The Saccharomyces cerevisiae Succinate-ubiquinone Oxidoreductase

Abstract

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 mitochondrial respiratory chain consists of four discrete prosthetic group-containing protein complexes. One of these, the succinate-ubiquinone oxidoreductase (complex II or succinate dehydrogenase (SDH) 1 ) donates electrons derived from its substrate, succinate, to the respiratory chain via the reduction of ubiquinone to ubiquinol. Generally, SDH is made up of two parts: a soluble catalytic dimer and a membrane domain (1-5). In the yeast Saccharomyces cerevisiae, the catalytic dimer is composed of the 67-kDa Sdh1p subunit, to which is attached a covalent FAD cofactor (6 -9), and the 28-kDa Sdh2p subunit (10), in which are located three iron-sulfur clusters. The membrane-anchoring domain is composed of the two hydrophobic subunits, Sdh3p and Sdh4p, of 16.7 and 16.6 kDa, respectively (11-13). Each subunit is believed to have its amino terminus in the matrix and three transmembrane segments.The catalytic dimer can catalyze electron transfer from succinate to artificial electron acceptors, such as phenazine methosulfate (PMS), but not to the physiological acceptor, ubiquinone. The presence of the membrane domain anchors the catalytic dimer subunits to the inner membrane, restores ubiquinone reductase activity, and renders the enzyme sensitive to quinone analog inhibitors (1, 3). Thus, the membrane domain is believed to contain at least one quinone-binding site.In the bovine heart mitochondrial SDH, quinone binding has been assigned to the QPs1 and QPs3 membrane domain subunits by cross-linking with photoaffinity analogs of ubiquinone (14, 15). The quinone-binding site in the QPs1 subunit, which corresponds to Sdh3p, is localized in the matrix-facing loop connecting transmembrane segments II and III...

The Quinone-binding Sites of the Saccharomyces cerevisiae Succinate-ubiquinone Oxidoreductase

“…Residues in the unusual carboxyl-terminal extension of the yeast Sdh4p subunit are necessary for maintaining a stable conformation of the anchor domain required for quinone reduction (22,24). We have identified residues in the yeast Sdh3p that are involved in quinone reduction (25). The location of these residues and inhibitor sensitivity analysis suggested that the yeast SDH contains at least two quinone-binding sites.…”

“…The SDH4 knockout strain, sdh4w2, is an isogenic derivative of MH125 (MH125, SDH4::TRP1) and has been described previously (22). All the yeast media used in this study (YPD, YPG, SG, SD, YPDG, and YPGal) and yeast culture conditions have been described (22,25). Plasmid loss was routinely monitored by plating out aliquots on YPD and selectable SD media.…”

“…We have carried out several studies to identify functionally important residues in the yeast SDH (22,24,25). Residues in the unusual carboxyl-terminal extension of the yeast Sdh4p subunit are necessary for maintaining a stable conformation of the anchor domain required for quinone reduction (22,24).…”

“…The membrane-intrinsic domain is composed of two hydrophobic subunits, Sdh3p and Sdh4p, of 16.7 and 16.6 kDa, respectively (19 -21). The anchor domain contains a b-type heme and the active site for ubiquinone reduction (22)(23)(24)(25). The anchor domain was proposed to contain a core of four antiparallel helices comprising helices I, II, IV, and V (4,26).…”

“…The crystal structure of the Escherichia coli FRD (27) revealed two bound quinones. However, the crystal structure of the Wolinella succinogenes FRD (28) shows no such bound quinone, although there are two distal cavities in subunit C that could potentially bind quinone.We have carried out several studies to identify functionally important residues in the yeast SDH (22,24,25). Residues in the unusual carboxyl-terminal extension of the yeast Sdh4p subunit are necessary for maintaining a stable conformation of the anchor domain required for quinone reduction (22,24).…”