The Saccharomyces cerevisiae mitochondrial succinate:ubiquinone oxidoreductase

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.     

The Saccharomyces cerevisiae succinate dehydrogenase anchor subunit, Sdh4p: mutations at the C-terminal lys-132 perturb the hydrophobic domain

The yeast succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits, Sdh1p-Sdh4p, that couples the oxidation of succinate to the transfer of electrons to ubiquinone. One of the membrane anchor subunits, Sdh4p, has an unusual 30 amino acid extension at the C-terminus that is not present in SDH anchor subunits of other organisms. We identify Lys-132 in the Sdh4p C-terminal region as necessary for enzyme stability, ubiquinone reduction, and cytochrome b562 assembly in SDH. Five Lys-132 substituted SDH4 genes were constructed by site-directed mutagenesis and introduced into an SDH4 knockout strain. The mutants, K132E, K132G, K132Q, K132R, and K132V were characterized in vivo for respiratory growth and in vitro for ubiquinone reduction, enzyme stability, and cytochrome b562 assembly. Only the K132R substitution, which conserves the positive charge of Lys-132, produces a wild-type enzyme. The remaining four mutants do not affect the ability of SDH to oxidize succinate in the presence of the artificial electron acceptor, phenazine methosulfate, but impair quinone reductase activity, enzyme stability, and heme insertion. Our results suggest that the presence of a positive charge on residue 132 in the C-terminus of Sdh4p is critical for establishing a stable conformation in the SDH hydrophobic domain that is compatible with ubiquinone reduction and cytochrome b562 assembly. In addition, our data suggest that heme does not play an essential role in quinone reduction.     

Flavinylation and Assembly of Succinate Dehydrogenase Are Dependent on the C-terminal Tail of the Flavoprotein Subunit

The enzymatic function of succinate dehydrogenase (SDH) is dependent on covalent attachment of FAD on the ∼70-kDa flavoprotein subunit Sdh1. We show presently that flavinylation of the Sdh1 subunit of succinate dehydrogenase is dependent on a set of two spatially close C-terminal arginine residues that are distant from the FAD binding site. Mutation of Arg⁵⁸² in yeast Sdh1 precludes flavinylation as well as assembly of the tetrameric enzyme complex. Mutation of Arg⁶³⁸ compromises SDH function only when present in combination with a Cys⁶³⁰ substitution. Mutations of either Arg⁵⁸² or Arg⁶³⁸/Cys⁶³⁰ do not markedly destabilize the Sdh1 polypeptide; however, the steady-state level of Sdh5 is markedly attenuated in the Sdh1 mutant cells. With each mutant Sdh1, second-site Sdh1 suppressor mutations were recovered in Sdh1 permitting flavinylation, stabilization of Sdh5 and SDH tetramer assembly. SDH assembly appears to require FAD binding but not necessarily covalent FAD attachment. The Arg residues may be important not only for Sdh5 association but also in the recruitment and/or guidance of FAD and or succinate to the substrate site for the flavinylation reaction. The impaired assembly of SDH with the C-terminal Sdh1 mutants suggests that FAD binding is important to stabilize the Sdh1 conformation enabling association with Sdh2 and the membrane anchor subunits.     

Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect

Succinate dehydrogenase (SDH), also named as complex II or succinate:quinone oxidoreductases (SQR) is a critical enzyme in bioenergetics and metabolism. This is because the enzyme is located at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA); the two major pathways involved in generating energy within cells. SDH is composed of 4 subunits and is assembled through a multi-step process with the aid of assembly factors. Not surprisingly malfunction of this enzyme has marked repercussions in metabolism leading to devastating tumors such as paraganglioma and pheochromocytoma. It is already known that mutations in the genes encoding subunits lead to tumorigenesis, but recent discoveries have indicated that mutations in the genes encoding the assembly factors also contribute to tumorigenesis. The mechanisms of pathogenesis of tumorigenesis have not been fully understood. However, a multitude of signaling pathways including succinate signaling was determined. We, here discuss how defective SDH may lead to tumor development at the molecular level and describe how yeast, as a model system, has contributed to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH.     

Issue Information

Complex II [succinate dehydrogenase (succinate‐ubiquinone oxidoreductase); EC 1.3.5.1; SDH] is the only enzyme shared by both the electron transport chain and the tricarboxylic acid (TCA) cycle in mitochondria. Complex II in plants is considered unusual because of its accessory subunits (SDH5–SDH8), in addition to the catalytic subunits of SDH found in all eukaryotes (SDH1–SDH4). Here, we review compositional and phylogenetic analysis and biochemical dissection studies to both clarify the presence and propose a role for these subunits. We also consider the wider functional and phylogenetic evidence for SDH assembly factors and the reports from plants on the control of SDH1 flavination and SDH1–SDH2 interaction. Plant complex II has been shown to influence stomatal opening, the plant defense response and reactive oxygen species‐dependent stress responses. Signaling molecules such as salicyclic acid (SA) and nitric oxide (NO) are also reported to interact with the ubiquinone (UQ) binding site of SDH, influencing signaling transduction in plants. Future directions for SDH research in plants and the specific roles of its different subunits and assembly factors are suggested, including the potential for reverse electron transport to explain the succinate‐dependent production of reactive oxygen species in plants and new avenues to explore the evolution of plant mitochondrial complex II and its utility.     

The Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase. Identification of Sdh3p amino acid residues involved in ubiquinone binding.

Succinate dehydrogenase (SDH) participates in the mitochondrial electron transport chain by oxidizing succinate to fumarate and transferring the electrons to ubiquinone. In yeast, it is composed of a catalytic dimer, comprising the Sdh1p and Sdh2p subunits, and a membrane domain, comprising two smaller hydrophobic subunits, Sdh3p and Sdh4p, which anchor the enzyme to the mitochondrial inner membrane. To investigate the role of the Sdh3p anchor polypeptide in enzyme assembly and catalysis, we isolated and characterized seven mutations in the SDH3 gene. Two mutations are premature truncations of Sdh3p with losses of one or three transmembrane segments. The remaining five are missense mutations that are clustered between amino acids 103 and 117, which are proposed to be located in transmembrane segment II or the matrix-localized loop connecting segments II and III. Three mutations, F103V, H113Q, and W116R, strongly but specifically impair quinone reductase activities but have only minor effects on enzyme assembly. The clustering of the mutations strongly suggests that a ubiquinone-binding site is associated with this region of Sdh3p. In addition, the biphasic inhibition of quinone reductase activity by a dinitrophenol inhibitor supports the hypothesis that two distinct quinone-binding sites are present in the yeast SDH.     

The Saccharomyces cerevisiae succinate dehydrogenase does not require heme for ubiquinone reduction

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.     

The Saccharomyces cerevisiae succinate dehydrogenase does not require heme for ubiquinone reduction

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 b₅₆₂. 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.     

The Saccharomyces cerevisiae succinate-ubiquinone reductase contains a stoichiometric amount of cytochrome b562

The Saccharomyces cerevisiae succinate-ubiquinone reductase or succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. In most organisms, SDH contains one or two endogenous b-type hemes. However, it is widely believed that the yeast SDH does not contain heme. In this report, we demonstrate the presence of a stoichiometric amount of cytochrome b₅₆₂ in the yeast SDH. The cytochrome is detected as a peak present in fumarate-oxidized, dithionite-reduced mitochondria. The peak is centered at 562 nm and is present at a heme:covalent FAD molar ratio of 0.92±0.11. The cytochrome is not detectable in mitochondria isolated from SDH3 and SDH4 deletion strains. These observations strongly support our conclusion that cytochrome b₅₆₂ is a component of the yeast SDH.     

Identification of the heme axial ligands in the cytochrome b562 of the Saccharomyces cerevisiae succinate dehydrogenase

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.     

SDH mutations in cancer

The SDHA, SDHB, SDHC, SDHD genes encode the four subunits of succinate dehydrogenase (SDH; mitochondrial complex II), a mitochondrial enzyme involved in two essential energy-producing metabolic processes of the cell, the Krebs cycle and the electron transport chain. Germline loss-of-function mutations in any of the SDH genes or assembly factor (SDHAF2) cause hereditary paraganglioma/phaeochromocytoma syndrome (HPGL/PCC) through a mechanism which is largely unknown. Owing to the central function of SDH in cellular energy metabolism it is important to understand its role in tumor suppression. Here is reported an overview of genetics, clinical and molecular progress recently performed in understanding the basis of HPGL/PCC tumorigenesis.     

The Quinone-binding sites of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase

The Saccharomyces cerevisiae succinate dehydrogenase (SDH) of the mitochondrial electron transport chain oxidizes succinate and reduces ubiquinone. Using a random mutagenesis approach, we identified functionally important amino acid residues in one of the anchor subunits, Sdh4p. We analyzed three point mutations (F69V, S71A, and H99L) and one nonsense mutation (Y89OCH) that truncates the Sdh4p subunit at the third predicted transmembrane segment. The F69V and the S71A mutations result in greatly impaired respiratory growth in vivo and quinone reductase activities in vitro, with negligible effects on enzyme stability. In contrast, the Y89OCH and the H99L mutations elicit large structural perturbations that impair assembly as evidenced by reduced covalent FAD levels, membrane-associated succinate-phenazine methosulfate reductase activities, and thermal stability. We propose that the Phe-69 and the Ser-71 residues are involved in the formation of a quinone-binding site, whereas the His-99 residue is at the interface of the peripheral and the membrane domains. In addition, the properties of the Y89OCH mutation are consistent with the interpretation that the third transmembrane segment is not involved in catalysis but rather plays an important structural role. The mutant enzymes are differentially sensitive to a quinone analog inhibitor, providing further evidence for a two-quinone binding model in the yeast SDH.     

Protein-mediated assembly of succinate dehydrogenase and its cofactors

Abstract

Succinate dehydrogenase (or complex II; SDH) is a heterotetrameric protein complex that links the tribarboxylic acid cycle with the electron transport chain. SDH is composed of four nuclear-encoded subunits that must translocate independently to the mitochondria and assemble into a mature protein complex embedded in the inner mitochondrial membrane. Recently, it has become clear that failure to assemble functional SDH complexes can result in cancer and neurodegenerative syndromes. The effort to thoroughly elucidate the SDH assembly pathway has resulted in the discovery of four subunit-specific assembly factors that aid in the maturation of individual subunits and support the assembly of the intact complex. This review will focus on these assembly factors and assess the contribution of each factor to the assembly of SDH. Finally, we propose a model of the SDH assembly pathway that incorporates all extant data.     

High-Throughput Screening for Growth Inhibitors Using a Yeast Model of Familial Paraganglioma

Classical tumor suppressor genes block neoplasia by regulating cell growth and death. A remarkable puzzle is therefore presented by familial paraganglioma (PGL), a neuroendocrine cancer where the tumor suppressor genes encode subunits of succinate dehydrogenase (SDH), an enzyme of the tricarboxylic acid (TCA) cycle of central metabolism. Loss of SDH initiates PGL through mechanisms that remain unclear. Could this metabolic defect provide a novel opportunity for chemotherapy of PGL? We report the results of high throughput screening to identify compounds differentially toxic to SDH mutant cells using a powerful S. cerevisiae (yeast) model of PGL. Screening more than 200,000 compounds identifies 12 compounds that are differentially toxic to SDH-mutant yeast. Interestingly, two of the agents, dequalinium and tetraethylthiuram disulfide (disulfiram), are anti-malarials with the latter reported to be a glycolysis inhibitor. We show that four of the additional hits are potent inhibitors of yeast alcohol dehydrogenase. Because alcohol dehydrogenase regenerates NAD⁺ in glycolytic cells that lack TCA cycle function, this result raises the possibility that lactate dehydrogenase, which plays the equivalent role in human cells, might be a target of interest for PGL therapy. We confirm that human cells deficient in SDH are differentially sensitive to a lactate dehydrogenase inhibitor.     

Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex.

The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of Bacillus subtilis was determined. This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB. The predicted molecular weights for the Fp and Ip subunits, 65,186 (585 amino acids) and 28,285 (252 amino acids), agreed with the values determined independently for the labeled Fp and Ip antigens, although it appeared that the B. subtilis Fp was not functional after expression of the sdhA gene in Escherichia coli. Both subunits closely resembled the corresponding Fp and Ip subunits of the succinate dehydrogenase (SDH) and fumarate reductase of E. coli in size, composition, and amino acid sequence. The sequence homologies further indicated that the B. subtilis SDH subunits are equally related to the SDH and fumarate reductase subunits of E. coli but are less closely related than are the corresponding pairs of E. coli subunits. The regions of highest sequence conservation were identifiable as the catalytically significant flavin adenine dinucleotide-binding sites and cysteine clusters of the iron-sulfur centers.     

Succinate dehydrogenase – Assembly,regulation and role in human disease

Succinate dehydrogenase (or Electron Transport Chain Complex II) has been the subject of a focused but significant renaissance. This complex, which has been the least studied of the mitochondrial respiratory complexes has seen renewed interest due to the discovery of its role in human disease. Under this heightened scrutiny, the succinate dehydrogenase complex has proven to be a fascinating machine, whose regulation and assembly requires additional factors that are beginning to be discovered. Mutations in these factors and in the structural subunits of the complex itself cause a variety of human diseases. The mechanisms underlying the pathogenesis of SDH mutations is beginning to be understood.     

Early steps in assembly of the yeast vacuolar H+-ATPase.

Vacuolar proton-translocating ATPases are composed of a complex of integral membrane proteins, the Vo sector, attached to a complex of peripheral membrane proteins, the V1 sector. We have examined the early steps in biosynthesis of the yeast vacuolar ATPase by biosynthetically labeling wild-type and mutant cells for varied pulse and chase times and immunoprecipitating fully and partially assembled complexes under nondenaturing conditions. In wild-type cells, several V1 subunits and the 100-kDa Vo subunit associate within 3-5 min, followed by addition of other Vo subunits with time. Deletion mutants lacking single subunits of the enzyme show a variety of partial complexes, including both complexes that resemble intermediates in the assembly pathway of wild-type cells and independent V1 and Vo sectors that form without any apparent V1Vo subunit interaction. Two yeast sec mutants that show a temperature-conditional block in export from the endoplasmic reticulum accumulate a complex containing several V1 subunits and the 100-kDa Vo subunit during incubation at elevated temperature. This complex can assemble with the 17-kDa Vo subunit when the temperature block is reversed. We propose that assembly of the yeast V-ATPase can occur by two different pathways: a concerted assembly pathway involving early interactions between V1 and Vo subunits and an independent assembly pathway requiring full assembly of V1 and Vo sectors before combination of the two sectors. The data suggest that in wild-type cells, assembly occurs predominantly by the concerted assembly pathway, and V-ATPase complexes acquire the full complement of Vo subunits during or after exit from the endoplasmic reticulum.     

Reconstitution of succinate dehydrogenase in Bacillus subtilis by protoplast fusion

Bacillus subtilis succinate dehydrogenase (SDH) is composed of two unequal subunits designated Fp (Mr, 65,000) and Ip (Mr. 28,000). The enzyme is structurally and functionally complexed to cytochrome b 558 (Mr, 19,000) in the membrane. A total of 21 B. subtilis SDH-negative mutants were isolated. The mutants fall into five phenotypic classes with respect to the presence and localization of the subunits of the SDH-cytochrome b558 complex. One class contains mutants with an inactive membrane-bound complex. Membrane-bound enzymatically active SDH could be reconstituted in fused protoplasts of selected pairs of SDH-negative mutants. Most likely reconstitution is due to the assembly of preformed subunits in the fused cells. On the basis of the reconstitution data, the mutants tested could be divided into three complementation groups. The combined data of the present and previous work indicate that the complementation groups correspond to the structural genes for the three subunits of the membrane-bound SDH-cytochrome b558 complex. A total of 31 SDH-negative mutants of B. subtilis have now been characterized. The respective mutations all map in the citF locus at 255 degrees on the B. subtilis chromosomal map. In the present paper, we have revised the nomenclature for the genetics of SDH in B. subtilis. All mutations which give an SDH-negative phenotype will be called sdh followed by an isolation number. The designation citF will be omitted, and the citF locus will be divided into three genes: sdhA, sdhB, and sdhC. Mutations in sdhA affect cytochrome b558, mutations in sdhB affect Fp, and mutations in sdhC affect Ip.     

Complementation of succinate dehydrogenase mutants in fused Bacillus subtilis protoplasts

Abstract Succinate dehydrogenase (SDH) and cytochrome b ₅₅₈ are present in Bacillus subtilis cytoplasmic membranes as a complex consisting of three different subunits. Complementation of membrane-bound SDH activity was studied in fused protoplasts of two SDH-negative mutants mutated in different subunits of the complex. The complementation was found to be rapid, temperature dependent and not sensitive to inhibitors of respiration. It is concluded that the complementation predominantly results from the assembly of preformed SDH subunits.