutant. In contrast, the value was 5.23±0.36 for the ΔcydAB mutant. The overexpression of cytochrome bd in the ΔctaD mutant caused further reduction of the value, 2.29±0.29 for the cytochrome bd overexpression mutant. Interestingly, the cells grown in the LB medium showed about 25% higher value compared to that of cells grown in the semi-synthetic medium except for the ΔctaD mutant. Secondly, we investigated the growth rate and cell yield with different nutrients; semi-synthetic medium containing 1% (w/v) glucose and LB medium. The ΔctaD and cytochrome bd overexpression mutants grew less than the wild-type in LB, while they grew about equally insemi-synthetic medium. In contrast, the lack of cytochrome bd oxidase did not largely affect to cell growth in both medium. These findings suggest that correlation between bioenergetics and cell growth is significantly affected by nutritional condition for the growth.