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PubMed1982

Cell-free synthesis of a larger-molecular-weight precursor of cytochrome c oxidase subunit V from rat liver and the distribution of its mRNA between free and membrane-bound polysomes.

Poly(A)-rich RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germs. The labeled translation products were incubated with an antiserum against cytochrome c oxidase subunit V. After immunoprecipitation and affinity chromatography with protein-A-Sepharose, the isolated antigen-immunoglobulin complexes were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. Only one protein with an apparent molecular weight of 15 500 was visualized. In immunocompetition experiments with unlabeled individual cytochrome c oxidase subunits IV, V, VI or VII only subunit V could compete with the 15 500-Mr protein synthesized in vitro. Two-dimensional fingerprints of cytochrome c oxidase subunit V and the polypeptide synthesized in vitro showed a high degree of similarity. It is concluded that the cytochrome c oxidase subunit V is synthesized as a precursor with an amino-terminal extension of about 25 amino acids. It was possible to convert the precursor of cytochrome c oxidase subunit V synthesized in vitro to its mature form by intact mitochondria as well as by submitochondrial particles. A chain length of 830 +/- 70 nucleotides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit V. Assuming a molecular weight of 15 500 for the precursor a non-coding region of about 300 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit V is found in free, loosely and tightly membrane-bound polyribosomes.

Animals↗
PubMed1983

Dicyclohexylcarbodiimide does not inhibit proton pumping by cytochrome c oxidase of Paracoccus denitrificans.

The effect of dicyclohexylcarbodiimide (DCCD) on the proton pumping two-subunit cytochrome c oxidase from Paracoccus denitrificans was investigated. Purified Paracoccus oxidase was reconstituted into phospholipid vesicles by cholate dialysis. Following incubation with increasing amounts of DCCD, proton ejection was recorded in response to reductant pulses with reduced cytochrome c. Concentrations of DCCD which greatly reduced proton pumping by bovine cytochrome c oxidase used as a control were found to exert only a minor effect on proton translocation by Paracoccus oxidase. Similarly, incubation of the bacterial enzyme with [14C]DCCD failed to reveal the specific covalent interaction previously demonstrated to occur with bovine cytochrome c oxidase, and here also shown for the oxidase of yeast. Thus, Paracoccus oxidase differs in its interaction with DCCD from the functionally analogous eukaryotic enzymes.

Animals↗
PubMed1989

Chronic stimulation of rat skeletal muscle induces coordinate increases in mitochondrial and nuclear mRNAs of cytochrome-c-oxidase subunits.

Fast-twitch tibialis anterior muscle of the rat was subjected to chronic low-frequency (10 Hz, 10 h daily) nerve stimulation in order to investigate the time course of changes in cytochrome-c-oxidase activity, as well as in tissue levels of specific mitochondrially and nuclear-encoded, cytochrome-c-oxidase-subunit mRNAs. Chronic stimulation induced a progressive increase in cytochrome-c-oxidase activity which was threefold elevated after 35 days. A similar increase was recorded for citrate-synthase activity. Glyceraldehyde-3-phosphate dehydrogenase, which was studied as a glycolytic reference enzyme, moderately decreased, as did the tissue level of its corresponding mRNA. There was a parallel increase in the tissue levels of the two cytochrome-c-oxidase-subunit mRNAs over the entire stimulation time course. The extent of increase (stimulated/control) was 2.4 +/- 0.3 and 1.8 +/- 0.2 (means +/- SEM) for the mitochondrial and nuclear subunit mRNAs, respectively. This parallel increase suggested a coordinate regulation of the two subunits. The increase in cytochrome-c-oxidase activity initially corresponded to the changes at the mRNA level. However, with longer stimulation times (beyond 14 days), the increase in cytochrome-c-oxidase activity clearly exceeded that of the two mRNAs. This divergence was progressive and was interpreted to indicate that the increase in cytochrome-c-oxidase content was brought about not only by changes in the levels of the specific mRNAs, but also by alterations at the level of translation.

Animals↗
PubMed1993

Rapid screening of cytochromes of respiratory mutants of Saccharomyces cerevisiae. Application to the selection of strains containing novel forms of cytochrome-c oxidase.

A technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates. This allows rapid semi-quantitative estimations of cytochromes c, b and oxidase and permits the identification of strains with impaired respiratory electron flow. Results of screening of 105 mutants are presented. There appears to be a correlation between the exonic location of the mutation in COX1 of oxidase and the level of optically detectable enzyme. Mutations in cytochrome b of the bc1 complex also affect the level of expression of cytochrome oxidase and can cause either an increased or decreased level of expression of oxidase relative to the wild-type strain. Twelve strains selected by the rapid level-1 screening were grown as lawns on sections of an agar plate and resuspended for a second level of screening. Quantitative estimates have been made of the concentrations of cytochromes, the turnover number of cytochrome oxidase and the kinetics of recombination of carbon monoxide with oxidase after flash photolysis. This confirmed the validity of the rapid screening procedure, and we have identified several strains which contain high levels of a mutant form of cytochrome oxidase with properties worthy of further investigation.

Cytochromes↗
PubMed1994

Large-scale purification and further characterization of rat pristanoyl-CoA oxidase.

The elution of pristanoyl-CoA oxidase from butyl-Sepharose required unusually high concentrations of ethylene glycol, enabling the large-scale purification of this oxidase in a single chromatographic step. The enzyme, the native molecular mass of which was estimated previously at 415 kDa by gel filtration (Van Veldhoven, P.P., Vanhove, G., Vanhoutte, F., Dacremont, G., Eyssen, H. J. & Mannaerts, G. P. (1991) J. Biol. Chem. 266, 24676-24683), migrated as a 513-kDa protein during native gel electrophoresis. It showed a typical flavoprotein spectrum and probably binds 4 mol FAD/mol enzyme. Its amino acid composition was different from those of other known acyl-CoA oxidases. Screening of different rat tissues, either for enzyme activity or by immunoblotting, revealed the highest level of pristanoyl-CoA oxidase in liver, followed by kidney, intestinal mucosa, spleen and lung. The oxidase activities, measured with 2-methylpalmitoyl-CoA as the substrate, in livers from other vertebrates including man were low compared to rat. This was also confirmed by immunoblotting which provided a clear signal only in rat liver, possibly indicating that pristanoyl-CoA oxidase might be a rat-specific oxidase.

Amino Acids↗
PubMed1994

A second terminal oxidase in Sulfolobus acidocaldarius.

We previously found that the soxABCD operon encodes a quinol oxidase complex in Sulfolobus acidocaldarius and this enzyme was purified and characterized. In this study, we have used a cloning procedure based on the conservation of oxidase sequences and the polymerase chain reaction to isolate a new gene (soxM) encoding a subunit of another terminal oxidase. This terminal oxidase is a fusion between two central components of cytochrome oxidases, subunits I and III. soxM forms a transcriptional unit which is expressed under heterotrophic growth conditions. The corresponding protein was detected by direct protein sequencing in a preparation enriched with a cytochrome absorbing light at 562 nm. This preparation contains a terminal oxidase which is able to oxidize the artificial substrate N,N,N',N'-tetramethyl-p-phenylenediamine. This preparation also contains SoxC, a protein homologous to the mitochondrial cytochrome b, and a Rieske iron-sulphur center. We suggest that SoxM is the core component of a second terminal oxidase complex and that this complex may share a subunit (SoxC) with the SoxABCD complex.

Amino Acid Sequence↗
PubMed1995

Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum. Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols.

The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed. Vanillyl-alcohol oxidase weakly interacts with 4-hydroxyphenylglycols and a series of catecholamines. These compounds are converted to the corresponding ketones. Both enantiomers of (nor)epinephrine are substrates for vanillyl-alcohol oxidase, but the R isomer is preferred. Vanillyl-alcohol oxidase is most active with chavicol and eugenol. These 4-allylphenols are converted to coumaryl alcohol and coniferyl alcohol, respectively. Isotopic labeling experiments show that the oxygen atom inserted at the C gamma atom of the side chain is derived from water. The 4-hydroxycinnamyl alcohol products and the substrate analog isoeugenol are competitive inhibitors of vanillyl alcohol oxidation. The binding of isoeugenol to the oxidized enzyme perturbs the optical spectrum of protein-bound FAD. pH-dependent binding studies suggest that vanillyl-alcohol oxidase preferentially binds the phenolate form of isoeugenol (pKa < 6, 25 degrees C). From this and the high pH optimum for turnover, a hydride transfer mechanism involving a p-quinone methide intermediate is proposed for the vanillyl-alcohol-oxidase-catalyzed conversion of 4-allylphenols.

1-Propanol↗
PubMed1996

Rat pristanoyl-CoA oxidase. cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor.

The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.

Amino Acid Sequence↗
PubMed1996

L-aspartate oxidase from Escherichia coli. II. Interaction with C4 dicarboxylic acids and identification of a novel L-aspartate: fumarate oxidoreductase activity.

L-Aspartate oxidase is a monomeric flavoprotein that catalyzes the first step in the de novo biosynthetic pathway for pyridine nucleotide formation under both aerobic and anaerobic conditions. In spite of the physiological importance of this biosynthesis in particular in facultative aerobic organisms, such as Escherichia coli, little is known about the electron acceptor of reduced L-aspartate oxidase in the absence of oxygen. In this report, evidence is presented which suggests that in vitro fumarate can play such a role. L-Aspartate oxidase binds succinate and fumarate with Kd values of 0.24 mM and 0.22 mM, respectively. A competitive behaviour was observed for these two dicarboxylic acids towards iminoaspartate and sulfite ions. Photoreduction experiments suggest that fumarate and succinate bind at or close to the active site of the molecule. A new fumarate reductase activity of L-aspartate oxidase is reported using benzylviologen or L-aspartate as reductants and fumarate as oxidant. Steady-state kinetics for the oxidase and the fumarate reductase activity of L-aspartate oxidase were obtained using either fumarate or oxygen as electron acceptor and L-aspartate as electron donor. Finally, succinate was identified as the product of the L-aspartate:fumarate oxidoreductase activity using radiolabeled fumarate under anaerobic conditions. The results suggest that fumarate can be a valuable alternative to oxygen as a substrate for L-aspartate oxidase.

Amino Acid Oxidoreductases↗
PubMed1999

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions.

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.

Aerobiosis↗
PubMed1972

Chemical and physical characteristics of the deoxycholate-soluble and magnesium-reaggregated membrane nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium.

The inactive components of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase present in the 0.4% deoxycholate-soluble fraction obtained from Bacillus megaterium KM membranes were reaggregated into active NADH oxidase by dilution in the presence of Mg(2+). The reaggregated oxidase was different from the original membrane with respect to sedimentation behavior in a sucrose gradient and morphological appearance. The deoxycholate-insoluble portion of the membrane had membrane-like structure whereas the reaggregated oxidase appeared to be a filamentous aggregate of small particles. The reaggregated oxidase and the deoxycholate-insoluble membrane residue were similar to the original membrane with respect to total protein and total lipid content. The inactive components of the NADH oxidase system exist in deoxycholate as two molecular species which were separable by sucrose density gradient centrifugation or gel filtration in deoxycholate-containing solutions. Both components and dilution in the presence of Mg(2+) were necessary for restoration of oxidase activity. The smaller-molecular-weight component contained all of the NADH-2,6-dichlorophenolindophenol oxidoreductase activity of the original membrane.

Bacillus megaterium↗
PubMed1976

Regulation of tyramine oxidase synthesis in Klebsiella aerogenes.

Tyramine oxidase in Klebsiella aerogenes is highly specific for tyramine, dopamine, octopamine, and norepinephrine, and its synthesis is induced specifically by these compounds. The enzyme is present in a membrane-bound form. The Km value for tyramine is 9 X 10(-4) M. Tyramine oxidase synthesis was subjected to catabolite repression by glucose in the presence of ammonium salts. Addition of cyclic adenosine 3',5'-monophosphate (cAMP) overcame the catabolite repression. A mutant strain, K711, which can produce a high level of beta-galactosidase in the presence of glucose and ammonium chloride, can also synthesize tyramine oxidase and histidase in the presence of inducer in glucose ammonium medium. Catabolite repression of tyramine oxidase synthesis was relieved when the cells were grown under conditions of nitrogen limitation, whereas beta-galactosidase was strongly repressed under these conditions. A cAMP-requiring mutant, MK54, synthesized tyramine oxidase rapidly when tyramine was used as the sole source of nitrogen in the absence of cAMP. However, a glutamine synthetase-constitutive mutant, MK94, failed to synthesize tyramine oxidase in the presence of glucose and ammonium chloride, although it synthesized histidase rapidly under these conditions. These results suggest that catabolite repression of tyramine oxidase synthesis in K. aerogenes is regulated by the intracellular level of cAMP and an unknown cytoplasmic factor that acts independently of cAMP and is formed under conditions of nitrogen limitation.

Amino Acid Oxidoreductases↗
PubMed1991

Cloning, characterization, and expression in Escherichia coli of the genes encoding the cytochrome d oxidase complex from Azotobacter vinelandii.

Azotobacter vinelandii is a free-living nitrogen-fixing bacterium that has one of the highest respiratory rates of all aerobic organisms. Based on various physiological studies, a d-type cytochrome has been postulated to be the terminal oxidase of a vigorously respiring but apparently uncoupled branch of the electron transport system in the membranes of this organism. We cloned and characterized the structural genes of the two subunits of this oxidase. The deduced amino acid sequences of both subunits of the A. vinelandii oxidase have extensive regions of homology with those of the two subunits of the Escherichia coli cytochrome d complex. Most notably, the histidine residues proposed to be the axial ligands for the b hemes of the E. coli oxidase and an 11-amino-acid stretch proposed to be part of the ubiquinone binding site are all conserved in subunit I of the A. vinelandii oxidase. The A. vinelandii cytochrome d was expressed in a spectrally and functionally active form in the membranes of E. coli, under the control of the lac or tac promoter. The spectral features of the A. vinelandii cytochrome d expressed in E. coli are very similar to those of the E. coli cytochrome d. The expressed oxidase was active as a quinol oxidase and could reconstitute an NADH to oxygen electron transport chain.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of NADH oxidase from Serpulina (Treponema) hyodysenteriae.

NADH oxidase (EC 1.6.99.3) was purified from cell lysates of Serpulina (Treponema) hyodysenteriae B204 by differential ultracentrifugation, ammonium sulfate precipitation, and chromatography on anion-exchange, dye-ligand-affinity, and size-exclusion columns. Purified NADH oxidase had a specific activity 119-fold higher than that of cell lysates and migrated as a single band during denaturing gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]). The enzyme was a monomeric protein with an estimated molecular mass of 47 to 48 kDa, as determined by SDS-PAGE and size-exclusion chromatography. Optimum enzyme activity occurred in buffers with a pH between 5.5 and 7.0. In the presence of oxygen, beta-NADH but not alpha-NADH, alpha-NADPH, or beta-NADPH was rapidly oxidized by the enzyme (Km = 10 microM beta-NADH; Vmax = 110 mumol beta-NADH min-1 mg of protein-1). Oxygen was the only identified electron acceptor for the enzyme. On isoelectric focusing gels, the enzyme separated into three subforms, with isoelectric pH values of 5.25, 5.35, and 5.45. Purified NADH oxidase had a typical flavoprotein absorption spectrum, with peak absorbances at wavelengths of 274, 376, and 448 nm. Flavin adenine dinucleotide was identified as a cofactor and was noncovalently associated with the enzyme at a molar ratio of 1:1. Assays of the enzyme after various chemical treatments indicated that a flavin cofactor and a sulfhydryl group(s), but not a metal cofactor, were essential for activity. Hydrogen peroxide and superoxide were not yielded in significant amounts by the S. hyodysenteriae NADH oxidase, indirect evidence that the enzyme produces water from reduction of oxygen with NADH. The N-terminal amino acid sequence of the NADH oxidase was determined to be MKVIVIGCHGAGTWAAK. In its biochemical properties, the NADH oxidase of S. hyodysenteriae resembles the NADH oxidase of another intestinal bacterium, Enterococcus faecalis.

Amino Acid Sequence↗
PubMed1993

Characterization of a cytochrome a1 that functions as a ubiquinol oxidase in Acetobacter aceti.

The terminal oxidase for ethanol oxidation in Acetobacter aceti was purified as a complex consisting of four subunits (subunits I, II, III, and IV) with molecular masses of 72, 34, 21, and 13 kDa, respectively. Spectrophotometric analysis and catalytic properties determined with the purified enzyme showed that it belonged to a family of cytochrome a1 (ba)-type ubiquinol oxidases. A polymerase chain reaction with two oligonucleotides designed for amino acid sequences that are conserved in subunit I of the aa3-type cytochrome c oxidases from various origins and of an Escherichia coli o (bo)-type ubiquinol oxidase was used for cloning the cytochrome a1 gene. A 0.5-kb fragment thus amplified was used as the probe to clone a 4.5-kb KpnI fragment that contained a putative open reading frame for the whole subunit I gene. The molecular weight and amino acid composition of the product of this open reading frame (cyaA) were the same as those of the purified protein from A. aceti. The amino acid sequence of CyaA was homologous to that of subunit I of the E. coli o-type ubiquinol oxidase. Nucleotide sequence analysis of the region neighboring the cyaA gene revealed that the genes (cyaB, cyaC, and cyaD) encoding the other three subunits (subunits II, III, and IV) were clustered upstream and downstream of the cyaA gene in the order cyaB, cyaA, cyaC, and cyaD and with the same transcription polarity, forming an operon. As expected from the enzymatic properties, CyaB, CyaC, and CyaD showed great similarity in amino acid sequence to the corresponding sununits of the E. coli o-type ubiquinol oxidase and as(3)-type cytochrome c oxidases.

Acetobacter↗
PubMed1993

Cloning, sequencing, expression, and regulation of the structural gene for the copper/topa quinone-containing methylamine oxidase from Arthrobacter strain P1, a gram-positive facultative methylotroph.

Deoxyoligonucleotides corresponding to amino acid sequences of methylamine oxidase and polyclonal anti-methylamine oxidase antibodies were used to probe Arthrobacter strain P1 plasmid and chromosomal DNA libraries. Two open reading frames, maoxI and maoxII, which are greater than 99% homologous, were cloned from the chromosomal library. The deduced amino acid sequences of the coding regions are identical except for two residues near the C termini. On the other hand, the 5'- and 3'-flanking regions of maoxI and maoxII are quite different. While either gene could code for methylamine oxidase, the dissimilarity in the 5'-flanking regions indicates that the genes are differently regulated. It was determined that maoxII alone encodes methylamine oxidase. The tyrosyl residue which is converted to topa quinone in the mature enzyme was located by comparison with amino acid sequences at the cofactor sites in other copper/topa quinone-containing amine oxidase. Transcriptional start sites and possible regulatory elements were identified in the 5' region of maoxI and maoxII, and stem-loop structures were found in the 3'-flanking regions. High levels of methylamine oxidase are produced when Arthrobacter strain P1 is grown on methylamine alone or on glucose plus methylamine, but growth on LB medium plus methylamine resulted in very low production of the enzyme. Expression of maoxII from its own promoter in Escherichia coli grown on glucose or LB medium with or without methylamine gave the same level of production of methylamine oxidase.

Amino Acid Sequence↗
PubMed2001

Mobile cytochrome c2 and membrane-anchored cytochrome cy are both efficient electron donors to the cbb3- and aa3-type cytochrome c oxidases during respiratory growth of Rhodobacter sphaeroides.

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.

Cell Membrane↗
PubMed2004

Hydrogen peroxide production in Streptococcus pyogenes: involvement of lactate oxidase and coupling with aerobic utilization of lactate.

Streptococcus pyogenes strains can be divided into two classes, one capable and the other incapable of producing H2O2 (M. Saito, S. Ohga, M. Endoh, H. Nakayama, Y. Mizunoe, T. Hara, and S. Yoshida, Microbiology 147:2469-2477, 2001). In the present study, this dichotomy was shown to parallel the presence or absence of H2O2-producing lactate oxidase activity in permeabilized cells. Both lactate oxidase activity and H2O2 production under aerobic conditions were detectable only after glucose in the medium was exhausted. Thus, the glucose-repressible lactate oxidase is likely responsible for H2O2 production in S. pyogenes. Of the other two potential H2O2-producing enzymes of this bacterium, NADH and alpha-glycerophosphate oxidase, only the former exhibited low but significant activity in either class of strains. This activity was independent of the growth phase, suggesting that the protein may serve in vivo as a subunit of the H2O2-scavenging enzyme NAD(P)H-linked alkylhydroperoxide reductase. The activity of lactate oxidase was associated with the membrane while that of NADH oxidase was in the soluble fraction, findings consistent with their respective physiological roles, i.e., the production and scavenging of H2O2. Analyses of fermentation end products revealed that the concentration of lactate initially increased with time and decreased on glucose exhaustion, while that of acetate increased during the culture. These results suggest that the lactate oxidase activity of H2O2-producing cells oxidizes lactate to pyruvate, which is in turn converted to acetate. This latter process proceeds presumably via acetyl coenzyme A and acetyl phosphate with formation of extra ATP.

Aerobiosis↗