Nitric oxide- and superoxide-dependent mitochondrial signaling in endotoxin-induced apoptosis in the rostral ventrolateral medulla of rats

This study evaluated the hypothesis that the repertoire of cellular events that underlie circulatory fatality during endotoxemia may entail mitochondrial respiratory enzyme dysfunction, followed by the release of cytochrome c to the cytosol that triggers the activation of caspase cascades, leading to apoptotic cell death in the rostral ventrolateral medulla (RVLM) where sympathetic premotor neurons responsible for maintaining vasomotor tone are located. In adult Sprague-Dawley rats maintained under propofol anesthesia, nucleosomal DNA fragmentation was detected in the RVLM in a temporal profile that coincided positively with the progression of cardiovascular depression during experimental endotoxemia induced by Escherichia coli lipopolysaccharide (LPS). LPS also induced nitric oxide (NO) and superoxide (O(2)(-)) production, depressed mitochondrial Complex I and IV activity, promoted the release of cytochrome c from mitochondria to cytosol, upregulated the cytosolic expression of activated caspase-9 and -3, or increased caspase-3 enzyme activity in the RVLM. Microinjection bilaterally into the RVLM of an inducible nitric oxide synthase (iNOS) blocker, S-methylisothiourea, or a superoxide dismutase mimetic, Tempol, significantly blunted these apoptotic cellular events and antagonized the cardiovascular depression during endotoxemia. We conclude that caspase-dependent apoptotic cell death that results from NO- and O(2)(-)-associated mitochondrial signaling in the RVLM may underlie fatal cardiovascular depression during endotoxemia.     

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.     

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.     

Orientation of complex III in the yeast mitochondrial membrane: Labeling with [125I] diazobenzenesulfonate and functional studies with the decyl analogue of coenzyme Q as substrate

Mitochondria (or mitoplasts) and submitochondrial particles from yeast were treated with [¹²⁵I] diazobenzenesulfonate to label selectively proteins exposed on the outer or inner surface of the inner mitochondrial membrane. Polyacrylamide gel analysis of the immunoprecipitates formed with antibodies against Complex III or cytochromeb revealed that the two core proteins and cytochromeb were labeled in both mitochondria and submitochondrial particles, suggesting that these proteins span the membrane. Cytochromec ₁ and the iron sulfur protein were labeled in mitochondria but not in submitochondrial particles, suggesting that these proteins are exposed on the cytosolic side of the inner membrane. The steady-state reduction of cytochromesb andc ₁ was determined with succinate and the decyl analogue of coenzyme Q as substrates. Addition of the coenzyme Q analogue to mitochondria caused reduction of 15–30% of the total dithionite-reducibleb and 100% of the cytochromec ₁: Addition of the coenzyme Q analogue to submitochondrial particles led to the reduction of 70% of the total dithionite-reducible cytochromeb but insignificant amounts of cytochromec ₁. A model to explain the topography of Complex III in the inner membrane is proposed based on these results.Abbreviations used: DABS, diazobenzene sulfonate; DBH₂, reduced form of decyl analogue of coenzyme Q (2,3-dimethoxy-5-methyl-6-n-decyl-1,4-benzoquinone); PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate.     

Threshold Effects and Control of Oxidative Phosphorylation in Nonsynaptic Rat Brain Mitochondria

Abstract: The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by ∼72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by ∼70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed.     

On the impermeability of the outer mitochondrial membrane to cytochromec

Summary Outer mitochondrial membranes isolated by the swelling-shrinkage sonication procedure of Sottocasaet al. [19, 20] forms small sealed vesicles. If cytochromec is present during the procedure it is trapped inside these vesicles and can not be washed out nor is accessible to external enzymes, e.g., cytochrome oxidase (EC 1.9.3.1) or succinate-cytochromec reductase present as contamination by the inner membrane, but is fully accessible to rotenone-insensitive NADH-cytochromec reductase of the outer membrane. This indicates the impermeability of the outer mitochondrial membrane to cytochromec.A modification of the original procedure for the separation of the outer mitochondrial membrane is described.     

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.     

The reactions of the oxidase and reductases ofParacoccus denitrificans with cytochromesc

Electron transport in theParacoccus denitrificans respiratory chain system is considerably more rapid when it includes the membrane-bound cytochromec ₅₅₂ than with either solubleParacoccus c ₅₅₀ or bovine cytochromec; a pool function for cytochromec is not necessary. Low concentrations ofParacoccus or bovine cytochromec stimulate the oxidase activity. This observation could explain the multiphasic Scatchard plots which are obtained. A negatively charged area on the back side ofParacoccus c which is not present in mitochondrialc could be a control mechanism forParacoccus reactions.Paracoccus oxidase and reductase reactions with bovinec show the same properties as mammalian systems; and this is true ofParacoccus oxidase reactions with its own soluble cytochromec if added polycation masks the negatively charged area. Evidence for different oxidase and reductase reaction sites on cytochromec include: (1) stimulation of the oxidase but not reductase by a polycation; (2) differences in the inhibition of the oxidase and reductases by monoclonal antibodies toParacoccus cytochromec; and (3) reaction of another bacterial cytochromec withParacoccus reductases but not oxidase. Rapid electron transport occurs in cytochromec-less mutants ofParacoccus, suggesting that the reactions result from collision of diffusing complexes.     

Structure of cytochromec oxidase from baker's yeast—a progress report. Preparation of four subunits for amino acid sequence determination and attempts to localize the cytochromec binding site

Summary Cytochromec oxidase from the inner membrane of yeast mitochondria consists of seven nonidentical protein subunits, three being synthesized on mitochondrial ribosomes (molecular weights I: 43 K, II: 34 K, and III: 24 K) and four being made on cytoplasmic ribosomes (molecular weights IV: 14 K, V: 12 K, VI: 12 K, and VII: 4.5 K).In the present study all four cytoplasmically synthesized subunits of the enzyme were isolated on a large scale using ion exchange chromatography and gel filtartion. Their amino acid composition as well as their amino- and carboxy-terminal amino acid residues have been determined. Sequence determinations of sub-units IV and VI are already in an advanced state. The sequence of subunit VI is characterized by a large amino-terminal stretch dominated by charged amino acid residues followed by a cluster of hydrophobic amino acids.The binding site of yeast cytochrome oxidase for cytochromec was studied by chemical crosslinking experiments. The formation of a disulfide bridge between the two proteins was observed by using cytochromec from yeast modified with 5-thionitrobenzoate at the cysteinyl residue in position 107. Alternatively, a disulfide between yeast cytochromec and the oxidase could be formed directly by oxidation with copper phenanthroline. Gel electrophoresis of the crosslinked complexes in sodium dodecyl sulfate revealed a new protein band with an apparent molecular weight of 38 K. This new band appears to be derived from cytochromec and from subunit III of cytochrome oxidase.Recipient of a fellowship from the Swiss National Science Foundation. Present address: Department of Biology, University of California at San Diego, La Jolla, Calif. 92037 (USA).     

The bifunctional cytochromec reductase/processing peptidase complex from plant mitochondria

Cytochromec reductase from potato has been extensively studied with respect to its catalytic activities, its subunit composition, and the biogenesis of individual subunits. Molecular characterization of all 10 subunits revealed that the high-molecular-weight subunits exhibit striking homologies with the components of the general mitochondrial processing peptidase (MPP) from fungi and mammals. Some of the other subunits show differences in the structure of their targeting signals or in their molecular composition when compared to their counterparts from heterotrophic organisms. The proteolytic activity of MPP was found in the cytochromec reductase complexes from potato, spinach, and wheat, suggesting that the integration of the protease into this respiratory complex is a general feature of higher plants.     

Structural and functional organization of the mitochondrial respiratory chain: A dynamic super-assembly

The structural organization of the mitochondrial oxidative phosphorylation (OXPHOS) system has received large attention in the past and most investigations led to the conclusion that the respiratory enzymatic complexes are randomly dispersed in the lipid bilayer of the inner membrane and functionally connected by fast diffusion of smaller redox components, Coenzyme Q and cytochrome c. More recent investigations by native gel electrophoresis, however, have shown the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis has demonstrated that Complexes I and III in mammalian mitochondria and Complexes I, III, and IV in plant mitochondria kinetically behave as single units with control coefficients approaching unity for each single component, suggesting the existence of substrate channelling within the supercomplexes. The reasons why the presence of substrate channelling for Coenzyme Q and cytochrome c was overlooked in the past are analytically discussed. The review also discusses the forces and the conditions responsible for the formation of the supramolecular units. The function of the supercomplexes appears not to be restricted to kinetic advantages in electron transfer: we discuss evidence on their role in the stability and assembly of the individual complexes and in preventing excess oxygen radical formation. Finally, there is increasing evidence that disruption of the supercomplex organization leads to functional derangements responsible for pathological changes.     

A durohydroquinone oxidation site in the mitochondrial transport chain

Although duroquinone had little effect upon NADH oxidation in neutral lipid depleted mitochondria, durohydroquinone was oxidized by ETP at a rate sensitive to antimycin A. Fractionation of mitochondria into purified enzyme systems showed durohydroquinone: cytochromec reductase to be concentrated in NADH: cytochromec reductase, absent in succinate:cytochromec reductase, and decreased in reduced coenzyme Q:cytochromec reductase. Durohydroquinone oxidation could be restored by recombining reduced coenzyme Q:cytochromec reductase with NADH:coenzyme Q reductase. Pentane extraction had no effect upon either durohydroquinone or reduced coenzyme Q₁₀ oxidation, indicating lack of a quinone requirement between cytochromesb andc. Both chloroquine diphosphate and acetone (96%) treatment irreversibly inhibited NADH but not succinate oxidation. Neither reagents had any effect upon durohydroquinone oxidation but both inhibited reduced coenzyme Q₁₀ oxidation 50%, indicating a site of action between Q₁₀ and duroquinone sites. Loss of chloroquine sensitive reduced coenzyme Q₁₀ oxidation after acetone extraction suggests two sites for Q₁₀ before cytochromeb.     

Effect of ubiquinone extraction on the reaction of the mitochondrialbc 1 complex with ferricyanide

Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochromeb extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone-depleted membranes, even if cytochromec was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/molbc ₁ complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochromeb extrareduction. Since the initial rates of cytochromeb reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochromeb was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone.     

NADPH oxidase- and mitochondrion-derived superoxide at rostral ventrolateral medulla in endotoxin-induced cardiovascular depression

We evaluated the contribution of superoxide anion (O2*-) generated by NADPH oxidase or mitochondria in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for arterial pressure maintenance are located, on cardiovascular depression induced by inducible nitric oxide synthase-derived NO after Escherichia coli lipopolysaccharide (LPS) treatment. In Sprague-Dawley rats maintained under propofol anesthesia, microinjection of LPS bilaterally into the RVLM induced progressive hypotension, bradycardia, and reduction in sympathetic vasomotor outflow over our 240-min observation period. This was accompanied by an increase in O2*- production (60-240 min) in the RVLM, alongside phosphorylation of p47(phox) or p67(phox), upregulation of gp91(phox) or p47(phox) protein, and increase in Rac-1 or NADPH oxidase activity (60-120 min), and a depression of mitochondrial respiratory enzyme activity (120-240 min). Whereas inhibition of NADPH oxidase or knockdown of the gp91(phox) or p47(phox) gene blunted the early phase (60-150 min), coenzyme Q10 or mitochondrial K(ATP) channel inhibitor antagonized the delayed phase (120-240 min) of LPS-induced increase in O2*- production in RVLM and cardiovascular depression. We conclude that, whereas NADPH oxidase-derived O2*- in RVLM participates predominantly in the early phase, O2*- generated by depression in mitochondrial respiratory enzyme activity or opening of mitoK(ATP) channels mediates the delayed phase of LPS-induced cardiovascular depression.     

Biochemical,genetic and molecular characterization of new respiratory-deficient mutants inChlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt ^- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.     

Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochromec oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochromec reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochromec oxidoreductase is strongly stimulated by added CoQ₃, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of theK _m for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ₉ and CoQ₁₀.     

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.     

Modes of coenzyme Q function in electron transfer

Summary In the mitochondrial respiratory chain, coenzyme Q acts in different ways. A diffusable coenzyme Q pool as a common substrate-like intermediate links the low-potential complexes with complex III. Its diffusion in the lipids is not rate-limiting for electron transfer, but its content is not saturating for maximal rate of NADH oxidation. Protein-bound coenzyme Q is involved in energy conservation, and may be part of enzyme supercomplexes, as in succinate cytochromec reductase. The reason for lack of kinetic saturation of the respiratory chain by quinone concentration is in the low extent of solubility of monomeric coenzyme Q in the membrane lipids. Assays of respiratory enzymes are performed using water soluble coenzyme Q homologs and analogs; several problems exist in using oxidized quinones as acceptors of coenzyme Q reductases. In particular, for complex I no acceptor appears to favorably substitute the endogenous quinone. In addition, quinone reduction sites in complex III compete with the sites in the dehydrogenases, particularly when using duroquinone. The different extent by which these sites operate when different donor substrates (NADH, succinate, glycerol-3-phosphate) are used is best explained by different exposure of the quinone acceptor sites in the dehydrogenases.     

Experimental observations on the structure and function of mitochondrial Complex III that are unresolved by the protonmotive ubiquinone-cycle hypothesis

The current model of the protonmotive ubiquinone cycle as applied to mitochondrial ubiquinol-cytochromec reductase complex (Complex III) is able to explain a number of previously puzzling observations concerning electron-transfer and proton translocating functions of the complex. However, a number of pertinent experimental observations concerning the structure and function of this complex cannot as yet be incorporated into the present version of the ubiquinone cycle. The yet unresolved problems of electron transfer uncovered by these observations include some kinetic and thermodynamic problems, uncertainties in the binding site(s) and mode of binding of ubiquinol and inhibitors, the observed multiple spectroscopic, electrochemical, and kinetic forms of cytochromesb, iron-sulfur protein, and cytochromec ₁, the multiple and overlapping effects of inhibitors, and the functional role of conformational changes in the complex. It is concluded that although the Q cycle is a valuable base for the design of future experiments, its mechanism must be reconciled with the above uncertainties as well as with the accumulated evidence that Complex III can exist in two or more interchangeable forms, exhibiting different properties with respect to electron-transfer pathways, inhibitor binding, and spectral and electrochemical properties of the electron-carrier subunits.