Redox Bohr-effects in isolated cytochrome bc1 complex and cytochrome c oxidase from beef-heart mitochondria.
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Animals↗
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The recognition and redox properties of a component, possibly a quinone, which determines electron transfer rate in ubiquinone-cytochrome c oxidoreductase of mitochondria.
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Animals↗
Inhibition of the mitochondrial bc1 complex by dibromothymoquinone.
We have studied the effects of dibromothymoquinone (DBMIB) in various redox activities of the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations higher than 50 mol/mol of cytochrome c1 the inhibitor produces a bypass of electron transfer on the substrate side of the bc1 complex, because of its autooxidation capability. This induces an artifactual overestimation of the real inhibition titer of the redox activity of this enzyme, which has been found to be 3-6 mol/mol of cytochrome c1 by following the ubiquinol-cytochrome c reductase activity. This action is reversed by addition of excess of sulphydryl compounds like cysteine.
Animals↗
Reductive titration of CoQ-depleted Complex III from Baker's yeast. Evidence for an exchange-coupled complex between QH . and low-spin ferricytochrome b.
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Cytochrome b Group↗
Relationships of respiratory chain and ATP-synthetase in energized mitochondria.
The present study reveals that the previously described effect of ATP-synthetase inhibition concomitant with inhibition of respiratory chain functioning may be observed at different absolute values of delta psi on the mitochondrial membrane. This fact points out that the membrane potential is not a unique regulator in coupling of ATP-synthetase and respiratory chain activities. We found, using the double-inhibitor titration technique, that ATP-synthetase inhibition induces proportional inhibition of respiratory chain enzymes and vice versa respiratory chain inhibition induces proportional inhibition of ATP-synthetase. This effect is shown to exist only when osmolarity is close to 150-300 (mosM) (in the physiological range). The coupling effectivity (ADP/O) of mitochondria under these conditions is maximal. Under conditions of high osmolarity (400-600 mosM) the respiratory chain and ATP-synthetase behave as if they were coupled by bulk phase delta -mu H+, from the kinetic point of view.
ATP Synthetase Complexes↗
PRE3, highly homologous to the human major histocompatibility complex-linked LMP2 (RING12) gene, codes for a yeast proteasome subunit necessary for the peptidylglutamyl-peptide hydrolyzing activity.
20S proteasomes are multifunctional proteinase complexes ubiquitous in eucaryotes. We have cloned the yeast PRE3 gene by complementation of the pre3-2 mutation, which leads to a defect in the peptidylglutamyl-peptide hydrolyzing activity of the 20S proteasome. The PRE3 gene, a beta-type member of the proteasomal gene family, is essential for cellular life and codes for a 193-amino acid proteasomal subunit with a predicted molecular mass of 21.2 kDa. The Pre3 protein shows striking homology to the human proteasome subunits Hs delta and Lmp2 (Ring12). Lmp2 is encoded in the major histocompatibility complex class II region implicating proteasomes in antigen processing.
Amino Acid Sequence↗
A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast.
We report genetic and biochemical evidence that the RNA polymerase II carboxy-terminal domain (CTD) interacts with a large multisubunit complex that contains TATA-binding protein (TBP) and is an integral part of the transcription initiation complex. The isolation and characterization of extragenic suppressors of S. cerevisiae RNA polymerase II CTD truncation mutations led us to identify SRB2, SRB4, SRB5, and SRB6 as genes involved in CTD function in vivo. SRB2 was previously isolated and shown to encode a 23 kd TBP-binding protein. The four SRB proteins and a portion of cellular TBP are components of a high molecular weight multisubunit complex that is tightly bound to RNA polymerase II. This SRB-TBP complex binds specifically to recombinant CTD protein. In vitro transcription and template commitment assays confirm that SRB2 and SRB5 are components of a functional preinitiation complex and are required for efficient transcription initiation.
Amino Acid Sequence↗
Stabilized ubisemiquinone in reconstituted succinate ubiquinone reductase.
QP-S, a ubiquinone (Q) protein, accepts electrons from succinate through succinate dehydrogenase (SDH). A new method has produced a preparation of QP-S which has a different amino acid composition and SDS gel electrophoretic pattern from that of the old preparation (Biochemistry 19, 3579-3585 (1980)). The new preparation contains less than 1 nmol heme/mg protein; the activity of the preparation was not proportional to its heme content. A thenoyltrifluoroacetone sensitive free radical signal was detected by EPR spectroscopy in succinate-Q reductase reconstituted from this QP-S and SDH; the characteristics of this species identify it as ubisemiquinone. At pH 7.4, the Em of the two electron step was about 70 mV with E1 = 5 mV and E2 = 125 mV. The properties of the radical differed slightly from those of "Qs" radical in more intact preparations (e.g. submitochondrial particles). The present is the simplest system in which such a succinate reducible ubisemiquinone free radical has been demonstrated.
Coenzymes↗
Low-resolution structural studies of mitochondrial ubiquinol:cytochrome c reductase in detergent solutions by neutron scattering.
Mitochondrial ubiquinol:cytochrome c reductase (Mr approximately 600,000) was cleaved into a complex (Mr approximately 280,000) of the subunits III (cytochrome b), IV (cytochrome c1) and VI to IX, a complex (Mr approximately 300,000) of the subunits I and II, and the single subunit V (iron-sulphur subunit, Mr approximately 25,000). Neutron scattering was applied to the whole enzyme, the cytochrome bc1 complex, both in hydrogenated and deuterated alkyl (phenyl) polyoxyethylene detergents, and the complex of subunits I and II in detergent-free solution. The neutron parameters were compared with the structures of the enzyme and the cytochrome bc1 complex previously determined by electron microscopy. Using the method of hard spheres, comparison of the calculated and experimental radius of gyration implies that the length of the enzyme across the bilayer or the detergent micelle is between 150 and 175 A and of the cytochrome bc1 complex between 90 and 115 A. The subunit topography was confirmed. The cleavage plane between the cytochrome bc1 complex and the complex of subunits I and II lies at the centre of the enzyme and runs parallel to the membrane just outside the bilayer. The detergent uniformly surrounds the protein as a belt, which is displaced by 30 to 40 A from the protein centre of the enzyme and by about 20 A from the protein centre of the cytochrome bc1 complex. The low protein matchpoint of the whole enzyme as compared to the subunit complexes is accounted for in terms of the non-exchange of about 30 to 60% of the exchangeable protons within the intact enzyme. Polar residues are, on average, at the protein surface and non-polar residues and polar residues with non-exchanged protons are buried within the enzyme.
Amino Acids↗
On earlier states of the biochemical system.
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Amino Acid Sequence↗
Deficits in a tricarboxylic acid cycle enzyme in brains from patients with Parkinson's disease.
Parkinson's disease (PD) is associated with mitochondrial dysfunction, specifically a deficiency of complex I of the electron transport chain. Most, although not all, studies indicate that this deficiency is limited to brain regions with neurodegeneration. The current studies tested for deficiencies in other mitochondrial components in PD brain in a neuropathologically unaffected region where the abnormality cannot be attributed to secondary effects of neurodegeneration. The activity of a key (and arguably rate-limiting) tricarboxylic acid cycle enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), was measured in the cerebellum of patients with PD. Activity in 19 PD brains was 50.5% of that in 18 controls matched for age, sex, post-mortem interval, and method of preservation (P<0.0019). The protein subunits of KGDHC were present in normal amounts in PD brains, indicating a relatively discrete abnormality in the enzyme. The activities of another mitochondrial enzyme, glutamate dehydrogenase (GDH), were normal in PD brains. These results demonstrate that specific reductions in KGDHC occur even in pathologically unaffected areas in PD, where the decline is unlikely to be a non-specific result of neurodegeneration. Reductions in the activity of this enzyme, if widespread in the brain, may predispose vulnerable regions to further damage.
Adolescent↗
Protection by thiols of the mitochondrial complexes from 4-hydroxy-2-nonenal.
In the present study, the effects of 4-hydroxy-2-nonenal (HNE) on highly purified pyruvate dehydrogenase complex (PDC) and its catalytic components in vitro and on PDC, alpha-ketoglutarate dehydrogenase complex (KGDC), and the branched-chain alpha-keto acid dehydrogenase complex (BCKDC) activities in cultured human HepG2 cells were investigated. Among the PDC components, the activity of the dihydrolipoamide acetyltransferase-E3-binding protein subcomplex (E2-E3BP) only was decreased by HNE. Dihydrolipoamide dehydrogenase (E3) protected the E2-E3BP subcomplex from HNE inactivation in the absence of the substrates. In the presence of E3 and NADH, when lipoyl groups were reduced, higher inactivation of the E2-E3BP subcomplex by HNE was observed. Purified PDC was protected from HNE-induced inactivation by several thiol compounds including lipoic acid plus [LA-plus; 2-(N,N-dimethylamine)ethylamidolipoate(.)HCl]. Treatment of cultured HepG2 cells with HNE resulted in a significant reduction of PDC and KGDC activities, whereas BCKDC activity decreased to a lesser extent. Lipoyl compounds afforded protection from HNE-induced inhibition of PDC. This protection was higher in the presence of cysteine and reduced glutathione. Cysteine was able to restore PDC activity to some extent after HNE treatment. These findings show that thiols, including lipoic acid, provide protection against HNE-induced inactivation of lipoyl-containing complexes in the mitochondria.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
Proteasome components with reciprocal expression to that of the MHC-encoded LMP proteins.
BACKGROUND: Intracellular proteins are processed into small peptides that bind HLA class I molecules of the major histocompatibility complex (MHC) in order to be presented to T lymphocytes. The proteasome, a multi-subunit protease, has recently been implicated in the generation of these peptides. Two genes encoding proteasome subunits, LMP2 and LMP7, are tightly linked to the TAP peptide transport loci in the class II region of the human MHC. Inclusion of the LMP subunits may alter proteasome activity, biasing it towards the production of peptides with carboxyl termini appropriate for binding HLA class I molecules. Nevertheless, mutant cells that lack the LMP genes are able to process and present antigens at the cell surface at similar levels to wild-type cells. These results raise questions about the role of the proteasome, and in particular of the LMP subunits, in antigen processing. RESULTS: We have cloned the genes encoding a new proteasome subunit, MB1, which is closely related to LMP7, and that encoding a second subunit, Delta, which is closely related to LMP2. Expression of the MB1 and delta genes is reciprocal to that of the LMP genes: MB1 and delta are up-regulated in mutant cell lines lacking LMPs and down-regulated in the presence of gamma-interferon. The MB1 and delta genes are found to be located on chromosomes 14 and 17, respectively, raising interesting evolutionary questions about how the LMP genes independently became incorporated into the MHC. CONCLUSIONS: We suggest that the subtle phenotype of LMP-deficient cell lines results from the compensatory expression in these lines of two other proteasome subunits, MB1 and Delta.
Amino Acid Sequence↗
Photoinduced electron transfer in the cytochrome c/cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c. Optical multichannel detection.
Intramolecular electron transfer in the electrostatic cytochrome c oxidase/cytochrome c complex was investigated using a novel photoactivatable dye. Laser photolysis of thiouredopyrenetrisulfonate (TUPS), covalently linked to cysteine 102 on yeast iso-1-cytochrome c, generates a triplet state of the dye, which donates an electron to cytochrome c, followed by electron transfer to cytochrome c oxidase. Time-resolved optical absorption difference spectra were collected at delay times from 100 ns to 200 ms between 325 and 650 nm. On the basis of singular value decomposition (SVD) and multiexponential fitting, three apparent lifetimes were resolved. A sequential kinetic mechanism is proposed from which the microscopic rate constants and spectra of the intermediates were determined. The triplet state of TUPS donates an electron to cytochrome c with a forward rate constant of approximately 2.0 x 10(4) s(-1). A significant fraction of the triplet returns back to the ground state on a similar time scale. The reduction of cytochrome c is followed by faster electron transfer from cytochrome c to Cu(A), with the equilibrium favoring the reduced cytochrome c. Subsequently, Cu(A) equilibrates with heme a with an apparent rate constant of approximately 1 x 10(4) s(-1). On a millisecond time scale, the oxidized TUPS returns to the ground state and heme a becomes reoxidized. The extracted intermediate spectra are in excellent agreement with model spectra of the postulated intermediates, supporting the proposed mechanism.
Animals↗
Functional orientation of the acyltransferase domain in a module of the erythromycin polyketide synthase.
Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multienzymes are organized into a series of functional units known as modules. Each dimeric module contains two catalytically independent clusters of active sites homologous to those of vertebrate fatty acid synthases. Earlier studies have shown that modules consist of head-to-tail homodimers in which ketosynthase (KS) and acyl carrier protein (ACP) domains are contributed by opposite subunits to form a catalytic center. Here, we probe the functional topology of the acyltransferase (AT) domain which transfers the methylmalonyl moiety of methylmalonyl-CoA onto the phosphopantetheine arm of the ACP domain. Using a bimodular derivative of DEBS, the AT domain of module 2 (AT2) was inactivated by site-directed mutagenesis. Heterodimeric protein pairs were generated in vitro between the inactivated AT2 (AT2 degrees) polypeptide and an inactive KS1 (KS1 degrees) or KS2 (KS2 degrees) protein. Both of these hybrid proteins supported polyketide synthesis, suggesting that AT2 can perform its function from either subunit. The apparent catalytic rate constants for each of the two hybrid protein pairs, KS1 degrees/AT2 degrees and KS2 degrees/AT2 degrees, were identical, indicating that no significant kinetic preference exists for a particular AT2-ACP2 combination. These results suggest that the AT domain can be shared between the two clusters of active sites within the same dimeric module. Such a novel structural organization might provide a functional advantage for the efficient biosynthesis of polyketides.
Acyltransferases↗
Chromatin deacetylation by an ATP-dependent nucleosome remodelling complex.
The dynamic assembly and remodelling of eukaryotic chromosomes facilitate fundamental cellular processes such as DNA replication and gene transcription. The repeating unit of eukaryotic chromosomes is the nucleosome core, consisting of DNA wound about a defined octamer of histone proteins. Two enzymatic processes that regulate transcription by targeting elements of the nucleosome include ATP-dependent nucleosome remodelling and reversible histone acetylation. The histone deacetylases, however, are unable to deacetylate oligonucleosomal histones in vitro. The protein complexes that mediate ATP-dependent nucleosome remodelling and histone acetylation/deacetylation in the regulation of transcription were considered to be different, although it has recently been suggested that these activities might be coupled. We report here the identification and functional characterization of a novel ATP-dependent nucleosome remodelling activity that is part of an endogenous human histone deacetylase complex. This activity is derived from the CHD3 and CHD4 proteins which contain helicase/ATPase domains found in SWI2-related chromatin remodelling factors, and facilitates the deacetylation of oligonucleosomal histones in vitro. We refer to this complex as the nucleosome remodelling and deacetylating (NRD) complex. Our results establish a physical and functional link between the distinct chromatin-modifying activities of histone deacetylases and nucleosome remodelling proteins.
Acetylation↗
MHC-linked LMP gene products specifically alter peptidase activities of the proteasome.
Proteasomes are highly conserved macromolecular structures which function as endopeptidases. They are found in the cytoplasm and nucleus of eukaryotic tissues and consist of at least 14 non-identical subunits with molecular masses ranging from approximately 20 to 32K. Proteasomes are essential in the selective degradation of ubiquitinated and certain non-ubiquitinated proteins, acting as the proteolytic core of an energy-dependent 26S (1,500K) proteolytic complex. Two proteasome subunits, LMP2 and LMP7 (refs 4-7), are encoded within the major histocompatibility complex (MHC), implicating proteasomes in antigen processing. Here we determine the function of these two MHC-linked subunits by comparing the proteolytic activities of purified proteasomes containing (LMP+) or lacking (LMP-) these components. We find that proteasomes of both types have endopeptidase activity against substrates bearing hydrophobic, basic or acidic residues immediately preceding the cleavage site (the P1 position) and at sites following asparagine, glycine and proline residues. The activity of LMP+ proteasomes is much higher than that of LMP- proteasomes against substrates with hydrophobic, basic or asparagine residues at P1, whereas their activities are comparable when acidic and glycine residues are present at P1. The MHC-linked LMP2 and LMP7 subunits therefore function to amplify specific endopeptidase activities of the proteasome.
Amino Acid Sequence↗
Induction of the branched-chain 2-oxo acid dehydrogenase complex in 3T3-L1 adipocytes during differentiation.
The activities of 2-oxo acid dehydrogenase complexes were measured during hormone-mediated differentiation of 3T3-L1 preadipocytes into adipocytes. Specific activity of leucine-activated branched-chain 2-oxo acid dehydrogenase complex increased approx. 10-fold in 3T3-L1 adipocytes compared with 3T3-L1 preadipocytes. In contrast, specific activity of the 2-oxoglutarate dehydrogenase complex increased by only 3-fold in 3T3-L1 adipocytes. The three catalytic component enzymes of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex showed concomitant increases in their specific activities. A close similarity in kinetics of induction of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex in 3T3-L1 adipocytes suggests that a common mechanism may be involved in hormone-dependent increases in the activities of the catalytic components of these two complexes in 3T3-L1 adipocytes during differentiation.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗