Fasciola hepatica: cytochrome c oxidoreductases and effects of oxygen tension and inhibitors

Studies were made on the mechanism of respiration in Fasciola hepatica (Trematoda). Respiration was found to be dependent on the oxygen tension. The respiratory enzyme systems, NADH-cytochrome c oxidoreductase (EC 1.6.2.1), succinate-cytochrome c oxidoreductase (EC 1.3.99.1) NADH oxidase and cytochrome c-oxygen oxidoreductase (EC 1.9.3.1) were detected in a mitochondrial preparation, the NADH oxidase activity being markedly stimulated by addition of mammalian cytochrome c. Amytal and rotenone inhibited NADH oxidase activity. Antimycin A inhibited succinoxidase activity only at relatively high concentrations. Azide was inhibitory at high concentrations. However, cyanide was found to stimulate respiration. Hydrogen peroxide was found to be an end product of respiration in F. hepatica.     

Generation of reducing power in chemosynthesis. VI. Energy-linked reactions in the chemoautotroph,Thiobacillus neapolitanus

Electron donors such as thiosulfate, sulfite, and ascorbate have been shown to enter the respiratory chain ofT. neapolitanus at the level of cytochromec. The enzymatic oxidation of these substrates catalyzed by the cytochrome oxidase (E. C. 1.9.3.1.) ofT. neapolitanus cell-free extracts was coupled to the generation of energy which could be utilized to drive the reverse electron flow from cytochromec to pyridine nucleotides.The reduction of endogenous or added flavin by thiosulfate or ascorbate has been shown to be ATP-dependent; likewise the reduction of cytochromeb by these electron donors also required energy. The rate of ATP-driven reversal of electron transfer from cytochromec to the pyridine nucleotides was much faster compared with the rate of electron reversal catalyzed by the substrate-linked generated energy. The pathway of energy-linked reversal of electron transfer from cytochrome c to pyridine nucleotides involved cytochromeb and flavoproteins.NADH oxidation byT. neapolitanus cell-free extracts is mediated by the flavoprotein and cytochrome systems and this process also appears to be coupled with energy generation. The NADH oxidase (NADH₂: cytochromec oxidoreductase) was partially inhibited by amytal or rotenone, antimycin A or HOQNO, and was relatively insensitive to cyanide or azide.This investigation was supported in part by a National Science Foundation Grant No. GB 6649 and in part by the Department of Interior, Office of Water Resources Research No. A-016-KY.     

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

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

     

Respiratory terminal oxidases in the facultative chemoheterotrophic and dinitrogen fixing cyanobacterium Anabaena variabilis strain ATCC 29413: characterization of the cox2 locus

Upon nitrogen step-down, some filamentous cyanobacteria differentiate heterocysts, cells specialized for dinitrogen fixation, a highly oxygen sensitive process. Aerobic respiration is one of the mechanisms responsible for a microaerobic environment in heterocysts and respiratory terminal oxidases are the key enzymes of the respiratory chains. We used Anabaena variabilis strain ATCC 29413, because it is one of the few heterocyst-forming facultatively chemoheterotrophic cyanobacteria amenable to genetic manipulation. Using PCR with degenerate primers, we found four gene loci for respiratory terminal oxidases, three of which code for putative cytochrome c oxidases and one whose genes are homologous to cytochrome bd-type quinol oxidases. One cytochrome c oxidase, Cox2, was the only enzyme whose expression, tested by RT-PCR, was evidently up-regulated in diazotrophy, and therefore cloned, sequenced, and characterized. Up-regulation of Cox2 was corroborated by Northern and primer extension analyses. Strains were constructed lacking Cox1 (a previously characterized cytochrome c oxidase), Cox2, or both, which all grew diazotrophically. In vitro cytochrome c oxidase and respiratory activities were determined in all strains, allowing for the first time to estimate the relative contributions to total respiration of the different respiratory electron transport branches under different external conditions. Especially adding fructose to the growth medium led to a dramatic enhancement of in vitro cytochrome c oxidation and in vivo respiratory activity without significantly influencing gene expression.     

Thiosulfate Oxidation and Electron Transport in Thiobacillus novellus.

Aleem, M. I. H. (Research Institute for Advanced Studies, Baltimore, Md.). Thiosulfate oxidation and electron transport in Thiobacillus novellus. J. Bacteriol. 90:95-101. 1965.-A cell-free soluble enzyme system capable of oxidizing thiosulfate was obtained from Thiobacillus novellus adapted to grow autotrophically. The enzyme systems of autotrophically grown cells brought about the transfer of electrons from thiosulfate to molecular oxygen via cytochromes of the c and a types; the reactions were catalyzed jointly by thiosulfate oxidase and thiosulfate cytochrome c reductase. The levels of both of these enzymes were markedly reduced in the heterotrophically grown organism. Cell-free extracts from the autotrophically grown T. novellus catalyzed formate oxidation and enzymatically reduced cytochrome c with formate. Both formate oxidation and cytochrome c reduction activities were abolished under heterotrophic conditions. The thiosulfate-activating enzyme S(2)O(3) (-2)-cytochrome c reductase, as well as thiosulfate oxidase, was localized chiefly in the soluble cell-free fractions, and the former enzyme was purified more than 200-fold by ammonium sulfate fractionation and calcium phosphate gel adsorption procedures. Optimal activity of the purified enzyme occurred at pH 8.0 in the presence of 1.67 x 10(-1)m S(2)O(3) (-2) and 2.5 x 10(-4)m cytochrome c. The thiosulfate oxidase operated optimally at pH 7.5 and thiosulfate concentrations of 1.33 x 10(-3) to 3.33 x 10(-2)m in the presence of added cytochrome c at a concentration of 5 x 10(-4)m. Both enzymes were markedly sensitive to cyanide and to a lesser extent to some metal-binding agents. Although a 10(-3)m concentration of p-hydroxymercuribenzoate had no effect on S(2)O(3) (-2)-cytochrome c reductase, it caused a 50% inhibition of S(2)O(3) (-2) oxidase, which was completely reversed in the presence of 10(-3)m reduced glutathione. Carbon monoxide also inhibited S(2)O(3) (-2) oxidase; the inhibition was completely reversed by light.     

Superoxide anion generation by the cytochrome bc1 complex

We have measured the rates of superoxide anion generation by cytochrome bc₁ complexes isolated from bovine heart and yeast mitochondria and by cytochrome bc₁ complexes from yeast mutants in which the midpoint potentials of the cytochrome b hemes and the Rieske iron-sulfur cluster were altered by mutations in those proteins. With all of the bc₁ complexes the rate of superoxide anion production was greatest in the absence of bc₁ inhibitor and ranged from 3% to 5% of the rate of cytochrome c reduction. Stigmatellin, an inhibitor that binds to the ubiquinol oxidation site in the bc₁ complex, eliminated superoxide anion formation, while myxothiazol, another inhibitor of ubiquinol oxidation, allowed superoxide anion formation at a low rate. Antimycin, an inhibitor that binds to the ubiquinone reduction site in the bc₁ complex, also allowed superoxide anion formation and at a slightly greater rate than myxothiazol. Changes in the midpoint potentials of the cytochrome b hemes had no significant effect on the rate of cytochrome c reduction and only a small effect on the rate of superoxide anion formation. A mutation in the Rieske iron-sulfur protein that lowers its midpoint potential from +285 to +220 mV caused the rate of superoxide anion to decline in parallel with a decline in cytochrome c reductase activity. These results indicate that superoxide anion is formed by similar mechanisms in mammalian and yeast bc₁ complexes. The results also show that changes in the midpoint potentials of the redox components that accept electrons during ubiquinol oxidation have only small effects on the formation of superoxide anion, except to the extent that they affect the activity of the enzyme.     

Parallel electron donation pathways to cytochrome cz in the type I homodimeric photosynthetic reaction center complex of Chlorobium tepidum

We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome c_z bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome c_z. It was also shown that the rereduction rate of cytochrome c_z by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center.     

Salsolinol,a catechol neurotoxin,induces oxidative modification of cytochrome c

Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), an endogenous neurotoxin, is known to perform a role in the pathogenesis of Parkinson’s disease (PD). In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with salsolinol. When cytochrome c was incubated with salsolinol, protein aggregation increased in a dosedependent manner. The formation of carbonyl compounds and the release of iron were obtained in salsolinol- treated cytochrome c. Salsolinol also led to the release of iron from cytochrome c. Reactive oxygen species (ROS) scavengers and iron specific chelator inhibited the salsolinol-mediated cytochrome c modification and carbonyl compound formation. It is suggested that oxidative damage of cytochrome c by salsolinol might induce the increase of iron content in cells, subsequently leading to the deleterious condition which was observed. This mechanism may, in part, provide an explanation for the deterioration of organs under neurodegenerative disorders such as PD. [BMB Reports 2013; 46(2): 119-123]     

Inhibition of membrane-bound cytochrome c oxidase by zinc ions: High-affinity Zn-binding site at the P-side of the membrane

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn²⁺. Inhibition of liposome-bound bovine cytochrome oxidase by external Zn²⁺ titrates with a K_i of 1 ± 0.3 μM. Presumably, the Zn²⁺-binding group at the positively charged side is not reactive in the oxidized enzyme, but becomes accessible to the cation in some partially reduced state(s) of the oxidase; reduction of Cu_B is tentatively proposed to be responsible for the effect.     

Physical and catalytic properties of a peroxidase derived from cytochrome c

Except for its redox properties, cytochrome c is an inert protein. However, dissociation of the bond between methionine-80 and the heme iron converts the cytochrome into a peroxidase. Dissociation is accomplished by subjecting the cytochrome to various conditions, including proteolysis and hydrogen peroxide (H₂O₂)-mediated oxidation. In affected cells of various neurological diseases, including Parkinson's disease, cytochrome c is released from the mitochondrial membrane and enters the cytosol. In the cytosol cytochrome c is exposed to cellular proteases and to H₂O₂ produced by dysfunctional mitochondria and activated microglial cells. These could promote the formation of the peroxidase form of cytochrome c. In this study we investigated the catalytic and cytolytic properties of the peroxidase form of cytochrome c. These properties are qualitatively similar to those of other heme-containing peroxidases. Dopamine as well as sulfhydryl group-containing metabolites, including reduced glutathione and coenzyme A, are readily oxidized in the presence of H₂O₂. This peroxidase also has cytolytic properties similar to myeloperoxidase, lactoperoxidase, and horseradish peroxidase. Cytolysis is inhibited by various reducing agents, including dopamine. Our data show that the peroxidase form of cytochrome c has catalytic and cytolytic properties that could account for at least some of the damage that leads to neuronal death in the parkinsonian brain.     

HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ_m) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.     

Cytochrome c interaction with membranes. Interaction of cytochrome c with isolated membrane fragments and purified enzymes

The midpoint redox potential of cytochrome c and the electron paramagnetic resonance spectra of nitroxide labeled cytochromes c were measured as a function of binding to purified cytochrome c oxidase, cytochrome c peroxidase, cytochrome b₅ and succinate—cytochrome c reductase. The midpoint redox potential of horse heart cytochrome c is lowered in the presence of cytochrome c oxidase and succinate-cytochrome c reductase, but is unchanged in the presence of cytochrome c peroxidase or cytochrome b₅. Further evidence of binding is afforded by an increase in correlation time, T_c, of the spin-labeled cytochrome c at methionine 65 upon binding to cytochrome c peroxidase, cytochrome c oxidase and succinate—cytochrome c reductase. The changes in midpoint redox potential and electron paramagnetic resonance spectrum of the spin-labeled derivative upon binding can either be the consequence of specific interaction leading to formation of ES complexes, or it can be due to nonspecific electrostatic interaction between positively charged groups on cytochrome c and negatively charged groups on the isolated cytochrome preparations.     

Sulfite-oxido-reductase is involved in the oxidation of sulfite in Desulfocapsa sulfoexigens during disproportionation of thiosulfate and elemental sulfur

The enzymatic pathways of elemental sulfur and thiosulfate disproportionation were investigated using cell-free extract of Desulfocapsa sulfoexigens. Sulfite was observed to be an intermediate in the metabolism of both compounds. Two distinct pathways for the oxidation of sulfite have been identified. One pathway involves APS reductase and ATP sulfurylase and can be described as the reversion of the initial steps of the dissimilatory sulfate reduction pathway. The second pathway is the direct oxidation of sulfite to sulfate by sulfite oxidoreductase. This enzyme has not been reported from sulfate reducers before. Thiosulfate reductase, which cleaves thiosulfate into sulfite and sulfide, was only present in cell-free extract from thiosulfate disproportionating cultures. We propose that this enzyme catalyzes the first step in thiosulfate disproportionation. The initial step in sulfur disproportionation was not identified. Dissimilatory sulfite reductase was present in sulfur and thiosulfate disproportionating cultures. The metabolic function of this enzyme in relation to elemental sulfur or thiosulfate disproportionation was not identified. The presence of the uncouplers HQNO and CCCP in growing cultures had negative effects on both thiosulfate and sulfur disproportionation. CCCP totally inhibited sulfur disproportionation and reduced thiosulfate disproportionation by 80% compared to an unamended control. HQNO reduced thiosulfate disproportionation by 80% and sulfur disproportionation by 90%.     

Oxidation of sulfur compounds and electron transport in Thiobacillus denitrificans

Summary Intact cells of Thiobacillus denitrificans catalyzed the oxidation of thiosulfate, sulfide and sulfite with nitrate or oxygen as the terminal acceptor. The anaerobic oxidation of thiosulfate, sulfide and sulfite was sensitive to the inhibitors of the flavoprotein system. Under aerobic conditions the oxidation of sulfide and sulfite was sensitive to these inhibitors but the thiosulfate oxidation was unaffected. Cyanide and azide inhibited the aerobic and anaerobic respiration when thiosulfate, sulfide or sulfite served as electron donors. The oxidation of thiosulfate by cell-free preparations was mediated by cytochromes of c, a and o-types. The cell-free extracts also catalyzed the oxidation of NADH and succinate, involving flavoproteins and b, c, a and o-type cytochromes. In addition, a cytochrome oxidase sensitive to cyanide and azide was also present.Non-Standard Abbreviations TTFA Thenoyltrifluoroacetone - HQNO 2-heptyl-4-hydroxyquonoline N-oxide Aspirant van het Nationaal Fonds voor Wetenschappelijk Onderzoek (Belgian National Science Foundation).     

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a2+3 · CO

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity.Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a₃ in the mixed-valence enzyme after photodissociation.Upon addition of CO to partially reduced formate cytochrome c oxidase (a²⁺a³⁺₃ · HCOOH) the cytochrome a²⁺₃ · CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a₃ and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a³⁺₃ · HCOOH compound is dissociated slowly with a concomitant formation of the a²⁺₃ · CO compound and oxidation of cytochrome a.When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a₃ and the a²⁺₃ · CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.     

The participation of cytochromes in the process of nitrate respiration in Klebsiella (Aerobacter) aerogenes

1. The participation of cytochromes in the membrane-bound, nitrate and oxygen respiratory systems of Klebsiella (Aerobacter) aerogenes has been investigated. The membrane preparations contained the NADH, succinate, lactate and formate oxidase systems, and in addition a high respiratory nitrate reductase activity.2. Difference spectra indicated the presence of cytochromes b, a₁, d, and o. Cytochromes of the c-type could not be detected in these membranes. Both cytochrome b content and respiratory nitrate reductase activity were the highest in bacteria grown anaerobically in the presence of nitrate.3. Cytochrome b was the only cytochrome which, after being reduced by NADH, could be partially reoxidized anaerobically in the presence of nitrate. Furthermore, nitrate caused a lower aerobic steady state reduction only of cytochrome b.4. NADH oxidase and NADH-linked respiratory nitrate reductase activities were both inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and KCN. NADH oxidase activity was selectively inhibited by CO, while azide was found to inhibit only the respiratory nitrate reductase. In the presence of azide, nitrate did not affect the level of reduction of cytochrome b.5. The evidence presented suggests that cytochrome b is a carrier in the electron transport systems to both nitrate and oxygen; from cytochrome b branching occurs, with one branch linked to the respiratory nitrate reductase and one branch linked to oxidase systems, containing the cytochromes a₁, d and o.     

Electron transport chains and bioenergetics of respiratory nitrogen metabolism in Wolinella succinogenes and other Epsilonproteobacteria

Recent phylogenetic analyses have established that the Epsilonproteobacteria form a globally ubiquitous group of ecologically significant organisms that comprises a diverse range of free-living bacteria as well as host-associated organisms like Wolinella succinogenes and pathogenic Campylobacter and Helicobacter species. Many Epsilonproteobacteria reduce nitrate and nitrite and perform either respiratory nitrate ammonification or denitrification. The inventory of epsilonproteobacterial genomes from 21 different species was analysed with respect to key enzymes involved in respiratory nitrogen metabolism. Most ammonifying Epsilonproteobacteria employ two enzymic electron transport systems named Nap (periplasmic nitrate reductase) and Nrf (periplasmic cytochrome c nitrite reductase). The current knowledge on the architecture and function of the corresponding proton motive force-generating respiratory chains using low-potential electron donors are reviewed in this article and the role of membrane-bound quinone/quinol-reactive proteins (NapH and NrfH) that are representative of widespread bacterial electron transport modules is highlighted. Notably, all Epsilonproteobacteria lack a napC gene in their nap gene clusters. Possible roles of the Nap and Nrf systems in anabolism and nitrosative stress defence are also discussed. Free-living denitrifying Epsilonproteobacteria lack the Nrf system but encode cytochrome cd₁ nitrite reductase, at least one nitric oxide reductase and a characteristic cytochrome c nitrous oxide reductase system (cNosZ). Interestingly, cNosZ is also found in some ammonifying Epsilonproteobacteria and enables nitrous oxide respiration in W. succinogenes.     

Molybdenum Oxidation by Thiobacillus ferrooxidans

Thiobacillus ferrooxidans AP19-3 oxidized molybdenum blue (Mo⁵⁺) enzymatically. Molybdenum oxidase in the plasma membrane of this bacterium was purified ca. 77-fold compared with molybdenum oxidase in cell extract. A purified molybdenum oxidase showed characteristic absorption maxima due to reduced-type cytochrome oxidase at 438 and 595 nm but did not show absorption peaks specific for c-type cytochrome. The optimum pH of molybdenum oxidase was 5.5. The activity of molybdenum oxidase was completely inhibited by sodium cyanide (5 mM) or carbon monoxide, and an oxidized type of cytochrome oxidase in a purified molybdenum oxidase was reduced by molybdenum blue, indicating that cytochrome oxidase in the enzyme plays a crucial role in molybdenum blue oxidation.     

Quantum chemistry as a tool in bioenergetics

Recent developments of quantum chemical methods have made it possible to tackle crucial questions in bioenergetics. The most important systems, cytochrome c oxidase in cellular respiration and photosystem II (PSII) in photosynthesis will here be used as examples to illustrate the power of the quantum chemical tools. One main contribution from quantum chemistry is to put mechanistic suggestions onto an energy scale. Accordingly, free energy profiles can be constructed both for reduction of molecular oxygen in cytochrome c oxidase and water oxidation in PSII, including O-O bond cleavage and formation, and also proton pumping in cytochrome c oxidase. For the construction of the energy diagrams, the computational results sometimes have to be combined with experimental information, such as reduction potentials and rate constants for individual steps in the reactions.