ABSTRACT
The structures of the NADH dehydrogenases from Bos taurus and Aquifex aeolicus have been determined by 3D electron microscopy, and have been analyzed in comparison with the previously determined structure of Complex I from Yarrowia lipolytica. The results show a clearly preserved domain structure in the peripheral arm of complex I, which is similar in the bacterial and eukaryotic complex. The membrane arms of both eukaryotic complexes show a similar shape but also significant differences in distinctive domains. One of the major protuberances observed in Y. lipolytica complex I appears missing in the bovine complex, while a protuberance not found in Y. lipolytica connects in bovine complex I a domain of the peripheral arm to the membrane arm. The structural similarities of the peripheral arm agree with the common functional principle of all complex Is. The differences seen in the membrane arm may indicate differences in the regulatory mechanism of the enzyme in different species.
Subject(s)
Electron Transport Complex I/chemistry , Animals , Bacteria/metabolism , Cattle , Electron Transport Complex I/metabolism , Electron Transport Complex I/ultrastructure , Eukaryota , Microscopy, Electron , Models, Molecular , NADH Dehydrogenase/chemistry , NADH Dehydrogenase/metabolism , NADH Dehydrogenase/ultrastructure , Protein Structure, Tertiary , Yarrowia/metabolismABSTRACT
Cardiomyopathy associated with a mitochondrial DNA depletion syndrome is a rare condition. We report on a child with a hypertrophic cardiomyopathy and a mitochondrial depletion syndrome who was successfully treated by heart transplantation, given the tissue-specific nature of her mitochondrial disorder.
Subject(s)
Cardiomyopathy, Hypertrophic, Familial/genetics , Cardiomyopathy, Hypertrophic, Familial/surgery , DNA, Mitochondrial/analysis , Heart Transplantation , Electron Transport Complex IV/analysis , Female , Humans , Infant , Muscle, Skeletal/cytology , Myocardium/enzymology , Myocardium/pathology , Succinate Dehydrogenase/analysisABSTRACT
The ratios of the oxidative phosphorylation complexes NADH:ubiquinone reductase (complex I), succinate:ubiquinone reductase (complex II), ubiquinol:cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and F1F0-ATP synthase (complex V) from bovine heart mitochondria were determined by applying three novel and independent approaches that gave consistent results: 1) a spectrophotometric-enzymatic assay making use of differential solubilization of complexes II and III and parallel assays of spectra and catalytic activities in the samples before and after ultracentrifugation were used for the determination of the ratios of complexes II, III, and IV; 2) an electrophoretic-densitometric approach using two-dimensional electrophoresis (blue native-polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis) and Coomassie blue-staining indices of subunits of complexes was used for determining the ratios of complexes I, III, IV, and V; and 3) two electrophoretic-densitometric approaches that are independent of the use of staining indices were used for determining the ratio of complexes I and III. For complexes I, II, III, IV, and V in bovine heart mitochondria, a ratio 1.1 +/- 0.2:1.3 +/- 0.1:3:6.7 +/- 0.8:3.5 +/- 0.2 was determined.
Subject(s)
Mitochondria, Heart/enzymology , NADH, NADPH Oxidoreductases/metabolism , Animals , Catalysis , Cattle , Electron Transport , Electrophoresis, Gel, Two-Dimensional , Glucosides/chemistry , Oxidative Phosphorylation , Spectrophotometry, UltravioletABSTRACT
Hereditary Spastic Paraplegias (HSPs) are heterogeneous neurodegenerative disorders whose etiopathogenesis is still unclear. The identification of pathogenic mutations in a gene (SPG7) encoding a mitochondrial metalloprotease suggested that oxidative phosphorylation (OXPHOS) alterations might underlie HSP in a subgroup of patients. We performed clinical, morphological, biochemical, and molecular genetic studies in six HSP patients and in six sporadic patients to investigate OXPHOS in muscle biopsies. Complicated and pure forms were included in our study. Morphological alterations of the type seen in OXPHOS-related disorders were found in three patients. Five patients showed an isolated defect of complex I activity. No mutations in the SPG7 gene were detected. Our results suggest that OXPHOS defects in HSP patients are more common than previously believed.
Subject(s)
Electron Transport/genetics , Paraplegia/genetics , Paraplegia/metabolism , Adolescent , Adult , Biopsy , Child , Female , Humans , Male , Mitochondria/metabolism , Oxidative Phosphorylation , Paraplegia/pathology , PedigreeABSTRACT
The low-resolution structure and overall dimensions of the A(3)B(3)CDF complex of the A(1) ATPase from Methanosarcina mazei Gö1 in solution is analyzed by synchrotron X-ray small-angle scattering. The radius of gyration and the maximum size of the complex are 5.03 +/- 0.1 and 18.0 +/- 0.1 nm, respectively. The low-resolution shape of the protein determined by two independent ab initio approaches has a knob-and-stalk-like feature. Its headpiece is approximately 9.4 nm long and 9.2 nm wide. The stalk, which is known to connect the headpiece to its membrane-bound A(O) part, is approximately 8.4 nm long. Limited tryptic digestion of the A(3)B(3)CDF complex was used to probe the topology of the smaller subunits (C-F). Trypsin was found to cleave subunit C most rapidly at three sites, Lys(20), Lys(21), and Arg(209), followed by subunit F. In the A(3)B(3)CDF complex, subunit D remained protected from proteolysis.
Subject(s)
Adenosine Triphosphatases/chemistry , Archaeal Proteins/chemistry , Methanosarcina/enzymology , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Archaeal Proteins/metabolism , Hydrolysis , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Scattering, Radiation , Structure-Activity Relationship , Trypsin/metabolism , X-RaysSubject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Membrane Proteins/isolation & purification , Mitochondria, Muscle/chemistry , Mitochondria/chemistry , Animals , Buffers , Cattle , Coloring Agents/chemistry , Humans , Intracellular Membranes/chemistry , Macromolecular Substances , Membrane Proteins/chemistry , Molecular WeightABSTRACT
The authors identified a novel mtDNA mutation (T9176G) in the ATPase 6 gene in a family in which a 10-year-old girl had a severe neurodegenerative disorder, her elder sister had died of Leigh syndrome (LS), and a maternal uncle had a spinocerebellar disorder. Biochemical studies disclosed a reduced rate of ATP synthesis in skin fibroblast cultures from the proposita as the likely explanation of her severe illness. The findings expand the genetic variants associated with LS.
Subject(s)
Adenosine Triphosphate/biosynthesis , DNA, Mitochondrial/genetics , Leigh Disease/etiology , Leigh Disease/genetics , Mutation/genetics , Child , DNA, Mitochondrial/metabolism , Female , Humans , PedigreeABSTRACT
The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium Thauera aromatica. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown cells did not utilize 3-hydroxybenzoate. The first step in 3-hydroxybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate-CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the N-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate-CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of beta-oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways.
Subject(s)
Coenzyme A Ligases/isolation & purification , Hydroxybenzoates/metabolism , Oxidoreductases Acting on CH-CH Group Donors , Thauera/metabolism , Acyl Coenzyme A/metabolism , Anaerobiosis , Cloning, Molecular , Enzyme Induction , Genes, Bacterial , Models, Genetic , Molecular Sequence Data , Nitrates/metabolism , Nitrites , Oxidation-Reduction , Oxidoreductases/isolation & purification , Oxidoreductases/metabolism , Sequence Analysis, DNA , Sequence Analysis, Protein , Substrate SpecificityABSTRACT
Respiratory chain supercomplexes have been isolated from mammalian and yeast mitochondria, and bacterial membranes. Functional roles of respiratory chain supercomplexes are catalytic enhancement, substrate channelling, and stabilization of complex I by complex III in mammalian cells. Bacterial supercomplexes are characterized by their relatively high detergent-stability compared to yeast or mammalian supercomplexes that are stable to sonication. The mobility of substrate cytochrome c increases in the order bacterial, yeast, and mammalian respiratory chain. In bacterial supercomplexes, the electron transfer between complexes III and IV involves movement of the mobile head of a tightly bound cytochrome c, whereas the yeast S. cerevisiae seems to use substrate channelling of a mobile cytochrome c, and mammalian respiratory chains have been described to use a cytochrome c pool. Dimeric ATP synthase seems to be specific for mitochondrial OXPHOS systems. Monomeric complex V was found in Acetobacterium woodii and Paracoccus denitrificans.
Subject(s)
Cell Respiration , Animals , Electron Transport , Macromolecular Substances , Mitochondrial Proton-Translocating ATPases/chemistry , Mitochondrial Proton-Translocating ATPases/metabolism , Oxidative Phosphorylation , YeastsABSTRACT
Mitochondrial complex I exhibits some peculiar and poorly understood features regarding the effects of detergents on activity and sensitivity to hydrophobic inhibitors that are not seen with other membrane complexes using ubiquinone as a substrate. Therefore, we investigated the interaction of complex I from bovine heart mitochondria with different types of detergents by monitoring activity, degree of inhibition and inhibitor binding in the presence of increasing concentrations of detergent. It is shown that apart from their nature as solubilizing and delipidating agents the polyoxyethylene-ether detergents Triton X-100, Brij-35 and Thesit act as specific inhibitors of complex I and compete with classical complex I inhibitors for a common binding domain. These findings were used to develop a novel large-scale chromatographic procedure for isolation of inhibitor-sensitive NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxylapatite, ion-exchange and gel-exclusion chromatography. By switching detergents from Triton X-100 to dodecylmaltoside after hydroxylapatite chromatography the procedure yields highly pure, monodisperse and fully inhibitor-sensitive enzyme.
Subject(s)
Detergents/pharmacology , Enzyme Inhibitors/pharmacology , Mitochondria, Heart/drug effects , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Amines/chemistry , Animals , Binding, Competitive , Catalysis , Cattle , Chromatography, Gel , Chromatography, Ion Exchange , Electron Spin Resonance Spectroscopy , Electron Transport Complex I , Hydrogen-Ion Concentration , Mitochondria, Heart/enzymology , NADH, NADPH Oxidoreductases/isolation & purification , TemperatureABSTRACT
Mitochondrial proton-translocating NADH-dehydrogenase (complex I) is one of the largest and most complicated membrane bound protein complexes. Despite its central role in eukaryotic oxidative phosphorylation and its involvement in a broad range of human disorders, little is known about its structure and function. Therefore, we have started to use the powerful genetic tools available for the strictly aerobic yeast Yarrowia lipolytica to study this respiratory chain enzyme. To establish Y. lipolytica as a model system for complex I, we purified and characterized the multisubunit enzyme from Y lipolytica and sequenced the nuclear genes coding for the seven central subunits of its peripheral part. Complex I from Y lipolytica is quite stable and could be isolated in a highly pure and monodisperse state. One binuclear and four tetranuclear iron-sulfur clusters, including N5, which was previously known only from mammalian mitochondria, were detected by EPR spectroscopy. Initial structural analysis by single particle electron microscopy in negative stain and ice shows complex I from Y. lipolytica as an L-shaped particle that does not exhibit a thin stalk between the peripheral and the membrane parts that has been observed in other systems.
Subject(s)
Genes, Fungal , NADH, NADPH Oxidoreductases/chemistry , Protons , Yeasts/enzymology , Yeasts/genetics , Amino Acid Sequence , Biological Transport , Cloning, Molecular , Cold Temperature , Electron Spin Resonance Spectroscopy , Electron Transport Complex I , Microscopy, Electron , Mitochondria/enzymology , Molecular Sequence Data , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/isolation & purification , Polymerase Chain ReactionABSTRACT
The Na(+)-F(1)F(0)-ATPase operon of Acetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunit a and b, a gene encoding a 16-kDa proteolipid (subunit c(1)), and two genes encoding 8-kDa proteolipids (subunits c(2) and c(3)). Because subunits a, b, and c(1) were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a, b, and c(1) are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunits a, c(2), c(3), b, delta, alpha, gamma, beta, and epsilon. Biochemical and immunological analyses revealed that subunits c(1), c(2), and c(3) are all part of the c-oligomer, the first of a F(1)F(0)-ATPase that contains 8- and 16-kDa proteolipids.
Subject(s)
Gram-Positive Rods/enzymology , Proton-Translocating ATPases/chemistry , Cytoplasm/enzymology , Escherichia coli/genetics , Immune Sera/immunology , Protein Subunits , Proton-Translocating ATPases/genetics , Proton-Translocating ATPases/immunologyABSTRACT
The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CF(O)F(1)) and its subcomplexes CF(O) and CF(1) were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569-575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CF(O)F(1), CF(O) or CF(1) and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF(1) and ATP synthesis activity of reconstituted CF(O)F(1) were determined before and after dye removal. The secondary structure of CF(O) was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CF(O)F(1) and CF(1) nor affects the subunit composition and the high alpha-helical content of CF(O). In crystallization attempts, 2D arrays of CF(O)F(1) and of CF(O) before and after dye removal were obtained. In the aggregates of CF(O), circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization.
Subject(s)
Chloroplasts/enzymology , Indicators and Reagents , Proton-Translocating ATPases/chemistry , Rosaniline Dyes , Spinacia oleracea/enzymology , Catalysis , Crystallization , Electrophoresis, Polyacrylamide Gel/methods , Indicators and Reagents/chemistry , Molecular Structure , Protein Structure, Secondary , Proton-Translocating ATPases/isolation & purification , Proton-Translocating ATPases/metabolism , Rosaniline Dyes/chemistryABSTRACT
A differential protein display screen resulted in the identification of a 27-kDa protein which strongly accumulates during the senescence of Podospora anserina cultures grown under standard conditions. After partial determination of the amino-acid sequence by mass-spectrometry analysis of trypsin-generated fragments, pairs of degenerated primers were deduced and used to amplify parts of the sequence coding for the protein. These PCR products were utilized to select specific cDNA and genomic clones from DNA libraries of P. anserina. A subsequent DNA-sequence analysis revealed that the 27-kDa protein is encoded by a discontinuous gene, PaMth1, capable of coding for 240 amino acids. The first three amino-terminal residues appear to be removed post-translationally. The deduced amino-acid sequence shows significant homology to S-adenosylmethionine (SAM)-dependent methyltransferases. We hypothesize that the 27-kDa protein, PaMTH1, is involved in age-related methylation reactions protecting aging cultures against increasing oxidative stress.
Subject(s)
Ascomycota/genetics , Fungal Proteins/genetics , Methyltransferases/genetics , Amino Acid Sequence , Ascomycota/enzymology , Ascomycota/growth & development , Base Sequence , Cloning, Molecular , DNA, Fungal/chemistry , DNA, Fungal/genetics , Electrophoresis, Polyacrylamide Gel , Exons , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Genes, Fungal/genetics , Introns , Molecular Sequence Data , Molecular Weight , Sequence Alignment , Sequence Analysis, DNA , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Time FactorsABSTRACT
Around 30-40 years after the first isolation of the five complexes of oxidative phosphorylation from mammalian mitochondria, we present data that fundamentally change the paradigm of how the yeast and mammalian system of oxidative phosphorylation is organized. The complexes are not randomly distributed within the inner mitochondrial membrane, but assemble into supramolecular structures. We show that all cytochrome c oxidase (complex IV) of Saccharomyces cerevisiae is bound to cytochrome c reductase (complex III), which exists in three forms: the free dimer, and two supercomplexes comprising an additional one or two complex IV monomers. The distribution between these forms varies with growth conditions. In mammalian mitochondria, almost all complex I is assembled into supercomplexes comprising complexes I and III and up to four copies of complex IV, which guided us to present a model for a network of respiratory chain complexes: a 'respirasome'. A fraction of total bovine ATP synthase (complex V) was isolated in dimeric form, suggesting that a dimeric state is not limited to S.cerevisiae, but also exists in mammalian mitochondria.
Subject(s)
Electron Transport , Mitochondria/chemistry , Saccharomyces cerevisiae/chemistry , ATP Synthetase Complexes , Animals , Cattle , Detergents/metabolism , Digitonin/metabolism , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Mitochondria, Heart/chemistry , Models, Biological , Multienzyme Complexes/chemistry , Oxidative Phosphorylation , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Protein Binding , Saccharomyces cerevisiae/enzymologyABSTRACT
The molecular basis of glomerular permselectivity remains largely unknown. The congenital nephrotic syndrome of the Finnish type (CNF) characterized by massive proteinuria already present but without extrarenal symptoms is a unique human disease model of pure proteinuria. In search of genes and pathophysiologic mechanisms associated with proteinuria, we used differential display-PCR to identify differences in gene expression between glomeruli from CNF and control kidneys. A distinctly underexpressed PCR product of the CNF kidneys showed over 98% identity with a mitochondrially encoded cytochrome c oxidase (COX I). Using a full-length COX I cDNA probe, we verified down-regulation of COX I mRNA to 1/4 of normal kidney values on Northern blots. In addition, transcripts of other mitochondrially encoded respiratory chain complexes showed a similar down-regulation whereas the respective nuclearly encoded complexes were expressed at comparable levels. Additional studies using histochemical, immunohistochemical, in situ hybridization, RT-PCR, and biochemical and electron microscopic methods all showed a mitochondrial involvement in the diseased kidneys but not in extrarenal blood vessels. As a secondary sign of mitochondrial dysfunction, excess lipid peroxidation products were found in glomerular structures in CNF samples. Our data suggest that mitochondrial dysfunction occurs in the kidneys of patients with CNF, with subsequent lipid peroxidation at the glomerular basement membrane. Our additional studies have revealed similar down-regulation of mitochondrial functions in experimental models of proteinuria. Thus, mitochondrial dysfunction may be a crucial pathophysiologic factor in this symptom.
Subject(s)
Gene Expression , Mitochondria/physiology , Nephrotic Syndrome/physiopathology , Adolescent , Adult , Base Sequence , Blotting, Northern , Child , Down-Regulation , Electron Transport , Electron Transport Complex IV/biosynthesis , Electron Transport Complex IV/genetics , Humans , In Situ Hybridization , Kidney/blood supply , Kidney/enzymology , Kidney/ultrastructure , Lipid Peroxidation , Microscopy, Electron , Mitochondria/enzymology , Molecular Sequence Data , Nephrotic Syndrome/complications , Nephrotic Syndrome/enzymology , Polymerase Chain Reaction , Proteinuria/complicationsABSTRACT
Five patients with diminished activity of complex III of the mitochondrial respiratory chain have been screened for mutations in the mitochondrial cytochrome b (cyt b) gene. In 1 patient, a young boy with an akinetic rigid syndrome and a mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), a novel 4-base pair deletion was identified. This mutation in this highly conserved gene is considered to be pathogenic since it is a heteroplasmic frame shift mutation predicted to lead to a truncated protein.
Subject(s)
Cytochrome b Group/genetics , Gene Deletion , MELAS Syndrome/genetics , Mitochondria/genetics , Parkinson Disease, Secondary/genetics , Adult , Blotting, Southern , DNA Mutational Analysis , Electron Transport Complex III/genetics , Humans , MELAS Syndrome/complications , Male , Oxidative Phosphorylation , Parkinson Disease, Secondary/complications , PhenotypeABSTRACT
The subunit composition of the mitochondrial ATP synthase from Saccharomyces cerevisiae was analyzed using blue native gel electrophoresis and high resolution SDS-polyacrylamide gel electrophoresis. We report here the identification of a novel subunit of molecular mass of 6,687 Da, termed subunit j (Su j). An open reading frame of 127 base pairs (ATP18), which encodes for Su j, was identified on chromosome XIII. Su j does not display sequence similarity to ATP synthase subunits from other organisms. Data base searches, however, identified a potential homolog from Schizosaccharomyces pombe with 51% identity to Su j of S. cerevisiae. Su j, a small protein of 59 amino acid residues, has the characteristics of an integral inner membrane protein with a single transmembrane segment. Deletion of the ATP18 gene encoding Su j led to a strain (Deltasu j) completely deficient in oligomycin-sensitive ATPase activity and unable to grow on nonfermentable carbon sources. The presence of Su j is required for the stable expression of subunits 6 and f of the F0 membrane sector. In the absence of Su j, spontaneously arising rho- cells were observed that lacked also ubiquinol-cytochrome c reductase and cytochrome c oxidase activities. We conclude that Su j is a novel and essential subunit of yeast ATP synthase.
Subject(s)
Mitochondria/enzymology , Mitochondrial Proton-Translocating ATPases , Proton-Translocating ATPases/genetics , Proton-Translocating ATPases/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Schizosaccharomyces/enzymology , Amino Acid Sequence , Base Sequence , DNA Primers , Electrophoresis, Polyacrylamide Gel/methods , Gene Deletion , Intracellular Membranes/enzymology , Molecular Sequence Data , Open Reading Frames , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/genetics , Sequence Homology, Amino AcidABSTRACT
Using the technique of blue native gel electrophoresis, the oligomeric state of the yeast mitochondrial F1F0-ATP synthase was analysed. Solubilization of mitochondrial membranes with low detergent to protein ratios led to the identification of the dimeric state of the ATP synthase. Analysis of the subunit composition of the dimer, in comparison with the monomer, revealed the presence of three additional small proteins. These dimer-specific subunits of the ATP synthase were identified as the recently described subunit e/Tim11 (Su e/Tim11), the putative subunit g homolog (Su g) and a new component termed subunit k (Su k). Although, as shown here, these three proteins are not required for the formation of enzymatically active ATP synthase, Su e/Tim11 and Su g are essential for the formation of the dimeric state. Su e/Tim11 appears to play a central role in this dimerization process. The dimer-specific subunits are associated with the membrane bound F0-sector. The F0-sector may thereby be involved in the dimerization of two monomeric F1F0-ATP synthase complexes. We speculate that the F1F0-ATP synthase of yeast, like the other complexes of oxidative phosphorylation, form supracomplexes to optimize transduction of energy and to enhance the stability of the complex in the membrane.
Subject(s)
Mitochondria/enzymology , Proton-Translocating ATPases/chemistry , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Dimerization , Electrophoresis/methods , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Mutation , Proton-Translocating ATPases/biosynthesis , Proton-Translocating ATPases/genetics , Sequence Analysis , Sequence Deletion , Surface PropertiesABSTRACT
Many aromatic compounds are anaerobically oxidized to CO2 via benzoyl-CoA as the common aromatic intermediate. In Thauera aromatica, the central benzoyl-CoA pathway comprises the ATP-driven two-electron reduction of the benzene ring; this reaction uses a ferredoxin as electron donor and is catalyzed by benzoyl-CoA reductase. The first intermediate, cyclohex-1,5-diene-1-carboxyl-CoA, is subsequently hydrated by dienoyl-CoA hydratase to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA. Formation of the main product produced by cell extracts, 3-hydroxypimelyl-CoA, requires at least two further steps; the oxidation of a hydroxyl group and the hydrolytic carbon ring cleavage of a CoA-activated beta-oxoacid. In addition, enoyl-CoA hydratase may come into play. A cluster of eight adjacent genes, which are transcribed in the same direction and may form an operon, was found in this bacterium. The cluster codes for proven and postulated enzymes of the benzoyl-CoA pathway. The genes for the enzymes code for ferredoxin, four subunits of benzoyl-CoA reductase, dienoyl-CoA hydratase, 6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase (NAD+), and the ring hydrolyzing enzyme. The deduced amino acid sequences of these proteins were 35-86% similar to the corresponding sequences found in Rhodopseudomonas palustris. Benzoyl-CoA reductase subunits exhibit distinct similarities with 2-hydroxyglutaryl-CoA dehydratase and its ATP-hydrolysing activase protein of Acidaminococcus fermentans as well as with open reading frames of unknown function in other bacteria. Conversion of benzoyl-CoA to 3-hydroxypimelyl-CoA can be explained by a minimal model of the benzoyl-CoA pathway assuming the four enzymes whose genes were characterized and an additional enoyl-CoA hydratase. In R. palustris the dienoyl-CoA hydratase gene is lacking suggesting the operation of a modified benzoyl-CoA pathway with cyclohex-1-ene-1-carboxyl-CoA as intermediate.