Expression of Saccharomyces cerevisiae Sdh3p and Sdh4p paralogs results in catalytically active succinate dehydrogenase isoenzymes

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.     

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.     

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.     

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.     

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.     

Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408-->Glu/Lys939-->Arg mutant retained significant activity, while an Asp407-->Glu/Lys939-->Arg mutant exhibited only marginal function. An Asp407-->Glu/Asp408-->Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407-->Glu/Asp408-->Glu/Lys939-->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.     

The quaternary structure of the Saccharomyces cerevisiae succinate dehydrogenase. Homology modeling, cofactor docking, and molecular dynamics simulation studies

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.     

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.     

Lys691 and Asp714 of the Na+/K+-ATPase alpha subunit are essential for phosphorylation, dephosphorylation, and enzyme turnover

P-type ATPases such as the sodium pump appear to be members of a superfamily of hydrolases structurally typified by the L-2-haloacid dehalogenases. In the dehalogenase L-DEX-ps, Lys151 serves to stabilize the excess negative charge in the substrate/reaction intermediates and Asp180 coordinates a water molecule that is directly involved in ester intermediate hydrolysis. To investigate the importance of the corresponding Lys691 and Asp714 of the sodium pump alpha subunit, sodium pump mutants were expressed in yeast and analyzed for their properties. Lys691Ala, Lys691Asp, Asp714Ala, and Asp714Arg mutants were inactive, not only with respect to ATPase activity but also to interaction with the highly sodium pump-specific inhibitors ouabain or palytoxin (PTX). In contrast, conservative mutants Lys691Arg and Asp714Glu retained some of the partial activities of the wild-type enzyme, although they completely failed to display any ATPase activity. Yeast cells expressing Lys691Arg and Asp714Glu mutants are sensitive to the sodium pump-specific inhibitor PTX and lose intracellular K+. Their sensitivity to PTX, with EC50 values of 118 +/- 24 and 76.5 +/- 3.6 nM, respectively, was clearly reduced by almost 7- or 4-fold below that of the native sodium pump (17.8 +/- 2.7 nM). Ouabain was recognized under these conditions with low affinity by the mutants and inhibited the PTX-induced K+ efflux from the yeast cells. The EC50 for the ouabain effect was 183 +/- 20 microM for Lys691Arg and 2.3 +/- 0.08 mM for the Asp714Glu mutant. The corresponding value obtained with cells expressing the native sodium pump was 69 +/- 18 microM. In the presence of Pi and Mg2+, none of the mutant sodium pumps were able to bind ouabain. When Mg2+ was omitted, however, both Lys691Asp and Asp714Glu mutants displayed ouabain binding that was reduced by Mg2+ with an EC50 of 0.76 +/- 0.11 and 2.3 +/- 0.2 mM, respectively. In the absence of Mg2+, ouabain binding was also reduced by K+. The EC50 values were 1.33 +/- 0.23 mM for the wild-type enzyme, 0.93 +/- 0.2 mM for the Lys691Arg mutant, and 1.02 +/- 0.24 mM for the Asp714Glu enzyme. None of the neutral or nonconservative mutants displayed any ouabain-sensitive ATPase activity. Ouabain-sensitive phosphatase activity, however, was present in membranes containing either the wild-type (1105 +/- 100 micromol of p-nitrophenol phosphate hydrolyzed min(-1) mg of protein(-1)) or the Asp714Glu mutant (575 +/- 75 micromol min(-1) mg(-1)) sodium pump. Some phosphatase activity was also associated with the Lys691Arg mutant (195 +/- 63 micromol min(-1) mg(-1)). The results are consistent with Lys691 and Asp714 being essential for the phosphorylation/dephosphorylation process that allows the sodium pump to accomplish the catalytic cycle.     

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.     

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 role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase

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.     

The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase

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.     

Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit

It has recently been shown that replacement of the border residues (Gln-111 and Asn-122) of the H1-H2 extracellular domain of the sheep Na,K-ATPase alpha subunit with the charged amino acids Arg and Asp generates a ouabain-resistant enzyme (Price, E. M. and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to further study structure-function relationships in Na,K-ATPase, six additional mutations have been made at these border positions. Two of these mutants were single amino acid substitutions (Gln-111 to Arg or Asn-122 to Asp). These mutations change one or the other H1-H2 border residue to a charged amino acid. The remaining substitutions were double mutants in which both of the H1-H2 border residues were simultaneously changed to charged amino acids. Changes were made which introduced either positively charged amino acids (Lys at positions 111 and 122), negatively charged amino acids (Glu at positions 111 and 122) or oppositely charged amino acids (Lys at position 111 and Glu at 122; Asp at position 111 and Arg at 122) at the borders of the H1-H2 extracellular domain. HeLa cells transfected with any of these sheep Na,K-ATPase alpha subunit mutants were able to grow in concentrations of ouabain that were toxic to untransfected cells or cells transfected with the wild type sheep alpha subunit. Crude membranes isolated from the transfectants were analyzed for ouabain inhibitable Na,K-ATPase activity. All of the transfectants contained a relatively ouabain-resistant component of enzyme activity, with the ouabain I50 values ranging from 4 x 10(-3) M to 1 x 10(-6) M. The most resistant enzyme was the double mutant that contained Asp at position 111 and Arg at 122, whereas the least resistant were the enzymes containing the single amino acid substitutions. There was no correlation between the type of charged amino acid present at the border position and the degree of ouabain resistance. These data demonstrate the functional importance, in terms of ouabain binding, of the border positions of the H1-H2 extracellular domain of the Na,K-ATPase alpha subunit.     

The ubiquinone-binding site of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase is a source of superoxide

The mitochondrial succinate dehydrogenase (SDH) is a tetrameric iron-sulfur flavoprotein of the Krebs cycle and of the respiratory chain. A number of mutations in human SDH genes are responsible for the development of paragangliomas, cancers of the head and neck region. The mev-1 mutation in the Caenorhabditis elegans gene encoding the homolog of the SDHC subunit results in premature aging and hypersensitivity to oxidative stress. It also increases the production of superoxide radicals by the enzyme. In this work, we used the yeast succinate dehydrogenase to investigate the molecular and catalytic effects of paraganglioma- and mev-1-like mutations. We mutated Pro-190 of the yeast Sdh2p subunit to Gln (P190Q) and recreated the C. elegans mev-1 mutation by converting Ser-94 in the Sdh3p subunit into a glutamate residue (S94E). The P190Q and S94E mutants have reduced succinate-ubiquinone oxidoreductase activities and are hypersensitive to oxygen and paraquat. Although the mutant enzymes have lower turnover numbers for ubiquinol reduction, larger fractions of the remaining activities are diverted toward superoxide production. The P190Q and S94E mutations are located near the proximal ubiquinone-binding site, suggesting that the superoxide radicals may originate from a ubisemiquinone intermediate formed at this site during the catalytic cycle. We suggest that certain mutations in SDH can make it a significant source of superoxide production in mitochondria, which may contribute directly to disease progression. Our data also challenge the dogma that superoxide production by SDH is a flavin-mediated event rather than a quinone-mediated one.     

Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha2beta2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens. We converted 16 residues in the human alpha subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild-type beta subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the K(m) for Fe2+ 15-fold and that for 2-oxoglutarate 5-fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the K(m) for 2-oxoglutarate 15-fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85-95%; all these mutations increased the K(m) for 2-oxoglutarate 2- to 3-fold and enhanced the rate of uncoupled decarboxylation of 2-oxoglutarate as a percentage of the rate of the complete reaction up to 12-fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C-5 carboxyl group of 2-oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate.     

The importance of four histidine residues in isocitrate lyase from Escherichia coli.

By site-directed mutagenesis, substitutions were made for His-184 (H-184), H-197, H-266, and H-306 in Escherichia coli isocitrate lyase. Of these changes, only mutations of H-184 and H-197 appreciably reduced enzyme activity. Mutation of H-184 to Lys, Arg, or Leu resulted in an inactive isocitrate lyase, and mutation of H-184 to Gln resulted in an enzyme with 0.28% activity. Nondenaturing polyacrylamide gel electrophoresis demonstrated that isocitrate lyase containing the Lys, Arg, Gln, and Leu substitutions at H-184 was assembled poorly into the tetrameric subunit complex. Mutation of H-197 to Lys, Arg, Leu, and Gln resulted in an assembled enzyme with less than 0.25% wild-type activity. Five substitutions for H-266 (Asp, Glu, Val, Ser, and Lys), four substitutions for H-306 (Asp, Glu, Val, and Ser), and a variant in which both H-266 and H-306 were substituted for showed little or no effect on enzyme activity. All the H-197, H-266, and H-306 mutants supported the growth of isocitrate lyase-deficient E. coli JE10 on acetate as the sole carbon source; however, the H-184 mutants did not.     

Pairwise electrostatic interactions between alpha-neurotoxins and gamma, delta, and epsilon subunits of the nicotinic acetylcholine receptor

alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of approximately 1200 A(2) and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound alpha-neurotoxin. Three distinct regions on the gamma subunit, defined by Trp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica alpha-toxin, Lys(27) --> Glu, Arg(33) --> Glu, and Arg(36) --> Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six alpha-toxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II and gammaLeu(119) (-5.7 kcal/mol) and between Lys(27) and gammaGlu(176) (-5.9 kcal/mol). gammaTrp(55) couples strongly to both Arg(33) and Lys(27), whereas gammaAsp(174) couples minimally to charged alpha-toxin residues. Arg(36), despite strong energetic contributions, does not partner with any gamma subunit residues, perhaps indicating its proximity to the alpha subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at gamma176 and gamma119 can be distinguished from those at gamma55.     

Alternative sites for proton entry from the cytoplasm to the quinone binding site in Escherichia coli succinate dehydrogenase

Escherichia coli succinate dehydrogenase (Sdh) belongs to the highly conserved complex II family of enzymes that reduce ubiquinone. These enzymes do not generate a protonmotive force during catalysis and are electroneutral. Because of its electroneutrality, the quinone reduction reaction must consume cytoplasmic protons which are released stoichiometrically during succinate oxidation. The X-ray crystal structure of E. coli Sdh shows that residues SdhB (G227), SdhC (D95), and SdhC (E101) are located at or near the entrance of a water channel that has been proposed to function as a proton wire connecting the cytoplasm to the quinone binding site. However, the pig and chicken Sdh enzymes show an alternative entrance to the water channel via the conserved SdhD (Q78) residue. In this study, site-directed mutants of these four residues were created and characterized by in vivo growth assays, in vitro activity assays, and electron paramagnetic resonance spectroscopy. We show that the observed water channel in the E. coli Sdh structure is the functional proton wire in vivo, while in vitro results indicate an alternative entrance for protons. In silico examination of the E. coli Sdh reveals a possible H-bonding network leading from the cytoplasm to the quinone binding site that involves SdhD (D15). On the basis of these results we propose an alternative proton pathway in E. coli Sdh that might be functional only in vitro.