Exploration of Respiratory Chain of <Emphasis Type="Italic">Nocardia asteroides:</Emphasis> Purification of Succinate Quinone Oxidoreductase

Nocardia asteroides is a pathogenic bacterium that causes severe pulmonary infections and plays a vital role in HIV development. Its electron transport chain containing cytochromes as electron carriers is still undiscovered. Information regarding cytochromes is important during drug synthesis based on cytochrome inhibitions. In this study we explored the electron transport of N. asteroides. Spectroscopic analysis of cytoplasm and membranes isolated from N. asteroides indicates the presence of soluble cytochrome-c, complex-II and the modified a ₁ c ₁ complex as the terminal oxidase. The molecular weight of the respiratory complex-II isolated and purified from the given bacterium was 103 kDa and was composed of three subunits, of 14, 26 and 63 kDa. Complex-II showed symmetrical α-absorption peaks at 561 nm in the reduced state. Spectral analysis revealed the presence of only one heme b molecule (14-kDa subunit) in complex-II, which was confirmed by heme staining. Heme b content was found to be 9.5 nmol/mg in complex-II. The electron transport chain of N. asteroides showed the presence of soluble cytochrome-c, cytochrome-a ₁ c ₁ and cytochrome-b.     

Characterisation of Escherichia coli K-12 mutants defective in formate-dependent nitrite reduction: essential roles for hemN and the menFDBCE operon

Three Escherichia coli mutants defective in formate-dependent nitrite reduction (Nrf activity) were characterised. Two of the mutants, JCB354 and JCB356, synthesized all five c-type cytochromes previously characterised in anaerobic cultures of E. coli. The third mutant, JCB355, was defective for both cytochrome b and cytochrome c synthesis, but only during anaerobic growth. The insertion sites of the transposon in strains JCB354 and JCB356 mapped to the menFDBCE operon; the hemN gene was disrupted in strain JCB355. The mutation in strain JCB354 was complemented by a plasmid encoding only menD; strain JCB356 was complemented by a plasmid encoding only menBCE. A mutant defective in the methyltransferase activity involved in both ubiquinone synthesis and conversion of demethylmenaquinone to menaquinone expressed the same Nrf activity as the parental strain. The effects of men, ubiA and ubiE mutations on other cytochrome-c-dependent electron transfer pathways were also determined. The combined data establish that menaquinones are essential for cytochrome-c-dependent trimethylamine-N-oxide reductase (Tor) and Nrf activity, but that either menaquinone or ubiquinone, but not demethylmenaquinone, can transfer electrons to a third cytochrome-c-dependent electron transfer chain, the periplasmic nitrate reductase. Received: 9 December 1996 / Accepted: 11 June 1997     

Studies of the Electron Transport Chain of the Euryarcheon Halobacterium salinarum: Indications for a Type II NADH Dehydrogenase and a Complex III Analog

The components involved in the respiratory system of the euryarcheon Halobacterium salinarum were investigated by spectroscopic and polarographic techniques. Previous results about the cytochrome composition could be verified. However, under low oxygen tension, the expression of a d-type cytochrome was detected. Membranes exerted an NADH– and succinate–cytochrome-c oxidoreductase as well as an NADH and succinate oxidase activity. These activities could be blocked by the following inhibitors: 7-jodocarboxylic acid, giving evidence for the presence of a type II NADH dehydrogenase, antimycin A, and myxothiazol, indicating the presence of a complex III analog, and the typical succinate dehydrogenase (SDH) and terminal oxidase inhibitors. Complex I inhibitors like rotenone and annonine were inactive, clearly excluding the presence of a coupled NADH dehydrogenase. In addition, no [Fe-S] resonances in the region of the NADH dehydrogenase (NDH) clusters could be observed after NADH addition. One of the terminal oxidases could be shown to act as a cytochrome-c oxidase with a K _m value of 37 M and an activation energy of 23.7 kJ/mol. The relative molecular mass of the endogenous c-type cytochrome could be determined as 14.1 kD. The complex III analog could be enriched after detergent extraction with Triton X-100 and hydroxylapatite (HTP) chromatography. The partially purified complex contained a Rieske iron–sulfur cluster, b- and c-type cytochromes, and was catalytically active in the decylubiquinone–cytochrome-c oxidoreductase assay.     

The interaction of rubroskyrin, a modified bis-anthraquinone pigment fromPenicillium islandicum Sopp, with respiratory chain of liver mitochondria

Summary  Rubroskyrin, a modified bisanthraquinone pigment from an yellow rice moldPenicillium islandicum Sopp, was examined for its redox-interaction with the mitochondrial respiratory chain by using rat liver submitochondrial particles (SMP) and was compared with luteoskyrin and rugulosin. Rubroskyrin showed a redox interaction with the NAD-linked respiratory chain of SMP, promoting NADH oxidase in the presence of rotenone, a specific inhibitor to coupling site I of the respiratory chain. Rubroskyrin-mediated NADH oxidase was not inhibited by antimycin A and cyanide, inhibitors to coupling sites II and III, respectively, indicating a generation of an electron transport shunt from a rotenone-insensitive site of NADH dehydrogenase (complex I) to dissolved oxygen. An electrontransport shunt to cytochromec oxidase from complex I was also observed in the experiment with cytochromec and antimycin A. Rubroskyrin did not interact with succinate-linked respiratory chain. Such enzymatic redox response which generates electron transport shunt was not detected for luteoskyrin and rugulosin in the present study.     

“Oxygen regulation” of the respiratory chain composition in the yeast Debaryomyces hansenii under multiple stress

It was shown that two stress factors, hypoxia and hyperosmotic shock, if applied simultaneously to the yeast Debaryomyces hansenii, display an antagonistic mode of interaction, which results in an increased degree of halophily of this microorganism under microaerobic conditions. Studies of the effects of respiration inhibitors (sodium azide and salicyl hydroxamic acid, SHA) and of the pattern of changes in the composition of the respiratory chain of Debaryomyces hansenii under the stated stress conditions led to the suggestion of three (or four) chains of electron transfer functioning simultaneously in the cell: the classical respiratory chain involving cytochrome-c oxidase, an alternative respiratory chain involving a cyanide-and azide-resistant oxidase, and additional respiratory chains involving oxidases resistant to salt, azide and SHA. Thus, the antagonistic mode of interaction between hypoxia and hyperosmotic shock results from the redirection of the electron flow from the salt-susceptible respiratory systems to the salt-unsusceptible ones encoded by “the hypoxia genes” and activated (induced) under microaerobic conditions.     

Molecular identification of the ten subunits of cytochrome-c reductase from potato mitochondria

Cytochrome-c reductase (EC 1.10.2.2.) from Solanum tuberosum L. comprises ten subunits with apparent molecular sizes of 55, 53, 51, 35, 33, 25, 14, 12, 11 and 10 kDa on 14% SDS-PAGE. The identity of the subunits was analysed by direct amino-acid sequencing via cyclic Edman degradation. A large-scale purification procedure for the enzyme complex based on affinity chromatography and gelfiltraton is described. All subunits were enzymatically fragmented and the generated peptides were separated by reverse-phase HPLC. Complete or partial sequence determination of 33 peptides comprising a total of nearly 500 amino acids showed, that cytochrome-c reductase from potato contains three respiratory proteins (cytochrome b, cytochrome c ₁ and the Rieske iron-sulfur protein), four small proteins with molecular sizes below 15 kDa (so-called Q-binding, hinge, cytochrome-c ₁-linked and core-linked proteins) and three proteins in the 50-kDa range which show similarity to members of the core/PEP/MPP protein family (core/processing enhancing protein/mitochondrial processing peptidase). In fact these subunits show highest sequence identity either to MPP or PEP, which is in line with earlier findings, that isolated cytochrome-c reductase from potato exhibits processing activity towards mitochondrial precursor proteins.Abbreviations MPP mitochondrial processing peptidase - PEP processing enhancing protein This research was supported by the Deutsche Forschungsgemeinschaft.     

Cytochrome-c reductases from wild-type and mutant strains of Aspergillus nidulans

Summary The sedimentation coefficients of the NADPH: cytochrome-c oxidoreductase enzymes from wild-type and mutant strains of Aspergillus nidulans have been estimated by sucrose density gradient centrifugation. In the wild-type, two species of cytochrome-c reductase were found, with sedimentation coefficients of 13.7s and 7.6s respectively. The 13.7s species did not appear to be associated with the enzymes of nitrate reduction, whereas the 7.6s species was closely associated with NADPH: nitrate oxidoreductase. In mutant strains lacking nitrate reductase, a thir species of cytochrome-c reductase with a sedimentation coefficient of 4.5s was found. There is some evidence that this 4.5s cytochrome-c reductase is a subunit or breakdown product of nitrate reductase and a model is presented for the role of this 4.5s cytocnorome-c reductase in the assembly of the intact nitrate reductase molecule.     

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.

     

Respiration in energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus: I. Relation of the respiratory and photosynthetic electron transport

The thermophilic cyanobacterium Mastigocladus laminosus was grown at different CO₂ concentrations and temperatures. Respiratory and photosynthetic electron transport in isolated membranes were measured and their activities were compared. Cells grown at low CO₂ concentration showed respiratory electron transport, whereas Photosystem-II-dependent transport was optimal in cells grown at high CO₂ concentrations. The respiratory electron transport from NADH and succinate were KCN-sensitive, whereas NADPH-dependent O₂ uptake was not. It could be shown that NADH and succinate donate electrons in the photosynthetic electron pathway via Photosystem I. In cytochrome-c-553-depleted membranes added cytochrome c-553 could stimulate photosynthetic and respiratory electron transport. A common electron transport pathway between the quinone and cytochrome c is postulated.     

Complexity and tissue specificity of the mitochondrial respiratory chain

There is a renewed interest in the structure and functioning of the mitochondrial respiratory chain with the realization that a number of genetic disorders result from defects in mitochondrial electron transfer. These so-called mitochondrial myopathies include diseases of muscle, heart, and brain. The respiratory chain can be fractionated into four large multipeptide complexes, an NADH ubiquinone reductase (complex I), succinate ubiquinone reductase (complex II), ubiquinol oxidoreductase (complex III), and cytochromec oxidase (complex IV). Mitochondrial myopathies involving each of these complexes have been described. This review summarizes compositional and structural data on the respiratory chain proteins and describes the arrangement of these complexes in the mitochondrial inner membrane. This biochemical information is provided as a framework for the diagnosis and molecular characterization of mitochondrial diseases.     

Evidence for the presence of di- and triphospho pyridine nucleotide dehydrogenase derivatives as consistent contaminants of purified beef heart cytochrome-c oxidase

Purified beef heart cytochrome-c oxidase preparations derived by three different laboratories contain NADH-K₃ Fe(CN)₆, NADH-nitrobluetetrazolium, and NADPH-nitrobluetetrazolium reductases. This is true of preparations exhibiting heme aa₃ to protein ratios considered indicative of an excellent purity. An apparent association of cytochrome-c oxidase and one or more of the contaminants persists through immunodiffusion and nondenaturing electrophoresis and, in addition, in one instance copurification of NADH-K₃ Fe(CN)₆ reductase and cytochrome-c oxidase to a constant ratio of specific activities was demonstrated. Cytochrome-c oxidase can be freed of the contaminants by equilibration with an NAD⁺-affinity matrix. As a concomitant of equilibration with the matrix, theK _M of cytochrome-c oxidase for ferrocytochrome-c is invariably decreased. Rate constants at low ferrocytochrome-c concentrations are consistently enhanced in all oxidase preparations upon equilibration with the NAD⁺ matrix. However, the effects of such equilibrations on the extrapolatedV _max varies from one preparation to another. Polyacrylamide gel electrophoresis in SDS-urea systems establishes that each of the preparations contains a minimum of three contaminants, each of an apparent formula weight of greater than 40,000 Daltons. NADH-NBT reductase was found to have a formula weight of approximately 46,000 Daltons. Their properties establish that NADH-K₃ Fe(CN)₆ and NADH-NBT reductases are separate proteins; the separate identity of NADPH-NBT reductase has not yet been determined.     

The cytochromec reductase/oxidase respiratory pathway ofParacoccus denitrificans: Genetic and functional studies

Data are presented on three components of the quinol oxidation branch of theParacoccus respiratory chain: cytochromec reductase, cytochromec ₅₅₂, and thea-type terminal oxidase. Deletion mutants in thebc ₁ and theaa ₃ complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochromec ₅₅₂ in electron transport between complex III and IV is presented.     

Identification and Characterization of Human cDNAs Specific to BCS1, PET112, SCO1, COX15, and COX11, Five Genes Involved in the Formation and Function of the Mitochondrial Respiratory Chain

We have successfully applied a strategy based on the “cyberscreening” of the expressed sequence tags database using yeast protein sequences as “probes” to identify the human gene orthologs to BCS1, COX15, PET112, COX11, and SCO1, five yeast genes involved in the biogenesis of the mitochondrial respiratory chain complexes. In yeast, BCS1 is involved mainly in the assembly of complex III, while the other genes appear to control the structure/function of cytochrome-coxidase. Significant amino acid identity and similarity were demonstrated by comparison of the human with the corresponding yeast polypeptides. Sequence alignment revealed numerous colinear identical regions and the conservation of functional domains. Mitochondrial targeting of the human gene products, suggested by computer analysis of the protein sequences, was confirmed by anin vitroimport and protease-protection assay. These data strongly suggest that the human gene products share similar or identical functions with their yeast homologues. Genes controlling the structure/function of the respiratory chain complexes are attractive candidates for human mitochondrial disorders such as Leigh disease. However, both sequence analysis and functional complementation assays on an index patient do not support an etiological role for any of these genes.     

The metabolism ofStreptomyces griseus

The electron transfer pathway in the respiratory particles ofStreptomyces griseus was studied. Vitamins K₃ and K₅,α- andβ-naphthoquinones, served as the hydrogen acceptors in succinate oxidation, and succinate- and reduced nicotinamide adenine dinucleotide (NADH)-cytochromec reductase activities, but were ineffective for NADH oxidase activity. Vitamin K seemed to mediate the hydrogen from NADH-diaphorase to cytochromec. Chlorpromazine inhibited electron transfer in the respiratory particles. Cyanide completely inhibited the electron transfer system initially, however, oxygen consumption increased gradually with time. AlthoughS. griseus possesses cytochromesa, b, c and pigment 625 (probablyd), the electron transfer chain was complicated. Two terminal oxidase activities (cytochromec oxidase and cytochromec peroxidase activities) were detected in the respiratory particles ofS. griseus. Dedicated to Prof. Shoichiro Usami celebrating his sexagenary birthday.     

Effect of FeSO4 treatment on glucose metabolism in diabetic rats

Iron, the prosthetic group of haemoglobin, was found to lower serum glucose levels of diabetic rats. Its regulative mechanism and effects on enzymatic activities of glucose metabolism are still unknown. In this study, the correlation between iron supply and enzymatic activities of glucose metabolism and respiratory chain were evaluated in liver and kidney tissues of alloxan induced-diabetic rats. After FeSO₄ and metformin administration, serum samples were collected for serum glucose and fructosamine level measurements. Kidney and liver tissues were excised at the end of the study for assaying enzymatic activities of isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH-dehydrogenase and cytochrome-c-oxidase. Results showed significantly decreased serum glucose and fructosamine levels in treatment groups and enhanced enzymatic activities of several proteins as compared with the diabetic control group. Therefore, these data suggested that FeSO₄ administration could increase the supply of oxygen, enhance enzymatic activities of glucose metabolism and the respiratory chain, accelerate glucose metabolism and consequently decrease serum glucose levels.     

The effect of respiration on the phototactic behavior of the purple nonsulfur bacterium Rhodospirillum centenum

The effect of respiration on the positive phototactic movement of swarming agar colonies of the facultative phototroph Rhodospirillum centenum was studied. When the electron flow was blocked at the bc ₁ complex level by myxothiazol, the oriented movement of the colonies was totally blocked. Conversely, inhibition of respiration via the cytochrome c oxidase stimulated the phototactic response. No phototaxis was observed in a photosynthesis deficient mutant (YB707) lacking bacteriochlorophylls. Analyses of the respiratory activities as monitored by a oxygen microelectrode in single agar colonies during light/dark transitions showed a close functional correlation between the photosynthetic and respiratory apparatuses. The respiratory chain of Rsp. centenum was formed by two oxidative pathways: one branch leading to a cytochrome c oxidase inhibited by low cyanide concentrations and a second pathway formed by an oxidase less-sensitive to cyanide that also catalyzes the light-driven respiration. These results were interpreted to indicate that (1) there is a cyclic electron transport, and (2) photoinduced cyclic electron flow is required for the phototactic response of Rsp. centenum. Furthermore, under oxic conditions in the light, reducing equivalents may switch from photosynthetic to respiratory components so as to reduce both the membrane potential and the rate of locomotion. Received: 25 September 1996 / Accepted: 11 November 1996     

The application of a biotin-anti-biotin gold technique providing a significant signal intensification in electron microscopic immunocytochemistry: a comparison with the ultrasmall immunogold silver staining procedure

 A three-step biotin-anti-biotin gold-detection system (method A) has been applied for ultraimmunocytochemistry using ultrasmall colloidal gold (0.8 nm) linked to anti-biotin antibodies which were visualized and enhanced by silver reduction. The reactivity for glucagon in human pancreatic islets and for cytochrome-c oxidase in heart mitochondria has been compared to a two-step ultrasmall immunogold technique (method B). For both antigens, method A provided significantly higher labelling indices (P<0.001): the labelling density for cytochrome-c oxidase was 223/μm² using method A and 78/μm² using method B. For glucagon, the labelling density was 1455/μm² with method A and 322/μm² with method B. The results demonstrate that the silver-intensified biotin-anti-biotin gold-detection system is a valuable immunocytochemical method for signal enhancement. The method utilizes biotinylated antibodies from different species, allowing its broad application at the electron microscopic level. Accepted: 24 June 1997     

Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1

The respiratory chain in the inner mitochondrial membrane contains three large multi‐enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19‐Å 3D map of the 1.7‐MDa amphipol‐solubilized supercomplex I₁III₂IV₁ from bovine heart obtained by single‐particle electron cryo‐microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X‐ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol‐binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.     

Elucidation of the effects of lipoperoxidation on the mitochondrial electron transport chain using yeast mitochondria with manipulated fatty acid content

Lipoperoxidative damage to the respiratory chain proteins may account for disruption in mitochondrial electron transport chain (ETC) function and could lead to an augment in the production of reactive oxygen species (ROS). To test this hypothesis, we investigated the effects of lipoperoxidation on ETC function and cytochromes spectra of Saccharomyces cerevisiae mitochondria. We compared the effects of Fe²⁺ treatment on mitochondria isolated from yeast with native (lipoperoxidation-resistant) and modified (lipoperoxidation-sensitive) fatty acid composition. Augmented sensitivity to oxidative stress was observed in the complex III-complex IV segment of the ETC. Lipoperoxidation did not alter the cytochromes content. Under lipoperoxidative conditions, cytochrome c reduction by succinate was almost totally eliminated by superoxide dismutase and stigmatellin. Our results suggest that lipoperoxidation impairs electron transfer mainly at cytochrome b in complex III, which leads to increased resistance to antimycin A and ROS generation due to an electron leak at the level of the Q_O site of complex III.     

The role of individual lysine residues of horse cytochrome <Emphasis Type="Italic">c</Emphasis> in the formation of reactive complexes with components of the respiratory chain

A number of mutant forms of horse cytochrome c with single or double substitutions of lysine residues near the heme cavity involved in interaction of mitochondrial cytochrome c with ubiquinol:cytochrome c reductase (EC 1.10.2.2) (complex III) and cytochrome c oxidase (EC 1.9.3.1) (complex IV) were prepared.. The succinate:cytochrome c reductase and cytochrome c oxidase activities of mitoplasts of rat liver were measured in the presence of mutant forms of cytochrome c. The lysine residues in positions 8, 27, 72, 86, and 87 were shown to be the main contribution to the formation of a reactive complex with ubiquinol:cytochrome c reductase of the respiratory chain, whereas the lysine residues in positions 13, 79, 86, and 87 were predominantly responsible for the formation of a complex with cytochrome c oxidase.