RESUMEN
Staphylococcus warneri is a Gram-positive bacterium commonly found in human skin flora. The genome of a laboratory S. warneri isolate, strain SG1, was sequenced to explore its mechanism of solvent tolerance and its potential as a chassis for biofuel production.
RESUMEN
Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.
Asunto(s)
Antiportadores/genética , Antiportadores/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Antiportadores/química , Catálisis , Complejo II de Transporte de Electrones/química , Complejo II de Transporte de Electrones/genética , Complejo II de Transporte de Electrones/metabolismo , Activación Enzimática/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Metabolómica/métodos , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/enzimología , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Datos de Secuencia Molecular , Fenotipo , Subunidades de Proteína , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Succinato Deshidrogenasa/química , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMEN
The coupling of succinate oxidation to the reduction of ubiquinone by succinate dehydrogenase (SDH) constitutes a pivotal reaction in the aerobic generation of energy. In Saccharomyces cerevisiae, SDH is a tetramer composed of a catalytic dimer comprising a flavoprotein subunit, Sdh1p and an iron-sulfur protein, Sdh2p and a heme b-containing membrane-anchoring dimer comprising the Sdh3p and Sdh4p subunits. In order to investigate the role of heme in SDH catalysis, we constructed an S. cerevisiae strain expressing a mutant enzyme lacking the two heme axial ligands, Sdh3p His-106 and Sdh4p Cys-78. The mutant enzyme was characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction and for its heme b content. Replacement of both Sdh3p His-106 and Sdh4p Cys-78 with alanine residues leads to an undetectable level of cytochrome b(562). Although enzyme assembly is slightly impaired, the apocytochrome SDH retains a significant ability to reduce quinone. The enzyme has a reduced affinity for quinone and its catalytic efficiency is reduced by an order of magnitude. To better understand the effects of the mutations, we employed atomistic molecular dynamic simulations to investigate the enzyme's structure and stability in the absence of heme. Our results strongly suggest that heme is not required for electron transport from succinate to quinone nor is it necessary for assembly of the S. cerevisiae SDH.
Asunto(s)
Hemo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Succinato Deshidrogenasa/química , Ubiquinona/metabolismo , Sustitución de Aminoácidos/genética , Transporte de Electrón/genética , Hemo/fisiología , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrofotometría Ultravioleta , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismoRESUMEN
Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron-sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (Q(P)) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species. We have investigated the role of the yeast Sdh4p Tyr-89, which forms a hydrogen bond with ubiquinone in the Q(P) site. This tyrosine residue is conserved in all succinate:ubiquinone oxidoreductases studied to date. In the human SDH, mutation of this tyrosine to cysteine results in paraganglioma, tumors of the parasympathetic ganglia in the head and neck. We demonstrate that Tyr-89 is essential for ubiquinone reductase activity and that mutation of Tyr-89 to other residues does not increase the production of reactive oxygen species. Our results support a role for Tyr-89 in the protonation of ubiquinone and argue that the generation of reactive oxygen species is not causative of tumor formation.
Asunto(s)
Complejo II de Transporte de Electrones/metabolismo , Saccharomyces cerevisiae/enzimología , Tirosina/fisiología , Ubiquinona/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Complejo II de Transporte de Electrones/química , Complejo II de Transporte de Electrones/genética , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Oxidación-Reducción , Paraganglioma/genética , Feocromocitoma/genética , Especies Reactivas de Oxígeno/metabolismo , Tirosina/genéticaRESUMEN
Succinate dehydrogenase (SDH) plays a key role in energy generation by coupling the oxidation of succinate to the reduction of ubiquinone in the mitochondrial electron transport chain. The Saccharomyces cerevisiae SDH is composed of a catalytic dimer of the Sdh1p and Sdh2p subunits containing flavin adenine dinucleotide (FAD) and iron-sulfur clusters and a heme b-containing membrane-anchoring domain comprised of the Sdh3p and Sdh4p subunits. We systematically mutated all the histidine and cysteine residues in Sdh3p and Sdh4p to identify the residues involved in axial heme ligation. The mutants were characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction, for heme b content, and for heme spectral properties. Mutation of Sdh3p His-46 or His-113 leads to a marked reduction in the catalytic efficiency of the enzyme for quinone reduction, suggesting that these residues form part of a quinone-binding site. We identified Sdh3p His-106 and Sdh4p Cys-78 as the most probable axial ligands for cytochrome b(562). Replacement of His-106 or Cys-78 with an alanine residue leads to a marked reduction in cytochrome b(562) content and to altered heme spectral characteristics that are consistent with a direct perturbation of heme b environment. This is the first identification of a cysteine residue serving as an axial ligand for heme b in the SDH family of enzymes. Loss of cytochrome b(562) has no effect on enzyme assembly and quinone reduction; the role of the heme in enzyme structure and function is discussed.
Asunto(s)
Grupo Citocromo b/genética , Proteínas de Saccharomyces cerevisiae/genética , Succinato Deshidrogenasa/genética , Secuencia de Aminoácidos , Sitios de Unión , Grupo Citocromo b/química , Hemo/química , Ligandos , Datos de Secuencia Molecular , Oxidación-Reducción , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Succinato Deshidrogenasa/químicaRESUMEN
Succinate dehydrogenases and fumarate reductases are complex mitochondrial or bacterial respiratory chain proteins with remarkably similar structures and functions. Succinate dehydrogenase oxidizes succinate and reduces ubiquinone using a flavin adenine dinucleotide cofactor and iron-sulfur clusters to transport electrons. A model of the quaternary structure of the tetrameric Saccharomyces cerevisiae succinate dehydrogenase was constructed based on the crystal structures of the Escherichia coli succinate dehydrogenase, the E. coli fumarate reductase, and the Wolinella succinogenes fumarate reductase. One FAD and three iron-sulfur clusters were docked into the Sdh1p and Sdh2p catalytic dimer. One b-type heme and two ubiquinone or inhibitor analog molecules were docked into the Sdh3p and Sdh4p membrane dimer. The model is consistent with numerous experimental observations. The calculated free energies of inhibitor binding are in excellent agreement with the experimentally determined inhibitory constants. Functionally important residues identified by mutagenesis of the SDH3 and SDH4 genes are located near the two proposed quinone-binding sites, which are separated by the heme. The proximal quinone-binding site, located nearest the catalytic dimer, has a considerably more polar environment than the distal site. Alternative low energy conformations of the membrane subunits were explored in a molecular dynamics simulation of the dimer embedded in a phospholipid bilayer. The simulation offers insight into why Sdh4p Cys-78 may be serving as the second axial ligand for the heme instead of a histidine residue. We discuss the possible roles of heme and of the two quinone-binding sites in electron transport.
Asunto(s)
Saccharomyces cerevisiae/enzimología , Succinato Deshidrogenasa/química , Secuencia de Aminoácidos , Escherichia coli , Proteínas Fúngicas/química , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Saccharomyces cerevisiae/química , Succinato Deshidrogenasa/genéticaRESUMEN
The Saccharomyces cerevisiae succinate dehydrogenase (SDH) provides an excellent model system for studying the assembly, structure, and function of a mitochondrial succinate:quinone oxidoreductase. The powerful combination of genetic and biochemical approaches is better developed in yeast than in other eukaryotes. The yeast protein is strikingly similar to other family members in the structural and catalytic properties of its subunits. However, the membrane domain and particularly the role of the single heme in combination with two ubiquinone-binding sites need further investigation. The assembly of subunits and cofactors that occurs to produce new holoenzyme molecules is a complex process that relies on molecular chaperones. The yeast SDH provides the best opportunity for understanding the biogenesis of this family of iron-sulfur flavoproteins.