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1.
Hum Mol Genet ; 21(15): 3435-48, 2012 Aug 01.
Article in English | MEDLINE | ID: mdl-22611163

ABSTRACT

Riboflavin-responsive forms of multiple acyl-CoA dehydrogenation deficiency (RR-MADD) have been known for years, but with presumed defects in the formation of the flavin adenine dinucleotide (FAD) co-factor rather than genetic defects of electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). It was only recently established that a number of RR-MADD patients carry genetic defects in ETF-QO and that the well-documented clinical efficacy of riboflavin treatment may be based on a chaperone effect that can compensate for inherited folding defects of ETF-QO. In the present study, we investigate the molecular mechanisms and the genotype-phenotype relationships for the riboflavin responsiveness in MADD, using a human HEK-293 cell expression system. We studied the influence of riboflavin and temperature on the steady-state level and the activity of variant ETF-QO proteins identified in patients with RR-MADD, or non- and partially responsive MADD. Our results showed that variant ETF-QO proteins associated with non- and partially responsive MADD caused severe misfolding of ETF-QO variant proteins when cultured in media with supplemented concentrations of riboflavin. In contrast, variant ETF-QO proteins associated with RR-MADD caused milder folding defects when cultured at the same conditions. Decreased thermal stability of the variants showed that FAD does not completely correct the structural defects induced by the variation. This may cause leakage of electrons and increased reactive oxygen species, as reflected by increased amounts of cellular peroxide production in HEK-293 cells expressing the variant ETF-QO proteins. Finally, we found indications of prolonged association of variant ETF-QO protein with the Hsp60 chaperonin in the mitochondrial matrix, supporting indications of folding defects in the variant ETF-QO proteins.


Subject(s)
Electron-Transferring Flavoproteins/genetics , Genetic Variation , Iron-Sulfur Proteins/genetics , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/genetics , Oxidoreductases Acting on CH-NH Group Donors/genetics , Riboflavin/metabolism , Electron Transport , Electron-Transferring Flavoproteins/metabolism , Flavin-Adenine Dinucleotide/metabolism , HEK293 Cells , Humans , Iron-Sulfur Proteins/metabolism , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/metabolism , Mutation , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Protein Folding , Protein Structure, Tertiary , Transfection
2.
Protein Sci ; 20(3): 610-20, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21308847

ABSTRACT

Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid ß-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (αII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the αII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD-containing αII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in αI (A43C) were between 4.0 and 4.5 ± 0.35 nm and for ßIII (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the αII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open).


Subject(s)
Bacterial Proteins/chemistry , Electron Spin Resonance Spectroscopy/methods , Electron-Transferring Flavoproteins/chemistry , Flavin-Adenine Dinucleotide/chemistry , Protein Structure, Tertiary , Spin Labels , Bacterial Proteins/metabolism , Crystallography, X-Ray , Electron-Transferring Flavoproteins/metabolism , Glutaryl-CoA Dehydrogenase/chemistry , Humans , Models, Molecular , Molecular Dynamics Simulation , Molecular Structure , Oxidation-Reduction , Paracoccus denitrificans/chemistry
3.
Biometals ; 24(2): 335-47, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21184140

ABSTRACT

Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe-S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS.


Subject(s)
Cobalt/metabolism , Escherichia coli/drug effects , Escherichia coli/metabolism , Metalloporphyrins/metabolism , Cobalt/pharmacology , Protoporphyrins/metabolism
4.
Biochim Biophys Acta ; 1797(12): 1910-6, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20937244

ABSTRACT

Electron transfer flavoprotein: ubiqionone oxidoreductase (ETF-QO) is a component of the mitochondrial respiratory chain that together with electron transfer flavoprotein (ETF) forms a short pathway that transfers electrons from 11 different mitochondrial flavoprotein dehydrogenases to the ubiquinone pool. The X-ray structure of the pig liver enzyme has been solved in the presence and absence of a bound ubiquinone. This structure reveals ETF-QO to be a monotopic membrane protein with the cofactors, FAD and a [4Fe-4S](+1+2) cluster, organised to suggests that it is the flavin that serves as the immediate reductant of ubiquinone. ETF-QO is very highly conserved in evolution and the recombinant enzyme from the bacterium Rhodobacter sphaeroides has allowed the mutational analysis of a number of residues that the structure suggested are involved in modulating the reduction potential of the cofactors. These experiments, together with the spectroscopic measurement of the distances between the cofactors in solution have confirmed the intramolecular pathway of electron transfer from ETF to ubiquinone. This approach can be extended as the R. sphaeroides ETF-QO provides a template for investigating the mechanistic consequences of single amino acid substitutions of conserved residues that are associated with a mild and late onset variant of the metabolic disease multiple acyl-CoA dehydrogenase deficiency (MADD).


Subject(s)
Electron-Transferring Flavoproteins/chemistry , Iron-Sulfur Proteins/chemistry , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Protein Structure, Tertiary , Amino Acid Sequence , Animals , Crystallography, X-Ray , Electron Transport , Electron-Transferring Flavoproteins/genetics , Electron-Transferring Flavoproteins/metabolism , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Sequence Homology, Amino Acid , Swine
5.
J Am Chem Soc ; 131(44): 15978-9, 2009 Nov 11.
Article in English | MEDLINE | ID: mdl-19886689

ABSTRACT

The human mitochondrial electron transfer flavoprotein (ETF) accepts electrons from at least 10 different flavoprotein dehydrogenases and transfers electrons to a single electron acceptor in the inner membrane. Paracoccus denitrificans ETF has the identical function, shares the same three-dimensional structure and functional domains, and exhibits the same conformational mobility. It has been proposed that the mobility of the alphaII domain permits the promiscuous behavior of ETF with respect to a variety of redox partners. Double electron-electron resonance (DEER) measurements between a spin label and an enzymatically reduced flavin adenine dinucleotide (FAD) cofactor in P. denitrificans ETF gave two distributions of distances: a major component centered at 4.2 +/- 0.1 nm and a minor component centered at 5.1 +/- 0.2 nm. Both components had widths of approximately 0.3 nm. A distance of 4.1 nm was calculated using the crystal structure of P. denitrificans ETF, which agrees with the major component obtained from the DEER measurement. The observation of a second distribution suggests that ETF, in the absence of substrate, adopts some conformations that are intermediate between the predominant free and substrate-bound states.


Subject(s)
Electron-Transferring Flavoproteins/chemistry , Flavin-Adenine Dinucleotide , Spin Labels , Electron Spin Resonance Spectroscopy/methods , Humans , Protein Conformation , Quinones
6.
Biochemistry ; 47(34): 8894-901, 2008 Aug 26.
Article in English | MEDLINE | ID: mdl-18672901

ABSTRACT

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S] (2+,1+) and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S] (2+,1+) to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S] (+) at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF 1e (-) catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF.


Subject(s)
Electron-Transferring Flavoproteins/metabolism , Iron-Sulfur Proteins/metabolism , Iron/metabolism , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Sulfur/metabolism , Animals , Asparagine/chemistry , Asparagine/genetics , Asparagine/metabolism , Crystallography, X-Ray , Electron Spin Resonance Spectroscopy , Electron Transport , Electron-Transferring Flavoproteins/chemistry , Electron-Transferring Flavoproteins/genetics , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Hydrogen Bonding , Iron/chemistry , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Models, Molecular , Molecular Structure , Mutagenesis, Site-Directed , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Protein Structure, Secondary , Sulfur/chemistry , Swine , Temperature , Threonine/chemistry , Threonine/genetics , Threonine/metabolism
7.
Biochemistry ; 47(1): 92-100, 2008 Jan 08.
Article in English | MEDLINE | ID: mdl-18069858

ABSTRACT

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.


Subject(s)
Bacterial Proteins/metabolism , Electron-Transferring Flavoproteins/metabolism , Iron-Sulfur Proteins/metabolism , Mutation , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Catalysis , Electron Spin Resonance Spectroscopy , Electron Transport , Electron-Transferring Flavoproteins/chemistry , Electron-Transferring Flavoproteins/genetics , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Molecular Structure , Mutagenesis, Site-Directed , Oxidation-Reduction , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Potentiometry , Rhodobacter sphaeroides/enzymology , Rhodobacter sphaeroides/genetics , Rhodobacter sphaeroides/metabolism
8.
J Magn Reson ; 190(2): 222-32, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18037314

ABSTRACT

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a membrane-bound electron transfer protein that links primary flavoprotein dehydrogenases with the main respiratory chain. Human, porcine, and Rhodobacter sphaeroides ETF-QO each contain a single [4Fe-4S](2+,1+) cluster and one equivalent of FAD, which are diamagnetic in the isolated enzyme and become paramagnetic on reduction with the enzymatic electron donor or with dithionite. The anionic flavin semiquinone can be reduced further to diamagnetic hydroquinone. The redox potentials for the three redox couples are so similar that it is not possible to poise the proteins in a state where both the [4Fe-4S](+) cluster and the flavoquinone are fully in the paramagnetic form. Inversion recovery was used to measure the electron spin-lattice relaxation rates for the [4Fe-4S](+) between 8 and 18K and for semiquinone between 25 and 65K. At higher temperatures the spin-lattice relaxation rates for the [4Fe-4S](+) were calculated from the temperature-dependent contributions to the continuous wave linewidths. Although mixtures of the redox states are present, it was possible to analyze the enhancement of the electron spin relaxation of the FAD semiquinone signal due to dipolar interaction with the more rapidly relaxing [4Fe-4S](+) and obtain point-dipole interspin distances of 18.6+/-1A for the three proteins. The point-dipole distances are within experimental uncertainty of the value calculated based on the crystal structure of porcine ETF-QO when spin delocalization is taken into account. The results demonstrate that electron spin relaxation enhancement can be used to measure distances in redox poised proteins even when several redox states are present.


Subject(s)
Bacterial Proteins/chemistry , Electron Spin Resonance Spectroscopy , Electron-Transferring Flavoproteins/chemistry , Iron-Sulfur Proteins/chemistry , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Ubiquinone/chemistry , Animals , Electron Transport , Humans , Liver/enzymology , Oxidation-Reduction , Rhodobacter sphaeroides/enzymology , Swine
9.
Biochemistry ; 46(50): 14468-77, 2007 Dec 18.
Article in English | MEDLINE | ID: mdl-18020372

ABSTRACT

We have determined steady-state rate constants and net rate constants for the chemical steps in the catalytic pathway catalyzed by the E370D mutant of glutaryl-CoA dehydrogenase and compared them with those of the wild-type dehydrogenase. We sought rationales for changes in these rate constants in the structure of the mutant cocrystallized with the alternate substrate, 4-nitrobutyric acid. Substitution of aspartate for E370, the catalytic base, results in a 24% decrease in the rate constant for proton abstraction at C-2 of 3-thiaglutaryl-CoA as the distance between C-2 of the ligand and the closest carboxyl oxygen at residue 370 increases from 2.9 A to 3.1 A. The net rate constant for flavin reduction due to hydride transfer from C-3 of the natural substrate, which includes proton abstraction at C-2, to N5 of the flavin decreases by 81% due to the mutation, although the distance increases only by 0.7 A. The intensities of charge-transfer bands associated with the enolate of 3-thiaglutaryl-CoA, the reductive half-reaction (reduced flavin with oxidized form of substrate), and the dienolate following decarboxylation are considerably diminished. Structural investigation suggests that the increased distance and the change in angle of the S-C1(=O)-C2 plane of the substrate with the isoalloxazine substantially alter rates of the reductive and oxidative half-reactions. This change in active site geometry also changes the position of protonation of the four carbon dienolate intermediate to produce kinetically favorable product, vinylacetyl-CoA, which is further isomerized to the thermodynamically stable normal product, crotonyl-CoA.


Subject(s)
Glutaryl-CoA Dehydrogenase/chemistry , Glutaryl-CoA Dehydrogenase/metabolism , Mutation , Acyl Coenzyme A/metabolism , Amino Acid Substitution , Aspartic Acid/genetics , Aspartic Acid/metabolism , Chromatography, High Pressure Liquid , Crystallography , Glutamic Acid/genetics , Glutamic Acid/metabolism , Glutaryl-CoA Dehydrogenase/genetics , Kinetics , Mass Spectrometry , Mutagenesis, Site-Directed , Protons , Substrate Specificity
10.
Brain ; 130(Pt 8): 2045-54, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17584774

ABSTRACT

Multiple acyl-CoA dehydrogenation deficiency (MADD) is a disorder of fatty acid, amino acid and choline metabolism that can result from defects in two flavoproteins, electron transfer flavoprotein (ETF) or ETF: ubiquinone oxidoreductase (ETF:QO). Some patients respond to pharmacological doses of riboflavin. It is unknown whether these patients have defects in the flavoproteins themselves or defects in the formation of the cofactor, FAD, from riboflavin. We report 15 patients from 11 pedigrees. All the index cases presented with encephalopathy or muscle weakness or a combination of these symptoms; several had previously suffered cyclical vomiting. Urine organic acid and plasma acyl-carnitine profiles indicated MADD. Clinical and biochemical parameters were either totally or partly corrected after riboflavin treatment. All patients had mutations in the gene for ETF:QO. In one patient, we show that the ETF:QO mutations are associated with a riboflavin-sensitive impairment of ETF:QO activity. This patient also had partial deficiencies of flavin-dependent acyl-CoA dehydrogenases and respiratory chain complexes, most of which were restored to control levels after riboflavin treatment. Low activities of mitochondrial flavoproteins or respiratory chain complexes have been reported previously in two of our patients with ETF:QO mutations. We postulate that riboflavin-responsive MADD may result from defects of ETF:QO combined with general mitochondrial dysfunction. This is the largest collection of riboflavin-responsive MADD patients ever reported, and the first demonstration of the molecular genetic basis for the disorder.


Subject(s)
Acyl-CoA Dehydrogenase/deficiency , Electron-Transferring Flavoproteins/genetics , Iron-Sulfur Proteins/genetics , Mitochondrial Myopathies/genetics , Mutation , Oxidoreductases Acting on CH-NH Group Donors/genetics , Riboflavin/therapeutic use , Adolescent , Adult , Brain Diseases, Metabolic/enzymology , Brain Diseases, Metabolic/genetics , Carnitine/analogs & derivatives , Carnitine/blood , Child , Child, Preschool , Electron Transport/physiology , Fatty Acids/metabolism , Female , Humans , Male , Metabolism, Inborn Errors/genetics , Metabolism, Inborn Errors/metabolism , Metabolism, Inborn Errors/pathology , Mitochondria, Muscle/metabolism , Mitochondrial Myopathies/drug therapy , Mitochondrial Myopathies/metabolism , Mitochondrial Myopathies/pathology , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Oxidation-Reduction
11.
J Biol Chem ; 282(20): 14708-18, 2007 May 18.
Article in English | MEDLINE | ID: mdl-17369262

ABSTRACT

MitoQ(10) is a ubiquinone that accumulates within mitochondria driven by a conjugated lipophilic triphenylphosphonium cation (TPP(+)). Once there, MitoQ(10) is reduced to its active ubiquinol form, which has been used to prevent mitochondrial oxidative damage and to infer the involvement of reactive oxygen species in signaling pathways. Here we show MitoQ(10) is effectively reduced by complex II, but is a poor substrate for complex I, complex III, and electron-transferring flavoprotein (ETF):quinone oxidoreductase (ETF-QOR). This differential reactivity could be explained if the bulky TPP(+) moiety sterically hindered access of the ubiquinone group to enzyme active sites with a long, narrow access channel. Using a combination of molecular modeling and an uncharged analog of MitoQ(10) with similar sterics (tritylQ(10)), we infer that the interaction of MitoQ(10) with complex I and ETF-QOR, but not complex III, is inhibited by its bulky TPP(+) moiety. To explain its lack of reactivity with complex III we show that the TPP(+) moiety of MitoQ(10) is ineffective at quenching pyrene fluorophors deeply buried within phospholipid bilayers and thus is positioned near the membrane surface. This superficial position of the TPP(+) moiety, as well as the low solubility of MitoQ(10) in non-polar organic solvents, suggests that the concentration of the entire MitoQ(10) molecule in the membrane core is very limited. As overlaying MitoQ(10) onto the structure of complex III indicates that MitoQ(10) cannot react with complex III without its TPP(+) moiety entering the low dielectric of the membrane core, we conclude that the TPP(+) moiety does anchor the tethered ubiquinol group out of reach of the active site(s) of complex III, thus explaining its slow oxidation. In contrast the ubiquinone moiety of MitoQ(10) is able to quench fluorophors deep within the membrane core, indicating a high concentration of the ubiquinone moiety within the membrane and explaining its good anti-oxidant efficacy. These findings will facilitate the rational design of future mitochondria-targeted molecules.


Subject(s)
Antioxidants/chemistry , Electron Transport Complex I/chemistry , Lipid Bilayers/chemistry , Mitochondria, Heart/enzymology , Organophosphorus Compounds/chemistry , Phospholipids/chemistry , Ubiquinone/analogs & derivatives , Animals , Antioxidants/pharmacology , Cattle , Electron Transport Complex I/metabolism , Lipid Bilayers/metabolism , Organophosphorus Compounds/pharmacology , Oxidation-Reduction , Phospholipids/metabolism , Ubiquinone/chemistry , Ubiquinone/pharmacology
12.
Biochemistry ; 45(51): 15853-61, 2006 Dec 26.
Article in English | MEDLINE | ID: mdl-17176108

ABSTRACT

Glutaryl-CoA dehydrogenase (GCD) is a homotetrameric enzyme containing one noncovalently bound FAD per monomer that oxidatively decarboxylates glutaryl-CoA to crotonyl-CoA and CO2. GCD belongs to the family of acyl-CoA dehydrogenases that are evolutionarily conserved in their sequence, structure, and function. However, there are differences in the kinetic mechanisms among the different acyl-CoA dehydrogenases. One of the unanswered aspects is that of the rate-determining step in the steady-state turnover of GCD. In the present investigation, the major rate-determining step is identified to be the release of crotonyl-CoA product because the chemical steps and reoxidation of reduced FAD are much faster than the turnover of the wild-type GCD. Other steps are only partially rate-determining. This conclusion is based on the transit times of the individual reactions occurring in the active site of GCD.


Subject(s)
Glutaryl-CoA Dehydrogenase/chemistry , Glutaryl-CoA Dehydrogenase/metabolism , Acyl Coenzyme A/metabolism , Binding Sites , Catalysis , Decarboxylation , Deuterium Exchange Measurement , Electron Transport , Energy Transfer , Flavin-Adenine Dinucleotide/metabolism , Humans , Kinetics , Oxidation-Reduction , Protons , Substrate Specificity
13.
Proc Natl Acad Sci U S A ; 103(44): 16212-7, 2006 Oct 31.
Article in English | MEDLINE | ID: mdl-17050691

ABSTRACT

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a 4Fe4S flavoprotein located in the inner mitochondrial membrane. It catalyzes ubiquinone (UQ) reduction by ETF, linking oxidation of fatty acids and some amino acids to the mitochondrial respiratory chain. Deficiencies in ETF or ETF-QO result in multiple acyl-CoA dehydrogenase deficiency, a human metabolic disease. Crystal structures of ETF-QO with and without bound UQ were determined, and they are essentially identical. The molecule forms a single structural domain. Three functional regions bind FAD, the 4Fe4S cluster, and UQ and are closely packed and share structural elements, resulting in no discrete structural domains. The UQ-binding pocket consists mainly of hydrophobic residues, and UQ binding differs from that of other UQ-binding proteins. ETF-QO is a monotopic integral membrane protein. The putative membrane-binding surface contains an alpha-helix and a beta-hairpin, forming a hydrophobic plateau. The UQ-flavin distance (8.5 A) is shorter than the UQ-cluster distance (18.8 A), and the very similar redox potentials of FAD and the cluster strongly suggest that the flavin, not the cluster, transfers electrons to UQ. Two possible electron transfer paths can be envisioned. First, electrons from the ETF flavin semiquinone may enter the ETF-QO flavin one by one, followed by rapid equilibration with the cluster. Alternatively, electrons may enter via the cluster, followed by equilibration between centers. In both cases, when ETF-QO is reduced to a two-electron reduced state (one electron at each redox center), the enzyme is primed to reduce UQ to ubiquinol via FAD.


Subject(s)
Electron-Transferring Flavoproteins/chemistry , Electron-Transferring Flavoproteins/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Mitochondria, Liver/enzymology , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Ubiquinone/chemistry , Ubiquinone/metabolism , Animals , Cell Membrane/metabolism , Electron Transport , Iron/chemistry , Iron/metabolism , Models, Molecular , Oxidation-Reduction , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Static Electricity , Sulfur/chemistry , Sulfur/metabolism , Swine
14.
Biochemistry ; 44(42): 13932-40, 2005 Oct 25.
Article in English | MEDLINE | ID: mdl-16229482

ABSTRACT

The protonation of crotonyl-CoA dienolate following decarboxylation of glutaconyl-CoA by glutaryl-CoA dehydrogenase was investigated. Although it is generally held that the active sites of acyl-CoA dehydrogenases are desolvated when substrate binds, recent evidence has established that water has access to the active site in these binary complexes of glutaryl-CoA dehydrogenase. The present investigation shows that the dehydrogenase catalyzes (a) a rapid exchange of C-4 methyl protons of crotonyl-CoA with bulk solvent and (b) protonation of crotonyl-CoA dienolate by solvent-derived protons under single turnover conditions. Both of the reactions require the catalytic base, Glu370. These findings indicate that decarboxylation proceeds via a dienolate intermediate. The involvement of water in catalysis by glutaryl-CoA dehydrogenase was previously unrecognized and is in conflict with a classically held intramolecular 1,3-prototropic shift for protonation of crotonyl-CoA dienolate.


Subject(s)
Acyl Coenzyme A/chemistry , Glutaryl-CoA Dehydrogenase/chemistry , Solvents/chemistry , Humans , Kinetics , Mass Spectrometry , Nuclear Magnetic Resonance, Biomolecular , Protons
15.
Biochemistry ; 43(30): 9674-84, 2004 Aug 03.
Article in English | MEDLINE | ID: mdl-15274622

ABSTRACT

Acyl-CoA dehydrogenases (ACDs) are a family of flavoenzymes that metabolize fatty acids and some amino acids. Of nine known ACDs, glutaryl-CoA dehydrogenase (GCD) is unique: in addition to the alpha,beta-dehydrogenation reaction, common to all ACDs, GCD catalyzes decarboxylation of glutaryl-CoA to produce CO(2) and crotonyl-CoA. Crystal structures of GCD and its complex with 4-nitrobutyryl-CoA have been determined to 2.1 and 2.6 A, respectively. The overall polypeptide folds are the same and similar to the structures of other family members. The active site of the unliganded structure is filled with water molecules that are displaced when enzyme binds the substrate. The structure strongly suggests that the mechanism of dehydrogenation is the same as in other ACDs. The substrate binds at the re side of the FAD ring. Glu370 abstracts the C2 pro-R proton, which is acidified by the polarization of the thiolester carbonyl oxygen through hydrogen bonding to the 2'-OH of FAD and the amide nitrogen of Glu370. The C3 pro-R proton is transferred to the N(5) atom of FAD. The structures indicate a plausible mechanism for the decarboxylation reaction. The carbonyl polarization initiates decarboxylation, and Arg94 stabilizes the transient crotonyl-CoA anion. Protonation of the crotonyl-CoA anion occurs by a 1,3-prototropic shift catalyzed by the conjugated acid of the general base, Glu370. A tight hydrogen-bonding network involving gamma-carboxylate of the enzyme-bound glutaconyl-CoA, with Tyr369, Glu87, Arg94, Ser95, and Thr170, optimizes orientation of the gamma-carboxylate for decarboxylation. Some pathogenic mutations are explained by the structure. The mutations affect protein folding, stability, and/or substrate binding, resulting in inefficient/inactive enzyme.


Subject(s)
Oxidoreductases Acting on CH-CH Group Donors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Acyl Coenzyme A/chemistry , Amino Acid Sequence , Arginine/chemistry , Binding Sites , Catalytic Domain , Crystallization , Crystallography, X-Ray , Decarboxylation , Frameshift Mutation , Glutamic Acid/chemistry , Glutaryl-CoA Dehydrogenase , Humans , Hydrogen Bonding , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Folding , Protein Structure, Secondary/genetics , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Deletion , Substrate Specificity/genetics
16.
Biochem J ; 378(Pt 2): 633-40, 2004 Mar 01.
Article in English | MEDLINE | ID: mdl-14640977

ABSTRACT

Electron-transfer flavoprotein (ETF)-ubiquinone (2,3-dimethoxy-5-methyl-1,4-benzoquinone) oxidoreductase (ETF-QO) is a membrane-bound iron-sulphur flavoprotein that participates in an electron-transport pathway between eleven mitochondrial flavoprotein dehydrogenases and the ubiquinone pool. ETF is the intermediate electron carrier between the dehydrogenases and ETF-QO. The steady-state kinetic constants of human ETF-QO were determined with ubiquinone homologues and analogues that contained saturated n-alkyl substituents at the 6 position. These experiments show that optimal substrates contain a ten-carbon-atom side chain, consistent with a preliminary crystal structure that shows that only the first two of ten isoprene units of co-enzyme Q10 (CoQ10) interact with the protein. Derivatives with saturated alkyl side chains are very good substrates, indicating that, unlike other ubiquinone oxidoreductases, there is little preference for the methyl branches or rigidity of the CoQ side chain. Few of the compounds that inhibit ubiquinone oxidoreductases inhibit ETF-QO. Compounds found to act as inhibitors of ETF-QO include 2-n-heptyl-4-hydroxyquinoline N-oxide, a naphthoquinone analogue, 2-(3-methylpentyl)-4,6-dinitrophenol and pentachlorophenol. 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which inhibits the mitochondrial bc1 complex and the chloroplast b6 f complex in redox-dependent fashion, can serve as an electron acceptor for human ETF-QO. The observation of simple Michaelis-Menten kinetic patterns and a single type of quinone-binding site, determined by fluorescence titrations of the protein with DBMIB and 6-(10-bromodecyl)ubiquinone, are consistent with one ubiquinone-binding site per ETF-QO monomer.


Subject(s)
Electron-Transferring Flavoproteins/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Iron-Sulfur Proteins/metabolism , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Quinones/metabolism , Quinones/pharmacology , Ubiquinone/analogs & derivatives , Humans , Kinetics , Quinones/chemistry
17.
Mol Genet Metab ; 79(4): 245-56, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12948740

ABSTRACT

Inherited defects in glutaryl-CoA dehydrogenase cause the neurometabolic disease, glutaric acidemia type I. Five of over 80 mutations that have been identified are located in a carboxyl-terminal domain. The five mutations were generated by site directed mutagenesis and expressed in Escherichia coli. The mutant dehydrogenases were purified and characterized by circular dichroism and fluorescence spectroscopy, analytical size exclusion chromatography, thermal stability, and steady state kinetic analysis. There is no significant change in the alpha-helical content of the mutant proteins and little effect on tertiary structure; however, spectral properties of the mutant proteins indicate that the FAD prosthetic group can dissociate from the mutant proteins. Size exclusion chromatography shows that four mutant proteins dissociate to dimers or a mixture of monomers and dimers. Steady state kinetic analyses show that K(m) for glutaryl-CoA is affected by the mutations, but there is little effect on k(cat) compared with the wild type dehydrogenase. The lack of effects of the mutations on the K(m) for the electron acceptor, electron transfer flavoprotein, and on secondary structure suggests that the mutations do not result in long-range structural effects. The crystal structures of the acyl-CoA dehydrogenases show that their overall folding patterns are very similar and that the carboxyl-terminal domain is involved in substrate binding, FAD binding and intersubunit interactions. Investigations of mutations in the carboxyl-terminal domain of glutaryl-CoA dehydrogenase clearly illustrate these multiple roles of this domain. The results also indicate that a primary effect of the mutations is to cause alterations that promote aggregation.


Subject(s)
Oxidoreductases Acting on CH-CH Group Donors/genetics , Chromatography, Gel , Circular Dichroism , Escherichia coli , Glutaryl-CoA Dehydrogenase , Kinetics , Mutagenesis, Site-Directed , Mutation , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Spectrometry, Fluorescence , Substrate Specificity , Temperature
18.
J Biol Chem ; 278(29): 26342-50, 2003 Jul 18.
Article in English | MEDLINE | ID: mdl-12716879

ABSTRACT

2-Pentynoyl-CoA inactivates glutaryl-CoA dehydrogenase at a rate that considerably exceeds the rates of inactivation of short chain and medium chain acyl-CoA dehydrogenases by this inhibitor and related 2-alkynoyl-CoAs. To determine the rate of inactivation by 2-pentynoyl-CoA, we investigated the inactivation in the presence of a non-oxidizable analog, 3-thiaglutaryl-CoA, which competes for the binding site. The enhanced rate of inactivation does not reflect an alteration in specificity for the acyl group, nor does it reflect the covalent modification of a residue other than the active site glutamate. In addition to determining the inactivation of catalytic activity a spectral intermediate was detected by stopped-flow spectrophotometry, and the rate constants of formation and decay of this charge transfer complex (lambdamax approximately 790 nm) were determined by global analysis. Although the rate-limiting step in the inactivation of the other acyl-CoA dehydrogenases can involve the abstraction of a proton at C-4, this is not the case with glutaryl-CoA dehydrogenase. Glutaryl-CoA dehydrogenase is also differentiated from other acyl-CoA dehydrogenases in that the catalytic base must access both C-2 and C-4 in the normal catalytic pathway. Access to C-4 is not obligatory for the other dehydrogenases. Analysis of the distance from the closest carboxylate oxygen of the glutamate base catalyst to C-4 of a bound acyl-CoA ligand for medium chain, short chain, and isovaleryl-CoA dehydrogenases suggests that the increased rate of inactivation reflects the carboxylate oxygen to ligand C-4 distance in the binary complexes. This distance for wild type glutaryl-CoA dehydrogenase is not known. Comparison of the rate constants of inactivation and formation of a spectral species between wild type glutaryl-CoA dehydrogenase and a E370D mutant are consistent with the idea that this distance in glutaryl-CoA dehydrogenase contributes to the enhanced rate of inactivation and the 1,3-prototropic shift catalyzed by the enzyme.


Subject(s)
Acyl Coenzyme A/pharmacology , Enzyme Inhibitors/pharmacology , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases/antagonists & inhibitors , Acyl Coenzyme A/metabolism , Amino Acid Sequence , Binding Sites , Binding, Competitive , Glutaryl-CoA Dehydrogenase , Humans , In Vitro Techniques , Kinetics , Models, Biological , Molecular Sequence Data , Mutagenesis, Site-Directed , Oxidoreductases/chemistry , Oxidoreductases/genetics , Oxidoreductases/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
19.
Mol Genet Metab ; 77(1-2): 86-90, 2002.
Article in English | MEDLINE | ID: mdl-12359134

ABSTRACT

Glutaric acidemia type II is a human inborn error of metabolism which can be due to defects in either subunit of electron transfer flavoprotein (ETF) or in ETF:ubiquinone oxidoreductase (ETF:QO), but few disease-causing mutations have been described. The ETF:QO gene is located on 4q33, and contains 13 exons. Primers to amplify these exons are presented, together with mutations identified by molecular analysis of 20 ETF:QO-deficient patients. Twenty-one different disease-causing mutations were identified on 36 of the 40 chromosomes.


Subject(s)
Amino Acid Metabolism, Inborn Errors/enzymology , Amino Acid Metabolism, Inborn Errors/genetics , Electron-Transferring Flavoproteins , Fatty Acid Desaturases/deficiency , Fatty Acid Desaturases/genetics , Glutarates/blood , Iron-Sulfur Proteins , Lipid Metabolism, Inborn Errors/enzymology , Lipid Metabolism, Inborn Errors/genetics , Multienzyme Complexes/deficiency , Multienzyme Complexes/genetics , Mutation , Oxidoreductases Acting on CH-NH Group Donors , Amino Acid Metabolism, Inborn Errors/blood , Base Sequence , DNA Mutational Analysis , DNA, Complementary/genetics , Exons , Genotype , Humans , Introns , Lipid Metabolism, Inborn Errors/blood , Phenotype
20.
Biochem J ; 364(Pt 3): 659-67, 2002 Jun 15.
Article in English | MEDLINE | ID: mdl-12049629

ABSTRACT

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulphur flavoprotein and a component of an electron-transfer system that links 10 different mitochondrial flavoprotein dehydrogenases to the mitochondrial bc1 complex via electron transfer flavoprotein (ETF) and ubiquinone. ETF-QO is an integral membrane protein, and the primary sequences of human and porcine ETF-QO were deduced from the sequences of the cloned cDNAs. We have expressed human ETF-QO in Sf9 insect cells using a baculovirus vector. The cDNA encoding the entire protein, including the mitochondrial targeting sequence, was present in the vector. We isolated a membrane-bound form of the enzyme that has a molecular mass identical with that of the mature porcine protein as determined by SDS/PAGE and has an N-terminal sequence that is identical with that predicted for the mature holoenzyme. These data suggest that the heterologously expressed ETF-QO is targeted to mitochondria and processed to the mature, catalytically active form. The detergent-solubilized protein was purified by ion-exchange and hydroxyapatite chromatography. Absorption and EPR spectroscopy and redox titrations are consistent with the presence of flavin and iron-sulphur centres that are very similar to those in the equivalent porcine and bovine proteins. Additionally, the redox potentials of the two prosthetic groups appear similar to those of the other eukaryotic ETF-QO proteins. The steady-state kinetic constants of human ETF-QO were determined with ubiquinone homologues, a ubiquinone analogue, and with human wild-type ETF and a Paracoccus-human chimaeric ETF as varied substrates. The results demonstrate that this expression system provides sufficient amounts of human ETF-QO to enable crystallization and mechanistic investigations of the iron-sulphur flavoprotein.


Subject(s)
Electron-Transferring Flavoproteins , Fatty Acid Desaturases/genetics , Iron-Sulfur Proteins , Multienzyme Complexes/genetics , Oxidoreductases Acting on CH-NH Group Donors , Animals , Baculoviridae , Cell Line , Cell Membrane/enzymology , DNA Primers , Electron Spin Resonance Spectroscopy , Fatty Acid Desaturases/chemistry , Fatty Acid Desaturases/metabolism , Genetic Vectors , Humans , Kinetics , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Spectrophotometry , Spodoptera , Transfection
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