Purification and characterization of the cytochrome oxidase from alkalophilic Bacillus firmus RAB

A cytochrome oxidase was purified 52-fold from membranes of alkalophilic Bacillus firmus RAB by extraction with Triton X-100, ion-exchange and hydroxyapatite chromatography, and gel filtration. On denaturing gels, the purified enzyme dissociated into two subunits of 56,000 and 40,000 Mr as well as a cytochrome c with an Mr of approximately 14,000. Heme contents calculated for an enzyme with a molecular weight of 110,000 were found to be 2 mol of heme a and 1 mol of heme c per mol of cytochrome oxidase; approximately 2 mol of copper per mol of purified enzyme was also found. Enzyme activity was observed in assays using reduced yeast or horse heart cytochrome c. Activity of the purified enzyme was optimal at pH 6.0 and in the presence of added lipids. Impure, membrane-associated activity exhibited a broader pH range for optimal activity extending to alkaline values.     

Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans

A new type of sulfite oxidase which utilizes ferric ion (Fe3+) as an electron acceptor was found in iron-grown Thiobacillus ferrooxidans. It was localized in the plasma membrane of the bacterium and had a pH optimum at 6.0. Under aerobic conditions, 1 mol of sulfite was oxidized by the enzyme to produce 1 mol of sulfate. Under anaerobic conditions in the presence of Fe3+, sulfite was oxidized by the enzyme as rapidly as it was under aerobic conditions. In the presence of o-phenanthroline or a chelator for Fe2+, the production of Fe2+ was observed during sulfite oxidation by this enzyme under not only anaerobic conditions but also aerobic conditions. No Fe2+ production was observed in the absence of o-phenanthroline, suggesting that the Fe2+ produced was rapidly reoxidized by molecular oxygen. Neither cytochrome c nor ferricyanide, both of which are electron acceptors for other sulfite oxidases, served as an electron acceptor for the sulfite oxidase of T. ferrooxidans. The enzyme was strongly inhibited by chelating agents for Fe3+. The physiological role of sulfite oxidase in sulfur oxidation of T. ferrooxidans is discussed.     

Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans.

A new type of sulfite oxidase which utilizes ferric ion (Fe3+) as an electron acceptor was found in iron-grown Thiobacillus ferrooxidans. It was localized in the plasma membrane of the bacterium and had a pH optimum at 6.0. Under aerobic conditions, 1 mol of sulfite was oxidized by the enzyme to produce 1 mol of sulfate. Under anaerobic conditions in the presence of Fe3+, sulfite was oxidized by the enzyme as rapidly as it was under aerobic conditions. In the presence of o-phenanthroline or a chelator for Fe2+, the production of Fe2+ was observed during sulfite oxidation by this enzyme under not only anaerobic conditions but also aerobic conditions. No Fe2+ production was observed in the absence of o-phenanthroline, suggesting that the Fe2+ produced was rapidly reoxidized by molecular oxygen. Neither cytochrome c nor ferricyanide, both of which are electron acceptors for other sulfite oxidases, served as an electron acceptor for the sulfite oxidase of T. ferrooxidans. The enzyme was strongly inhibited by chelating agents for Fe3+. The physiological role of sulfite oxidase in sulfur oxidation of T. ferrooxidans is discussed.     

Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata.

After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity.     

On the binding of mammalian cytochrome c in NADH- and succinate-cytochrome c-reductase from Rhodospirillum rubrum

Summary The effect of a number of salts on the activity of the particulate NADH-cytochrome c-reductase of Rhodospirillum rubrum was investigated. The enzyme was shown to be inhibited by the presence of these salts. With monovalent anions a relationship between the size of the anion and its capacity of inhibition is observed. Di- and trivalent anions inhibit more than do monovalent anions. Di- and trivalent cations cause very strong inhibition of the enzymatic activity. With all ions a relationship of the competitive type exists between cytochrome c and the ion tested.Results identical to those described are obtained when the oxidation of succinate is measured with cytochrome c as the electron acceptor.With these inibition experiments it was shown that a complex between mammalian cytochrome c and the phospholipids of the electron transport particles of R. rubrum is likely to be formed and that this complex rather than cytochrome c itself is the electron acceptor for NADH- and succinate-cytochrome c-reductase.

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Zusammenfassung Die Wirkung einer Reihe von Salzen auf die Aktivität der partikelgebundenen NADH-Cytochrom c-Reduktase aus Rhodospirillum rubrum wurde untersucht. Das Enzym wird durch die verschiedenen Salze unterschiedlich stark gehemmt. Bei einwertigen Anionen ist ein Zusammenhang zwischen dem Ionenradius und der Stärke der Hemmung zu erkennen. Zwei- und dreiwertige Anionen hemmen das Enzym mehr als einwertige. Zwei- und dreiwertige Kationen sind vergleichsweise sehr starke Inhibitoren. Bei allen untersuchten Ionen besteht eine Beziehung kompetitiver Art zwischen Cytochrom c und dem betreffenden Ion.Völlig analoge Ergebnisse werden bei der Bestimmung der Oxydation von Succinat mit Cytochrom c als Elektronenacceptor erhalten.Mit diesen Hemmungsexperimenten kann die Bildung eines Komplexes zwischen Cytochrom c aus Herzmuskel und den Phospholipiden der Elektronentransportpartikel von R. rubrum wahrscheinlich gemacht werden. Daraus ist zu folgern, daß der Komplex und nicht Cytochrom c allein der wirksame Elektronenacceptor für NADH- und Succinat-Cytochrom c-Reduktase ist.

     

The oxidation of cytochrome c oxidase by hydrogen peroxide

The reaction of H2O2 with mixed-valence and fully reduced cytochrome c oxidase was investigated by photolysis of fully reduced and mixed-valence carboxy-cytochrome c oxidase in the presence of H2O2 under anaerobic conditions. The results showed that H2O2 reacted rapidly (k = (2.5-3.1) X 10(4) M-1 X s-1) with both enzyme species. With the mixed-valence enzyme, the fully oxidised enzyme was reformed. On the time-scale of our experiments, no spectroscopically detectable intermediate was observed. This demonstrates that mixed-valence cytochrome c oxidase is able to use H2O2 as a two-electron acceptor, suggesting that cytochrome c oxidase may under suitable conditions act as a peroxidase. Upon reaction of H2O2 with the fully reduced enzyme, cytochrome a was oxidised before cytochrome a3. From this observation it was possible to estimate that the rate of electron transfer from cytochrome a to a3 is about 0.5-5 s-1.     

Apparent oxygen-dependent inhibition by superoxide dismutase of the quinoprotein methanol dehydrogenase.

Methanol dehydrogenase activity, when assayed with phenazine ethosulfate (PES) as an electron acceptor, was inhibited by superoxide dismutase (SOD) and by Mn2+ only under aerobic conditions. Catalase, formate, and other divalent cations did not inhibit the enzyme. The enzyme also exhibited significantly higher levels of activity when assayed with PES under anaerobic conditions relative to aerobic conditions. The oxygen- and superoxide-dependent effects on methanol dehydrogenase were not observed when either Wurster's Blue or cytochrome c-55li was used as an electron acceptor. Another quinoprotein, methylamine dehydrogenase, which possesses tryptophan tryptophylquinone (TTQ) rather than pyrroloquinoline quinone (PQQ) as a prosthetic group, was not inhibited by SOD or Mn2+ when assayed with PES as an electron acceptor. Spectroscopic analysis of methanol dehydrogenase provided no evidence for any oxygen- or superoxide-dependent changes in the redox state of the enzyme-bound PQQ cofactor of methanol dehydrogenase. To explain these data, a model is presented in which this cofactor reacts reversibly with oxygen and superoxide, and in which oxygen is able to compete with PES as an electron acceptor for the reduced species.     

Binding of arylazidocytochrome c to yeast cytochrome c peroxidase

Cytochrome c derivatives modified with a photoactivatable arylazido group in selected lysine residues were irradiated in the presence of cytochrome c peroxidase (EC 1.11.1.5). A derivative modified at lysine 13 was able to cross-link to the enzyme and inhibit electron transfer activity. Complete inhibition of cytochrome c peroxidase activity was obtained when 1 mol of cytochrome c was covalently bound per mol of cytochrome c peroxidase. Chemical cleavage of the covalent complex has been used for a preliminary characterization of the site of cross-linking of cytochrome c to cytochrome c peroxidase. This linkage site was localized to the NH2 terminal part of cytochrome c peroxidase including residues 1-51.     

Characteristics and amino-acid composition of a c-type cytochrome in electron acceptor function during thiosulfate-linked photoautotrophic growth of Rhodopseudomonas palustris

The purple bacterium Rhodopseudomonas palustris (Rhodospirillaceae) was grown in the light with thiosulfatee as the only electron source and HCO theta 3/CO2 as carbon requirement. During thiosulfat oxidation, photolithoautotrophically growing cells transferred the electrons enzymatically towards an endogenous, soluble cytochrome of type c. The cytochrome c in electron acceptor function was purified to homogeneity and appeared as a single protein band in a dodecyl sulfate disc gel electrophoresis. Its molecular mass was determined to be about 16000 Da and its pI value 10.0. The determination of its amino-acid composition revealed a long-chained cytochrome represented by more than 120 amino-acid residues with a characteristic content of lysine and a lack of tryptophan.     

Interaction of cytochrome c with mitochondrial proteins and cybacrone-dextran

Interaction of cytochrome c with electron carriers in intact and damaged (with destroyed outer membrane) rat liver mitochondria was studied. It was shown that the increase in ionic strength causes changes in the respiration rate of damaged mitochondria due to the reduction of the cytochrome c affinity for its binding sites in the organelles. This suggests that cytochrome c concentration in the intermembrane space of intact mitochondria is increased by salts, whereas the increase in ionic strength has a slight influence on the rates of succinate oxidase and external rotenone-insensitive NADH-oxidase of intact mitochondria. At low ionic strength values, the Michaelis constant (KM) value of external NADH-oxidase for cytochrome c exceeds by one order of magnitude that for succinate oxidase, while the maximal activity of these two systems is nearly the same. The increase in ionic strength causes an increase in the KM value for both oxidases. Interaction of cytochrome c with mitochondrial proteins was modelled by cytochrome c interaction with cibacron-dextran anions. It was concluded that the ionic strength-sensitive electrostatic interactions play a decisive role in cytochrome c binding to electron carriers in mitochondrial membranes. However, cytochrome c content and its binding parameters in intact-mitochondrial membranes prevent the latent activity of external NADH oxidase to be revealed in intact mitochondria after the increase in the ionic strength of the surrounding medium.     

Occurrence and comparison of sulfite oxidase activity in mammalian tissues

Tissue extracts from six mammalian species have been assayed for sulfite oxidase (sulfite: ferricytochrome c oxidoreductase, EC 1.8.3.1) activity with cytochrome c as electron acceptor. Our results show a large distribution of sulfite oxidase activity in mammalian tissues. Liver, kidney, and heart tissues exhibit high activities whereas brain, spleen, and testis show very low activities. No significant species dependence was observed for the activity of this enzyme.     

The haem-copper oxygen reductase of Desulfovibrio vulgaris contains a dihaem cytochrome c in subunit II

The genome of the sulphate reducing bacterium Desulfovibrio vulgaris Hildenborough, still considered a strict anaerobe, encodes two oxygen reductases of the bd and haem-copper types. The haem-copper oxygen reductase deduced amino acid sequence reveals that it is a Type A2 enzyme, which in its subunit II contains two c-type haem binding motifs. We have characterized the cytochrome c domain of subunit II and confirmed the binding of two haem groups, both with Met-His iron coordination. Hence, this enzyme constitutes the first example of a ccaa3 haem-copper oxygen reductase. The expression of D. vulgaris haem-copper oxygen reductase was found to be independent of the electron donor and acceptor source and is not altered by stress factors such as oxygen exposure, nitrite, nitrate, and iron; therefore the haem-copper oxygen reductase seems to be constitutive. The KCN sensitive oxygen reduction by D. vulgaris membranes demonstrated in this work indicates the presence of an active haem-copper oxygen reductase. D. vulgaris membranes perform oxygen reduction when accepting electrons from the monohaem cytochrome c553, thus revealing the first possible electron donor to the terminal oxygen reductase of D. vulgaris. The physiological implication of the presence of the oxygen reductase in this organism is discussed.     

Preparation and kinetic studies of immobilised yeast cytochrome c peroxidase.

Yeast cytochrome c peroxidase (CcP) was purified from baker's yeast and immobilised onto a nylon membrane. The kinetics of the soluble and immobilised forms of the enzyme were investigated for the catalysed oxidation of potassium ferrocyanide in the presence of H2O2 and m-chloroperoxybenzoic acid. The pH dependence of the two forms of the enzyme differed. Although both the soluble and the immobilised enzymes showed optimal activity at pH 6.2, a different kinetic behaviour was demonstrated. Both forms of the enzyme showed similar activity toward H2O2, although when m-chloroperoxybenzoic acid was replaced as the electron acceptor, the immobilised form of the enzyme had a reduced turnover number and an increased Km. The activation energy of immobilised CcP was greater in the presence of both H2O2 [16.6 kJ mol-1] and m-chloroperoxybenzoic acid [37.9 kJ mol-1] than for soluble CcP [11.4 and 23.4 kJ mol-1, respectively]. The activities of both soluble and immobilised CcP were greatly reduced above 45 degrees C, although at higher temperatures the immobilised enzyme retained a relatively greater percentage of its maximum activity.     

Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni.

Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name 'quinohaemoprotein alcohol dehydrogenase'. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.     

Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans.

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.     

Dependence on membrane components of methanogenesis from methyl-CoM with formaldehyde or molecular hydrogen as electron donors

Methane formation from 2-(methylthio)-ethanesulfonate (methyl-CoM) and H2 by the soluble fraction from the methanogenic bacterium strain G?1 was stimulated up to tenfold by the addition of the membrane fraction. This stimulation was observed with membranes from various methanogenic species belonging to different phylogenetic families, but not with membranes from Escherichia coli or Acetobacterium woodii. Treatment of the membranes with strong oxidants, i.e. O2 and K3[Fe(CN)6], or with SH reagents, i.e. Ag+, p-chloromercuribenzoate or iodoacetamide, caused an irreversible decrease or loss in stimulatory activity, as did heat treatment at temperatures above 78 degrees C. Methanogenesis from methyl-CoM with formaldehyde instead of H2 as electron donor depended similarly on the membrane fraction. With membranes, 1 mol HCHO was oxidized to 1 mol CO2 and allowed the formation of 2 mol CH4 from 2 mol CH3-CoM. Without membranes, per mol of HCHO oxidized 1 mol H2 was formed and 1 mol CH4 was produced from CH3-CoM; the rate was 10-20% of that in the presence of membranes. When methyl-CoM was replaced by an artificial electron acceptor system consisting of methylviologen and metronidazole, the formaldehyde-oxidizing activity was no longer stimulated by the membrane fraction. These results demonstrate for the first time an essential function of membrane components in methanogenic electron transfer.     

Implications of the integrated rate law for the reactions of Paracoccus denitrificans nitrite reductase.

The integrated rate law for the reaction of the nitrite reductase of Paracoccus denitrificans, a cytochrome cd, has been established for turnover assays using donor ferrocytochromes c and either nitrite or molecular oxygen as the ultimate acceptor. The time course for the concentration of ferrocytochrome follows the law: formula: (see text), where S is the concentration of donor ferrocytochrome c, So is the initial concentration, t is time, and u1, u2, and u3 are empirical parameters that are constant for a given experiment but depend upon the initial substrate concentration. In particular, all the u1 increase with decreasing initial ferrocytochrome concentration. Saturation of reaction rates at high donor ferrocytochrome concentrations was not observed. The parameter u1 was proportional to the enzyme concentration while u2 and u3 were not. The form of the integrated rate law and the behavior of the u1 impose severe restrictions on possible kinetic schemes for the activity of the enzyme. Contemporary mechanisms that have been proposed for mitochondrial oxidase aa3 are examined and found to be inadequate to explain the reactivity of cytochrome cd. The simplest interpretations of the cytochrome cd data suggest that the enzyme does not bind the ferri and ferro forms of donor cytochromes c with equal affinity and that the enzyme is subject to inhibition by a product of reaction. Eucaryotic horse cytochrome c reacts with the Paracoccus cytochrome cd with 77% of the activity when Paracoccus cytochrome c550 is used as the electron donor.     

Characterization of the Membrane-Bound Succinic Dehydrogenase of Micrococcus lysodeikticus

The occurrence of succinic dehydrogenase [succinic:(acceptor) oxidoreductase, EC 1.3.99.1] in membrane fractions of Micrococcus lysodeikticus was investigated. The enzyme could be purified 10-fold, by deoxycholate treatment. Butanol extraction of membranes yielded an active fraction, nonsedimentable at 130,000 x g for 2 hr and altered in its phospholipid content relative to membranes. The activity of the enzyme in particulate preparations was decreased in the presence of competitive inhibitors and by compounds known to react with iron, sulfhydryl groups, and flavine. In this respect, the bacterial succinic dehydrogenase is similar to the enzyme derived from yeast and mammalian sources. In certain membrane fractions, Ca(2+) and Mg(2+) exhibited inhibitory effects whereas Triton X-100 caused activation. The enzyme could also be activated by substrate. In the phenazine reductase assay, incomplete reduction of electron acceptor was observed upon addition of divalent cations and iron binding agents.     

Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing alpha beta dimer

Azotobacter vinelandii hydrogenase has been purified to homogeneity from membranes. The enzyme was solubilized with Triton X-100 followed by ammonium sulfate-hexane extractions to remove lipids and detergent. The enzyme was then purified by carboxymethyl-Sepharose and octyl-Sepharose column chromatography. All purification steps were performed under anaerobic conditions in the presence of dithionite and dithiothreitol. The enzyme was purified 143-fold from membranes to a specific activity of 124 mumol of H2 uptake . min-1 . mg protein-1. Nondenaturing polyacrylamide gel electrophoresis of the hydrogenase revealed a single band which stained for both activity and protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to peptides of 67,000 and 31,000 daltons. Densitometric scans of the SDS-gel indicated a molar ratio of the two bands of 1.07 +/- 0.05. The molecular weight of the native enzyme was determined by three different methods. While gel permeation gave a molecular weight of 53,000, sucrose density gradient centrifugation and native polyacrylamide gel electrophoresis gave molecular weights of 98,600 +/- 10,000 and 98,600 +/- 2,000, respectively. We conclude that the A. vinelandii hydrogenase is an alpha beta dimer (98,000 daltons) with subunits of 67,000 and 31,000 daltons. Analyses for nickel and iron indicated 0.68 +/- 0.01 mol Ni/mol hydrogenase and 6.6 +/- 0.5 mol Fe/mol hydrogenase. The isoelectric point of the enzyme was 6.1 +/- 0.01. In addition, several catalytic properties of the enzyme have been examined. The Km for H2 was 0.86 microM, and H2 evolution was observed in the presence of reduced methyl viologen. The pH profile of enzyme activity with methylene blue as the electron acceptor has been determined, along with the Km and Vmax for various electron acceptors.     

Methylene blue competes with paraquat for reduction by flavo-enzymes resulting in decreased superoxide production in the presence of heme proteins

Methylene blue competes 100 to 600 times more effectively than paraquat for reduction by three different flavo-containing enzymes; xanthine oxidase, NADH cytochrome c reductase, and NADPH cytochrome c reductase. Paraquat and methylene blue both interact with deflavo xanthine oxidase, indicating that neither electron acceptor reacted at the FAD site of the enzyme where molecular oxygen is reduced to superoxide. As the paraquat radical also directly reduced acetylated cytochrome c the hemeprotein could not be utilized for measuring superoxide production in the presence of the herbicide. In the presence of cytochrome c the methylene blue caused a sharp decrease in both paraquat-induced superoxide and hydroxyl radical production.