Sulfite oxidase activity in Thiobacillus novellus.

Thiobacillus novellus shows a maximum induction of sulfite oxidase activity and a maximum growth rate as a result of supplementing the autotrophic growth medium with 4.0 microM ammonium molybdate. Cells grown in the presence of molybdate showed approximately 10-fold increases in the amount of enzyme-associated molybdenum and in the sulfite-to-cytochrome c and sulfite-to-ferricyanide reductase activities. The effect of exogenous molybdate was not discernible with cells grown in the absence of thiosulfate. Tungsten inhibited the growth of T. novellus and the expression of sulfite oxidase activity.     

Enzymic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobacillus ferrooxidans.

Activities of enzymes which mediate the oxidation of thiosulfate to sulfate and the assimilation of sulfate to sulfide were assayed in various cell-free fractions of Thiobacillus ferrooxidans grown autotrophically on either ferrous iron or thiosulfate or heterotrophically on glucose. There was no activity of the thiosulfate-oxidizing enzyme in extracts of bacteria grown with ferrous iron. Comparable activities for ATP-sulfurylase (EC 2.7.7.4), ADP-sulfurylase (EC 2.7.7.5), and adenylate kinase (EC 2.7.4.3) were found in the bacteria grown autotrophically with either Fe2+ or S2O32- or heterotrophically with glucose.     

A comparative survey of several bacterial aa3-type cytochrome c oxidases

The aa3-type cytochrome c oxidases purified from Nitrobacter agilis, Thiobacillus novellus, Nitrosomonas europaea, and Pseudomonas AM 1 were compared. They have haem a and copper atom as the prosthertic groups and show alpha and gamma absorption peaks at around 600 and 440 nm, respectively. Each oxidase molecule is composed of two kinds of subunits. The N. agilis oxidase has 2 moles of haem a and 2 atoms of copper in the minimal structural unit composed of one molecule each of the two kinds of subunits, while the T. novellus enzyme seems to contain one molecule of the haem and one atom of the metal in the unit. The N. europaea oxidase shows very low affinity for carbon monoxide. Each oxidase reacts rapidly with some eukaryotic cytochromes c as well as with its native cytochrome c. The cytochrome c oxidase activity of the N. agilis oxidase is 50% inhibited by 1 microM KCN, while 50% inhibition of the activity requires 100 microM KCN in the case of the N. europaea enzyme.     

Studies on partially reduced mammalian cytochrome oxidase. Reactions with carbon monoxide and oxygen

A number of methods were used to prepare a species of mammalian cytochrome oxidase (EC 1.9.3.1, ferrocytochrome c-oxygen oxidoreductase) in which only cytochrome a(3) is reduced and in combination with CO. The kinetics of CO binding by cytochrome a(3) (2+) in this species is significantly different from that exhibited by cytochrome a(3) (2+) in the fully reduced enzyme. The second-order rate constant for combination was 5x10(4)m(-1).s(-1) and the ;off' constant was 3x10(-2)s(-1). The kinetic difference spectra cytochrome a(3) (2+)-cytochrome a(3) (2+)-CO reveal further differences between the mixed-valence and the fully reduced enzyme. The reaction between cytochrome a(3) (2+) and oxygen in the mixed-valence species was followed in flow-flash experiments and reveals a fast, oxygen-dependent (8x10(7)m(-1).s(-1) at low oxygen) rate followed by a slow process, whose rate is independent of oxygen but whose amplitude is dependent on [O(2)]. The fast oxygen-dependent reaction yields as the first product the so-called ;oxygenated' enzyme. We conclude from these experiments that the ligand-binding behaviour of cytochrome a(3) depends on the redox state of its partners, a fact which represents clear evidence for site-site interaction in this enzyme. The fact that oxygen reacts rapidly with this enzyme species in which only one component, namely cytochrome a(3), is reduced represents clear and unequivocal evidence that this is indeed the O(2)-binding site in cytochrome oxidase and may indicate that reduction of oxygen can proceed via single electron steps.     

A comparison between Nitrosomas europaea and Thiobacillus novellus on the basis of their oxidation systems of inorganic compounds.

Nitrosomonas europaea and Thiobacillus novellus were compared with each other on the basis of the biochemical properties of their inorganic compound-oxidizing systems. Cytochromes c of the two organisms differ considerably from each other; N. europaea cytochrome c-552 belongs to the "bacterial-type" cytochrome c, while T. nouellus cytochrome c-550 resembles eucaryolic cytochrome c. The specificity of cytochrome oxidase for cytochrome c as the electron donor is different between the two organisms; T novellus oxidase reacts rapidly with cytochromes c of the organisms which seem to be higher than the organisms whose cytochromes c react rapidly with N. europaea oxidase. On the basis of these facts, N. europaea seems to be older organism than T. novellus in terms of evolution.     

Oxalate, formate, formamide, and methanol metabolism in Thiobacillus novellus.

Thiobacillus novellus was able to grow with oxalate, formate, formamide, and methanol as sole sources of carbon and energy. Extensive growth on methanol required yeast extract or vitamins. Glyoxylate carboligase was detected in extracts of oxalate-grown cells. Ribulose bisphosphate carboxylase was found in extracts of cells grown on formate, formamide, and thiosulfate. These data indicate that oxalate is utilized heterotrophically in the glycerate pathway, and formate and formamide are utilized autotrophically in the ribulose bisphosphate pathway. Nicotinamide adenine dinucleotide-linked formate dehydrogenase was present in extracts of oxalate-, formate-, formamide-, and methanol-grown cells but was absent in thiosulfate- and acetate-grown cells.     

Cytochromes c of Nitrobacter winogradskyi and Thiobacillus novellus: structure, function and evolution.

The amino acid sequences of Thiobacillus novellus and Nitrobacter winogradskyi cytochromes c have been compared with those of cytochromes c from several other organisms. The two bacterial cytochromes resemble eukaryotic cytochromes c; 49 amino-acid residues are identical between T. novellus and horse cytochromes c, and 50 residues identical between N. winogradskyi and horse cytochromes c. However, their reactivity with cow cytochrome c oxidase is about 80% lower than the reactivity of eukaryotic cytochromes c with the cow mitochondrial oxidase, while they react with yeast cytochrome c peroxidase as rapidly as eukaryotic cytochromes c. The numbers of identical amino-acid residues between T. novellus and animal cytochromes c are 45-53 and those between N. winogradskyi and animal cytochromes c 47-53, while those between the two bacterial cytochromes and yeast and protozoan cytochromes c are around 40. Thus, N. winogradskyi and T. novellus cytochromes c are more similar to animal cytochromes c than to yeast and protozoan cytochromes c on the basis of the amino-acid sequence.     

Molecular aspects of the electron transfer system which participates in the oxidation of ferrous ion by Thiobacillus ferrooxidans

Abstract: The enzymes and redox proteins, which participate in the oxidation of ferrous ion by the acidophilic iron-oxidizing bacterium Thiobacillus ferrooxidans , have been isolated and characterized. They are Fe(II)-cytochrome c oxidoreductase, cytochromes c -552(s), c -552(m) and c -550(m), rusticyanin, and cytochrome c oxidase. On the basis of the interactions of these components, an electron transfer system has been proposed which seems to function in the oxidation of ferrous ion by the bacterium.     

Regulation of reductase formation in Proteus mirabilis

Summary The levels of several redox enzymes in a chlorate-resistant mutant of Proteus mirabilis, which is partially affected in the formation of formate hydrogenlyase, thiosulfate reductase and tetrathionate reductase, were compared with those of the wild type. The composition of the electron transport system of both strains was almost the same in cells grown aerobically, but very different in cells grown anaerobically. In the mutant, the cytochrome content increased twofold, whereas the level of the anaerobic enzymes is strongly diminished. The anaerobic formation of electron transport components in the mutant was, in contrast to that of the wild type, not influenced significantly by azide. During anaerobic growth with nitrate low levels of a functional nitrate reductase system were formed in the mutant. Under these conditions the formation of formate dehydrogenase, formate hydrogenlyase, formate oxidase, thiosulfate reductase, tetrathionate reductase, cytochrome b_563,5 and partly that of cytochrome a₂, was repressed. The repressive effect of nitrate, however, was completely abolished by azide. Therefore, it seems likely that a functional nitrate reductase system, rather than nitrate, controls the formation of the enzymes repressible by nitrate.     

Evidence for two pathways of thiosulfate oxidation in Starkeya novella (formerly Thiobacillus novellus)

The pathway of thiosulfate oxidation in the facultatively chemolithotrophic, sulfur-oxidizing bacterium Starkeya novella (formerly Thiobacillus novellus) has not been established beyond doubt. Recently, isolation of the sorAB genes, which encode a soluble sulfite:cytochrome c oxidoreductase, has been reported, indicating that a thiosulfate-oxidizing pathway not involving a multienzyme complex may exist in this organism. Here we report the cloning and sequencing of the soxBCD genes from S. novella, which are closely related to the corresponding genes encoding the thiosulfate-oxidizing multienzyme complex from Paracoccus pantotrophus. These findings suggest two distinct pathways for thiosulfate oxidation in S. novella. The expression of sorAB and soxC in cells grown on thiosulfate- and/or glucose-containing media was studied by Western blot analysis. The results showed that the SorAB protein is synthesized in the presence of thiosulfate irrespective of the presence of glucose. In contrast, the SoxC protein is subject to repression by glucose; the repression, however, appears to be dependent on the relative amounts of glucose and thiosulfate present. The regulatory effects observed for the expression of sorAB are likely to be mediated by an extracytoplasmic function sigma factor encoded by the sigE gene identified upstream of sorAB.     

The role of high-potential iron protein and cytochrome c(8) as alternative electron donors to the reaction center of Chromatium vinosum

Under anaerobic conditions, intact cells of the purple sulfur bacterium Chromatium vinosum exhibit rapid photooxidation of the two low-potential hemes of the c-type cytochrome associated with the reaction center, after exposure to two short light flashes separated by a dark interval. Reduction of the photooxidized low-potential hemes is very slow under these conditions. On subsequent flashes, rapid photooxidation of a high-potential reaction center heme occurs and is followed by its rereduction on the millisecond time scale. Cells maintained under aerobic conditions exhibit the millisecond time scale reduction of the photooxidized high-potential heme after each flash. Cells grown autotrophically in the presence of Na(2)S and Na(2)S(2)O(3) appear to use the soluble [4Fe-4S]-containing protein, HiPIP, as the only direct electron donor to the reaction center heme under aerobic conditions. In contrast, cells grown in the presence of organic compounds, but in the absence of Na(2)S and Na(2)S(2)O(3), appear to use a soluble c-type cytochrome (most likely cytochrome c(8)) as the only electron donor to the reaction center heme under aerobic conditions. Cells grown autotrophically, in the presence of Na(2)S and Na(2)S(2)O(3), have a slightly higher ratio of HiPIP to cytochrome c(8) and a ratio of Rieske iron-sulfur protein to reaction center that is approximately one-half that of cells grown in the absence of Na(2)S and Na(2)S(2)O(3) but in the presence of organic compounds.     

Properties of an enzymatic complex active in sulfite and thiosulfate oxidation by Rhodotorula sp.

An enzymatic complex from Rhodotorula was characterized and it was indicated that it possessed thiosulfate-oxidizing activity, forming tetrathionate as well as sulfite oxidase activity. Both activities coupled with ferricyanide and native cytochrome c but no with mammalian cytochrome c. Activities of these enzymes were inhibited by thiol inhibitors. Chelating agents did not affect thiosulfate oxidizing activity and only moderately inhibited sulfite oxidase. Both activities disappeared after treatment with proteolytic enzymes or sodium deoxycholate which indicates an essential role played not only by protein but also by phospholipids in the enzymatic activity of the complex. Thiosulfate oxidizing enzyme had a K _m for thiosulfate of 0.16 mM with ferricyanide as electron acceptor and of 14 M with native cytochrome c and of 0.34 mM for ferricyanide. Optimum pH for this activity was 7.8. Other properties of this enzyme were similar to those of thiobacilli and heterotrophic bacteria. The activity of sulfite oxidase was inhibited by 50% with 10 M AMP. The K _m values of this enzyme were 1 mM with ferricyanide as electron acceptor and 60 M with native cytochrome c for sulfite and 0.42 mM for ferricyanide. The enzyme did not show a specific optimum pH value with ferricyanide as electron acceptor. However, with native cytochrome c optimum pH was 7.8 for its activity. In many properties the sulfite oxidase from Rhodotorula was similar to the enzyme from Thiobacillus ferrooxidans, T. concretivorus, T. thioparus and T. novellus.Abbreviations CSH reduced glutathion - APS reductase, adenosine-S-phosphosulfate reductase - pHMB p-hydroxymercuribenzoate - NEM N-ethylmalcimide - TCA trichloroacetic acid - PPO 2,5-diphenyloxazole - POPOP 2,2-p-phenylen-bis 5-phenyloxazol     

Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase.

A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities indicating efficient electron transfer from FAD to heme and retention of physiological function. This work represents the first successful bacterial expression of a soluble, catalytically competent, rat hepatic cytochrome b(5)-cytochrome b(5) reductase fusion protein that retains the functional properties characteristic of the individual heme and flavin domain.     

The octahaem SirA catalyses dissimilatory sulfite reduction in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a metal reducer that uses a large number of electron acceptors including thiosulfate, polysulfide and sulfite. The enzyme required for thiosulfate and polysulfide respiration has been recently identified, but the mechanisms of sulfite reduction remained unexplored. Analysis of MR-1 cultures grown anaerobically with sulfite suggested that the dissimilatory sulfite reductase catalyses six-electron reduction of sulfite to sulfide. Reduction of sulfite required menaquinones but was independent of the intermediate electron carrier CymA. Furthermore, the terminal sulfite reductase, SirA, was identified as an octahaem c cytochrome with an atypical haem binding site. The sulfite reductase of S. oneidensis MR-1 does not appear to be a sirohaem enzyme, but represents a new class of sulfite reductases. The gene that encodes SirA is located within a 10-gene locus that is predicted to encode a component of a specialized haem lyase, a menaquinone oxidase and copper transport proteins. This locus was identified in the genomes of several Shewanella species and appears to be linked to the ability of these organisms to reduce sulfite under anaerobic conditions.     

Assembly of the mitochondrial membrane system. Characterization of nuclear mutants of Saccharomyces cerevisiae with defects in mitochondrial ATPase and respiratory enzymes.

Mutants of Saccharomyces cereviaiae showing defects in cytochrome oxidase, coenzyme QH2-cytochrome c reductase, and rutamycin-sensitive ATPase are described. The mutations have been established to be nuclear, based on complementation with a cytoplasmic petite tester strain and 2:2 segregation of tetrads. Genetic analysis indicate the coenzyme QH2-cytochrome c reductase and cytochrome oxidase mutants fall into 9 and 10 different complementation groups, respectively. The mutants also form distinct classes based on absorption spectra of the mitochondrial cytochromes. Two of the ATPase mutants lack detectable F1 ATPase, while the third synthesizes F1 but does not integrate it into a membrane complex. The latter mutant is missing one of the mitochondrially synthesized subunits of the rutamycin-sensitive ATPase complex.     

The effects of several nucleotides on the molecular state and catalytic activity of Thiobacillus novellus cytochrome c oxidase. ATP affects the oxidase uniquely.

The catalytic activity and molecular aspects of Thiobacillus novellus cytpchrome c oxidase were affected by ATP. The steady-state kinetics in the oxidation of ferrocytochrome c by the oxidase varied with the presence or absence of ATP; the [S]-v curve of the reaction was sigmoid in the absence of ATP whereas it was a Michaelis-Menten-type hyperbola in the presence of 700 microM ATP. The oxidase was a dimer of the minimal structural subunit consisting of one molecule each of two subunits in the presence of Tween 20 and in the absence of ATP. The dimer dissociated into monomers in the presence of 700 microM ATP. The trough at 452 nm seen in the second derivative absorption spectrum of the CO compound of the oxidase in the absence of ATP, a characteristic of the cytochrome a component of cytochrome aa3, dissappeared in the presence of 700 microM ATP. However, ADP, AMP, GTP, CTP and UTP had little affect on both the [S]-v curve and the molecular mass of the oxidase when used in place of ATP.     

D-Ribulose-1,5-bisphosphate carboxylase and polyhedral inclusion bodies in Thiobacillus intermedius.

The growth-related parameters of Thiobacillus intermedius, cultured in glutamate-CO2-S2O32- medium, have been determined. After centrifugation at 48,000 X g for 1 h, 24% of the D-ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity of the disrupted-cell suspensions obtained from CO2-S2O32--and glutamate-CO2-S2O3(3)- grown cells could be sedimented, and the specific activities of this enzyme in the supernatant fractions were almost equivalent. The enzyme was stable in T. intermedius starved of thiosulfate in the presence and absence of glutamate, but a progressive decrease was evident in several growth cycles, each cycle supported by resupplementation of cells with thiosulfate. Polyhedral inclusion bodies were present in CO2-S2O3(2)- and glutamate-CO2S2O3(2)- grown cells. The number of polyhedral bodies per cell increased during mixotrophic growth approximately in proportion to the observed increase in the specific activity of RuBPCase. RuBPCase could not be detected in T. intermedius grown heterotrophically on yeast extract, nor could polyhedral bodies be found.     

Intermediates of denitrification in the chemoautotroph Thiobacillus denitrificans

Summary Intact cells obtained from Thiobacillus denitrificans grown autotrophically with thiosulfate as the oxidizable substrate and nitrate as the final electron acceptor catalyzed the reduction of nitrate, nitrite and nitric oxide stoichiometrically to nitrogen gas with the concomitant oxidation of thiosulfate. In addition, nitrous oxide was also capable of acting as the terminal oxidant of the respiratory chain with thiosulfate as the reductant. The anaerobic oxidation of thiosulfate by NO₃ ^-, NO, and N₂O was sensitive to the flavoprotein inhibitors, antimycin A or NHQNO, and cyanide or azide thus, implicating the participation of flavins, and cytochromes of b-, c-, and a-types in the denitrification process. The nitrite reductase system, however, was not markedly affected by the electron transport chain inhibitors. The experimental observations suggest that the dissimilatory nitrate reduction in the chemoautotroph T. denitrificans involves nitrite, nitric oxide, and nitrous oxide as theintermediates with nitrogen gas as the final reduction product.Non-Standard Abbreviations TTFA Thenoyltrifluoroacetone - NHQNO 2-n-nonyl-4-hydroxyquinoline N-oxide     

Thiobacillus novellus cytochrome c oxidase contains one heme alpha molecule and one copper atom per catalytic unit.

The minimal structural unit of cytochrome c oxidase purified from Thiobacillus novellus was composed of one molecule each of two subunits with molecular masses of 32 and 23 kDa, respectively, and the unit had one molecule of heme a and one atom of copper. In the presence of n-octyl-beta-D-thioglucoside, the oxidase existed as the monomeric form of the unit, while it occurred as the dimeric form of the unit in the presence of Tween 20. The monomeric form showed an active cytochrome c oxidizing activity and reduced molecular oxygen to water with ferrocytochrome c. Namely, it has been shown that the bacterial cytochrome c oxidase with one heme a molecule and one copper atom per molecule can catalyze oxidation of ferrocytochrome c with concomitant reduction of molecular oxygen to water.     

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.