Biochemical and molecular aspects of human mitochondrial respiratory chain disorders.
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Electron Transport Complex II↗
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Sphingolipid activator proteins (SAPs) in neuronal ceroid lipofuscinoses (NCL).
Based on the predominant component of the storage material the neuronal ceroid lipofuscinoses (NCL) can be divided into two categories: one storing mitochondrial ATP synthase subunit c and the other storing sphingolipid activator proteins (SAPs). The latter group is represented by the human infantile NCL (INCL), a congenital ovine NCL, and a canine NCL. Small amounts of SAPs also accumulate in most other forms of NCL. The SAPs, their functions and occurrence in different forms of NCL, as well as the relationship between SAPs and palmitoyl protein thioesterase, an enzyme implicated in INCL, are discussed.
ATP Synthetase Complexes↗
Mitochondrial myopathy with a defect of mitochondrial-protein transport.
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Adolescent↗
Identification of the ubiquinone-binding domain in QPs1 of succinate-ubiquinone reductase.
An azidoubiquinone derivative, 3-azido-2-methyl-5-methoxy [3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone-protein interaction and to identify ubiquinone-binding proteins in bovine heart mitochondrial succinate-ubiquinone reductase. When the reductase was incubated with [3H]azido-Q and illuminated with long wavelength UV light, the decrease in the enzymatic activity correlated with the amount of azido-Q incorporated into the protein. When the illuminated, [3H]azido-Q-treated reductase was extracted with organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity was found primarily in the QPs1 subunit. The [3H]azido-Q-labeled QPs1 was purified from labeled reductase by a procedure involving ammonium sulfate fractionation, dialysis, organic solvent extraction, lyophilization, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cold acetone precipitation. The purified, [3H]azido-Q-labeled QPs1 protein was subjected to reductive carboxymethylation prior to digestion by trypsin. One azido-Q-linked peptide, with a retention time of 66.9 min, was obtained by high performance liquid chromatographic separation. The partial amino-terminal sequence of this peptide is GLTISQL-, indicating that this tryptic peptide comprises amino acid residues 113-140 of the revised amino acid sequence of QPs1. The Q-binding domain, using the proposed structure of QPs1, is probably located in the stretch connecting transmembrane helices 2 and 3 that extrude from the surface of the M side of the inner membrane.
Affinity Labels↗
Assembly and function of the cytochrome cbb3 oxidase subunits in Bradyrhizobium japonicum.
The Bradyrhizobium japonicum cbb3-type cytochrome oxidase, which supports microaerobic respiration, is a multisubunit enzyme encoded by the genes of the fixNOQP operon. We investigated the contribution of the individual subunits to function and assembly of the membrane-bound complex. In-frame deletion mutants of fixN, fixO, and fixQ, and an insertion mutant of fixP were constructed. All mutants, except the fixQ mutant, showed clearly altered absorption difference spectra of their membranes and decreased oxidase activities, and they were unable to fix nitrogen symbiotically. The presence of the individual subunits was assayed by Western blot analysis, using subunit-specific antibodies, and by heme staining of the c-type cytochromes FixO and FixP. These analyses led to the following conclusions: (i) FixN and FixO are necessary for assembly of the multimeric oxidase, (ii) FixN and FixO assemble independently of FixP, and (iii) FixQ is not required for complex formation and, therefore, does not seem to be an essential subunit. The possible oxidase biogenesis pathway involves the formation of a primary core complex consisting of FixN and FixO, which allows the subsequent association with FixP to form the complete enzyme.
Bacterial Proteins↗
Resolution and reconstitution of succinate-ubiquinone reductase from Escherichia coli.
A modified procedure is developed for isolation of highly purified succinate-ubiquinone reductase from Escherichia coli NM256 containing a cloned sdh operon in a multicopy plasmid. Succinate-ubiquinone reductase is solubilized from the membrane by polyoxyethylene-9-lauryl ether and purified by DEAE-Sepharose CL-6B column chromatography. The isolated reductase is resolved into a reconstitutively active, two-subunit succinate dehydrogenase and a two-subunit membrane anchoring protein fraction (the SdhC-SdhD fraction) by alkaline (pH 10.2) treatment of the reductase in the presence of 1 M urea, followed by DEAE-Sepharose CL-6B column chromatography under anaerobic conditions. Isolated succinate dehydrogenase and the SdhC-SdhD fraction alone show no succinate-ubiquinone reductase activity. However, when a given amount of the SdhC-SdhD fraction is mixed with varying amounts of succinate dehydrogenase or vice versa succinate-ubiquinone reductase activity increases as the amount of succinate dehydrogenase or the SdhC-SdhD fraction added increases. Maximum reconstitution is obtained when the weight ratio of succinate dehydrogenase to the SdhC-SdhD fraction reaches 5.26. This ratio is slightly higher than the calculated value of 3.37, obtained by assuming 1 mol of succinate dehydrogenase reacts with 1 mol of SdhC and SdhD. The isolated SdhC-SdhD fraction contains 35 nmol cytochrome b556/mg protein. Unlike mitochondrial cytochrome b560, the cytochrome b556 is reducible by succinate in the isolated and complex forms. Furthermore, cytochrome b556 in the isolated SdhC-SdhD fraction has absorption properties, carbon monoxide reactivity, and EPR characteristics similar to those of cytochrome b556 in intact succinate-ubiquinone reductase, indicating that its heme environments are not affected by the presence of succinate dehydrogenase. However, the redox potential of cytochrome b556 in the SdhC-SdhD fraction (22 mV) increases slightly when complexed with succinate dehydrogenase (34 mV). No hybrid succinate-ubiquinone reductase is formed from mitochondrial QPs (the membrane-anchoring protein fraction of bovine heart mitochondrial succinate-ubiquinone reductase) and E. coli succinate dehydrogenase or vice versa. However, the cytochrome b556 in E. coli SdhC-SdhD fraction is reducible by succinate in the presence of mitochondrial succinate dehydrogenase, and the rate of cytochrome b556 reduction correlates with the reconstitutive activity of the mitochondrial succinate dehydrogenase.
Animals↗
The smallest membrane anchoring subunit (QPs3) of bovine heart mitochondrial succinate-ubiquinone reductase. Cloning, sequencing, topology, and Q-binding domain.
The cDNA encoding the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate-ubiquinone reductase was cloned and sequenced. This cDNA is 1330 base pairs long with an open reading frame of 474 base pairs that encodes the 103 amino acid residues of mature QPs3 and a 55-amino acid residue presequence. The cDNA insert has an 820-base pair long 3'-untranslated region, including a poly(A) tail. The molecular mass of QPs3, deduced from the nucleotide sequence, is 10,989 Da. QPs3 is a very hydrophobic protein; the hydropathy plot of the amino acid sequence reveals three transmembrane helices. Previous photoaffinity labeling studies of succinate-ubiquinone reductase, using 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), identified QPs3 as one of the putative Q-binding proteins in this reductase. An azido-Q-linked peptide with a retention time of 66 min is obtained by high performance liquid chromatography of the chymotrypsin digest of carboxymethylated and succinylated [3H]azido-Q-labeled QPs3 purified from labeled succinate-ubiquinone reductase by a procedure involving phenyl-Sepharose 4B column chromatography, preparative SDS-polyacrylamide gel electrophoresis, and acetone precipitation. The amino acid sequence of this peptide is NH2-L-N-P-C-S-A-M-D-Y-COOH, corresponding to residues 29-37. The structure of QPs3 in the inner mitochondrial membrane is proposed based on the hydropathy profile of the amino acid sequence, on the predicted tendencies to form alpha-helices and beta-sheets, and on immunobinding of Fab' fragmenthorseradish peroxidase conjugates prepared from antibodies against two synthetic peptides, corresponding to the NH2 terminus region and the loop connecting helices 2 and 3 of QPs3, in mitoplasts and submitochondrial particles. The ubiquinone-binding domain in the proposed model of QPs3 is probably located at the end of transmembrane helix 1 toward the C-side of the mitochondrial inner membrane.
Amino Acid Sequence↗
Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a.
We investigated the biochemical phenotype of the mtDNA T8993G point mutation in the ATPase 6 gene, associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in three patients from two unrelated families. All three carried >80% mutant genome in platelets and were manifesting clinically various degrees of the NARP phenotype. Coupled submitochondrial particles prepared from platelets capable of succinate-sustained ATP synthesis were studied using very sensitive and rapid luminometric and fluorescence methods. A sharp decrease (>95%) in the succinate-sustained ATP synthesis rate of the particles was found, but both the ATP hydrolysis rate and ATP-driven proton translocation (when the protons flow from the matrix to the cytosol) were minimally affected. The T8993G mutation changes the highly conserved residue Leu(156) to Arg in the ATPase 6 subunit (subunit a). This subunit, together with subunit c, is thought to cooperatively catalyze proton translocation and rotate, one with respect to the other, during the catalytic cycle of the F(1)F(0) complex. Our results suggest that the T8993G mutation induces a structural defect in human F(1)F(0)-ATPase that causes a severe impairment of ATP synthesis. This is possibly due to a defect in either the vectorial proton transport from the cytosol to the mitochondrial matrix or the coupling of proton flow through F(0) to ATP synthesis in F(1). Whatever mechanism is involved, this leads to impaired ATP synthesis. On the other hand, ATP hydrolysis that involves proton flow from the matrix to the cytosol is essentially unaffected.
ATP Synthetase Complexes↗
Nuclear gene dosage effects upon the expression of maize mitochondrial genes.
Each mitochondrion possesses a genome that encodes some of its own components. The nucleus encodes most of the mitochondrial proteins, including the polymerases and factors that regulate the expression of mitochondrial genes. Little is known about the number or location of these nuclear factors. B-A translocations were used to create dosage series for 14 different chromosome arms in maize plants with normal cytoplasm. The presence of one or more regulatory factors on a chromosome arm was indicated when variation of its dosage resulted in the alteration in the amount of a mitochondrial transcript. We used quantitative Northern analysis to assay the transcript levels of three mitochondrially encoded components of the cytochrome c oxidase complex (cox1, cox2, and cox3). Data for a nuclearly encoded component (cox5b) and for two mitochondrial genes that are unrelated to cytochrome c oxidase, ATP synthase alpha-subunit and 18S rRNA, were also determined. Two tissues, embryo and endosperm, were compared and most effects were found to be tissue specific. Significantly, the array of dosage effects upon mitochondrial genes was similar to what had been previously found for nuclear genes. These results support the concept that although mitochondrial genes are prokaryotic in origin, their regulation has been extensively integrated into the eukaryotic cell.
ATP Synthetase Complexes↗
Analyses of muscle proteins in a patient with a mitochondrial myopathy.
Using the small amounts of muscle available from biopsy (approximately 100 mg), from both normal controls and a patient with a previously identified defect of the mitochondrial electron transfer protein complex III, we analyzed both structural and mitochondrial proteins. The myosin light chains were found to be unchanged with respect to charge or size between patient and control. Two prominent proteins detected after two dimensional gel electrophoresis were present in the patient's total homogenised muscle protein but were not detected in the controls. One protein was positively identified as cytochrome c oxidase subunit II and the other tentatively as a component of the ATP synthetase. We suggest that the increased amounts of these proteins represents a response of the patients muscle cells to the ATP deficiency caused by the primary lesion in complex III.
ATP Synthetase Complexes↗
Genotoxic and functional consequences of transplacental zidovudine exposure in fetal monkey brain mitochondria.
Mitochondrial toxicity was assessed in the brains of developing Erythrocebus patas monkey fetuses exposed in utero to the nucleoside analogue drug zidovudine (3'-azido-3'deoxythymidine or AZT). Pregnant E. patas monkeys were given 0 (n = 5), 10 (n = 3), and 40 (n = 3) mg of AZT/day, equivalent to 21 and 86% of the human daily dose, for the last half (about 10 weeks) of gestation. Mitochondria were isolated from fetal cerebrum and cerebellum at birth and mitochondrial morphology was examined in these tissues by transmission electron microscopy (TEM). Oxidative phosphorylation (OXPHOS) enzyme specific activities were measured spectrophotometrically. Mitochondrial DNA (mtDNA) integrity and quantity were determined by Southern blot and slot blot analysis. In the cerebral mitochondria, reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (complex I) specific activity decreased by 25% in monkeys treated with 40 mg of AZT/day compared with unexposed monkeys (p > or = .05). At the same AZT dose in the cerebral mitochondria, succinate dehydrogenase (complex II) and cytochrome c reductase (complex IV)-specific activities showed dose-dependent increases (p > or = .05), compared with those in controls. In the cerebellum, no difference was seen in mitochondrial OXPHOS enzyme activities between unexposed and exposed fetuses. Furthermore, TEM demonstrated no difference in mitochondrial morphology in frontal cerebrum or cerebellum from unexposed and exposed fetuses, and all fetuses had similar amounts of mtDNA in both tissues. Cerebral mtDNA degradation was noted in the highest AZT dosage group, whereas mtDNA from cerebellum was uneffected. Thus, in fetal patas monkeys given a human equivalent daily dose of AZT during the last half of pregnancy, mitochondria in the fetal cerebrum appear to sustain moderate damage, while the fetal cerebellum mitochondria were not effected.
Animals↗
Chemical modification of the F0 part of the ATP synthase (F1F0) from Escherichia coli. Effects on proton conduction and F1 binding.
The purified F0 part of the ATP synthase complex from Escherichia coli was incorporated into liposomes and chemically modified by various reagents. The modified F0-liposomes were assayed for H+ uptake and, after reconstitution with F1, for total and dicyclohexylcarbodiimide-sensitive ATPase activity. The water-soluble carbodiimide, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide methiodide, (1.2 mM), inhibited H+ uptake to a great extent. Binding of F1 was almost unaffected, but the hydrolysis of ATP was uncoupled from H+ transport. This is reflected by the inhibition of dicyclohexylcarbodiimide-sensitive ATPase activity. Woodward's reagent K, N-ethyl-5-phenylisoxazolium-3'-sulfonate, inhibited both H+ uptake and total ATPase activity. Modification of arginine residues by phenylglyoxal (20 mM) was followed by inhibition of the F1 binding activity by 80% of the control. H+ translocation was reduced to 70%. Diethylpyrocarbonate (3 mM) exhibited a strong inhibiting effect on H+ uptake but not on F1 binding. Modification of tyrosine (by tetranitromethane) as well as lysine residues (by succinic anhydride) did not affect F0 functions. From the data presented we conclude that carboxyl-groups, different from the dicyclohexylcarbodiimide-binding site, are involved in H+ translocation through F0 and, in part, in the functional binding of F1. Furthermore, for the latter function, also arginine residues seem to be important. The role of histidine residues remains unclear at present.
ATP Synthetase Complexes↗
Simultaneous synthesis and hydrolysis of ATP regulated by the inhibitor protein in submitochondrial particles.
Coupled submitochondrial particles from bovine heart with ATP synthases devoid of control by the inhibitor protein of Pullman and Monroy [J. Biol. Chem. 238, 3762-3769 (1963)] can be prepared by incubation of Mg-ATP particles in 50 mM phosphate, 250 mM sucrose, and greater than 95% D2O (pD 7.8) at 38 degrees C. As monitored with oxonol, the respiring particles build up and maintain a delta psi about 5-10% lower than that of the starting preparation. With oligomycin delta psi of the two preparations is the same. In the presence of an ATP trap (hexokinase and glucose), the two types of particles carry out oxidative phosphorylation at comparable rates. Low concentrations of oligomycin induce a small enhancement of the rate of ATP synthesis in non-controlled particles. In the absence of an ATP trap, net accumulation of ATP, as driven by electron transport in particles without control by the inhibitor protein, is low. Apparently this is due to lack of control by the inhibitor protein of ATP hydrolysis that occurs during oxidative phosphorylation.
ATP Synthetase Complexes↗
Cloning and expression of the genes of two fumarate reductase subunits from Wolinella succinogenes.
The fumarate reductase complex of the anaerobic bacterium Wolinella succinogenes catalyzes the electron transfer from menaquinol to fumarate. Two structural genes coding for subunits of the enzyme have been cloned in Escherichia coli. The genes were isolated from a lambda EMBL3 phage gene bank by immunological screening and subcloned in an expression vector. The genes frdA and frdB, which encode the FAD protein (Frd A, Mr 79,000) and the iron-sulfur protein (Frd B, Mr 31,000) of the fumarate reductase complex, were cloned together with a W. succinogenes promoter. The gene order was promoter-frdA-frdB. The FAD protein and the iron-sulfur protein were expressed in the correct molar mass in E. coli from the clones. The identity of the frdA gene and the suggested polarity were confirmed by comparing the amino-terminal sequence of the Frd A protein with that predicted from the 5'-terminal nucleotide sequence of frdA. The frdA and frdB genes are present only once in the genome. A region downstream of frdB, possibly a gene encoding cytochrome b of the fumarate reductase complex, hybridizes with a second site in the genome.
Amino Acid Sequence↗
Hypochlorous acid and myeloperoxidase-catalyzed oxidation of iron-sulfur clusters in bacterial respiratory dehydrogenases.
Hypochlorous acid and related oxidants derived from myeloperoxidase-catalyzed reactions contribute to the microbicidal activities of phagocytosing neutrophils and monocytes. Microbial iron-sulfur (Fe/S) clusters have been suggested as general targets of myeloperoxidase-derived oxidations, but no susceptible Fe/S site has yet been identified. In this study, the effects of HOCl and myeloperoxidase-catalyzed peroxidation of chloride ion upon EPR-detectable Fe/S clusters in Escherichia coli and Pseudomonas aeruginosa were examined. Increasing amounts of oxidant produced progressive loss of signal amplitudes from the S-1 and S-3 Fe/S clusters of succinate:ubiquinone oxidoreductase in respiring membrane fragments. These changes were compared to loss of microbial viability, succinate uptake rates, succinate dehydrogenase activity and succinate-dependent respiration. The amounts of oxidant required to destroy Fe/S clusters exceeded the amounts required to kill organisms or inhibit respiratory function by factors of four or five. Power saturation characteristics of the S-1 signal indicated that the S-2 signal was also resistant to modification, even in highly oxidized membranes. Loss of succinate-dependent respiration was closely associated with HOCl and myeloperoxidase-mediated microbicidal activity against P. aeruginosa and was also an early event in the oxidant-mediated metabolic dysfunctions of E. coli. However, these effects were not caused by the destruction of the Fe/S clusters within the succinate:ubiquinone oxidoreductase. Rather, the major respiration-inhibiting lesion(s) appeared to reside at points in the respiratory chain between the Fe/S clusters and the ubiquinone reductase site.
Electron Spin Resonance Spectroscopy↗
Architecture of succinate dehydrogenase and reactive oxygen species generation.
The structure of Escherichia coli succinate dehydrogenase (SQR), analogous to the mitochondrial respiratory complex II, has been determined, revealing the electron transport pathway from the electron donor, succinate, to the terminal electron acceptor, ubiquinone. It was found that the SQR redox centers are arranged in a manner that aids the prevention of reactive oxygen species (ROS) formation at the flavin adenine dinucleotide. This is likely to be the main reason SQR is expressed during aerobic respiration rather than the related enzyme fumarate reductase, which produces high levels of ROS. Furthermore, symptoms of genetic disorders associated with mitochondrial SQR mutations may be a result of ROS formation resulting from impaired electron transport in the enzyme.
Aerobiosis↗
Structural biology. Complex II is complex too.
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Aerobiosis↗
Respiratory development in Saccharomyces cerevisiae grown at controlled oxygen tension.
Saccharomyces cerevisiae was grown in batch culture over a wide range of oxygen concentrations, varying from the anaerobic condition to a maximal dissolved oxygen concentration of 3.5 muM. The development of cells was assayed by measuring amounts of the aerobic cytochromes aa(3), b, c, and c(1), the cellular content of unsaturated fatty acids and ergosterol, and the activity of respiratory enzyme complexes. The half-maximal levels of membrane-bound cytochromes aa(3), b, and c(1), were reached in cells grown in O(2) concentrations around 0.1 muM; this was similar to the oxygen concentration required for half-maximal levels of unsaturated fatty acid and sterol. However, the synthesis of ubiquinone and cytochrome c and the increase in fumarase activity were essentially linear functions of the dissolved oxygen concentration up to 3.5 muM oxygen. The synthesis of the succinate dehydrogenase, succinate cytochrome c reductase, and cytochrome c oxidase complexes showed different responses to changes in O(2) concentration in the growth medium. Cyanide-insensitive respiration and P(450) cytochrome content were maximal at 0.25 muM oxygen and declined in both more anaerobic and aerobic conditions. Cytochrome c peroxidase and catalase activities in cell-free homogenates were high in all but the most strictly anaerobic cells.
Aerobiosis↗