Role of the Evolutionarily Conserved Cytochrome b Tryptophan 142 in the Ubiquinol Oxidation Catalyzed by the bc1 Complex in the Yeast Saccharomyces cerevisiae

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The membrane-bound mitochondrial bc 1 complex (b 6 f complex in chloroplasts and cyanobacteria) is a key component of the respiratory and photosynthetic electron transfer chains (see Refs. 1-5 for reviews). It catalyzes the transfer of electrons from ubiquinol to cytochrome c and couples this electron transfer to the vectorial translocation of protons across the inner mitochondrial membrane. It contributes to the proton-motive force, which is subsequently used by the ATP synthase to produce ATP. All bc complexes, from bacteria to higher eukaryotes, contain three subunits forming the catalytic core of the enzyme and carrying four redox centers as follows: the iron-sulfur protein (ISP) 1 with a [2Fe-2S] cluster; the monohemic cyt c 1 ; the dihemic mitochondrially encoded cytochrome b with a low potential b L heme (E m7 around Ϫ50 mV, where E m indicates equilibrium redox midpoint potential) located near the positive side of the membrane; and a high potential b H heme (E m7 around ϩ90 mV) located on the negative side of the membrane. In eukaryotic cells, in addition to these three conserved subunits, as many as eight additional subunits are found whose functions are poorly understood (5). The bc 1 complex catalytic activity is best described by the modified Q cycle (6 -9). Electrons are delivered into a bifurcated pathway at the Q O site. A first electron is transferred from quinol to the ISP (in the so-called high potential electron transfer pathway to cyt c 1 and the soluble cyt c), resulting in the formation of an unstable semiquinone which then transfers a second electron to hemes b L and b H (in the so-called low potential pathway) across the membrane. At the Q I site, on the negative side of the membrane, quinone is reduced to semiquinone by heme b H . Two molecules of ubiquinol at the Q O site are thus required to reduce a quinone to quinol at the Q I site. Although several models have been proposed to account for the bifurcated electron transfer at the Q O site (10 -15), the mechanism at the molecular level is not completely understood. Several threedimensional structures of eukaryotic bc 1 complexes have been obtained in the presence or absence of different inhibitors (16 -19), and more recently, the yeast bc 1 complex structure was obtained at 2.3 Å, including bound water molecules (20). These different structures as well as biochemical and spectroscopic data (21-29) suggest that the extrinsic carboxyl-terminal domain of the ISP carrying the [2Fe-2S] cluster moves between a position close to cyt b (proximal conformation or "b" state) and a position close to cyt c 1 (distal conformation or "c 1 " state), thus solving the apparent incompatibility between distances and rate of electron transfer between the redox centers (16 -19). This movement is made possible by the presence of a flexible "hinge" region (sequence 85 TADVLAMAK 93 in the yeast Saccharomyces cerevisiae) located after the transmembrane domain of the ISP. However, recent data (30, 31) obtained with the cyt b 6 f complex indicate that the ISP l...

Section: Table I Genetic Background and Growth Characteristics Of Thementioning

confidence: 77%

QO Site Deficiency Can Be Compensated by Extragenic Mutations in the Hinge Region of the Iron-Sulfur Protein in the bc1 Complex of Saccharomyces cerevisiae

Brasseur

Lemesle-Meunier

Reinaud

et al. 2004

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“…Brasseur et al (8) favor the explanation that the pleiotropic effects of an ABC1 deletion are due to indirect effects arising from interactions of the other respiratory complexes with an altered complex III. There is evidence for a close interaction between complexes II and III since some mutations in complex III subunits interfere with SDH function (56,57). The tcm62⌬2::HIS3 mutation results in an almost complete loss of SDH-dependent activities, with little or no loss of complex III function as judged by the NADHcytochrome c reductase activities (Table I).…”

Section: Discussionmentioning

confidence: 99%

The Saccharomyces cerevisiae TCM62 Gene Encodes a Chaperone Necessary for the Assembly of the Mitochondrial Succinate Dehydrogenase (Complex II)

Dibrov

Lemire

1998

The assembly of the mitochondrial respiratory chain is mediated by a large number of helper proteins. To better understand the biogenesis of the yeast succinate dehydrogenase (SDH), we searched for assembly-defective mutants. SDH is encoded by the SDH1, SDH2, SDH3, and SDH4 genes. The holoenzyme is composed of two domains. The membrane extrinsic domain, consisting of Sdh1p and Sdh2p, contains a covalent FAD cofactor and three iron-sulfur clusters. The membrane intrinsic domain, consisting of Sdh3p and Sdh4p, is proposed to bind two molecules of ubiquinone and one heme. We isolated one mutant that is respiration-deficient with a specific loss of SDH oxidase activity. SDH is not assembled in this mutant. The complementing gene, TCM62 (also known as SCYBR044C), does not encode an SDH subunit and is not essential for cell viability. It encodes a mitochondrial membrane protein of 64,211 Da. The Tcm62p sequence is 17.3% identical to yeast hsp60, a molecular chaperone. The Tcm62p amino terminus is in the mitochondrial matrix, whereas the carboxyl terminus is accessible from the intermembrane space. Tcm62p forms a complex containing at least three SDH subunits. We propose that Tcm62p functions as a chaperone in the assembly of yeast SDH.The respiratory chain of the mitochondrial inner membrane is typically composed of four multisubunit enzymes (complexes I-IV) and the ATP synthase (complex V). In Saccharomyces cerevisiae, the succinate dehydrogenase (SDH) 1 or complex II subunits are encoded by the SDH1, SDH2, SDH3, and SDH4 genes (1, 2). The four SDH subunits are as follows: a large flavoprotein (Sdh1p) subunit of 67 kDa to which is covalently attached an FAD cofactor (3), an iron-sulfur subunit (Sdh2p) of 28 kDa that contains three iron-sulfur clusters (4), and two small hydrophobic membrane subunits of 16.7 (Sdh3p) and 16.6 (Sdh4p) kDa (5, 6). The two small subunits serve as membrane anchors and are thought to contain two ubiquinonebinding sites and a b-type heme (7,8).A large number of gene products are necessary for the biogenesis of the respiratory chain. It is a complex process involving the expression of two genomes and the transport of hundreds of proteins into the organelle (9, 10). In addition to the many nuclear and to the seven (in yeast) mitochondrially encoded subunits of the five complexes, many gene products are necessary to replicate and express the mitochondrial DNA. Perhaps more surprising are the long lists of genes required for the post-translational assembly of the respiratory chain and the ATP synthase (9, 11-13). Some of these, like the COX10 and COX11 gene products, are involved in heme a synthesis for complex IV biogenesis (14, 15). The COX17 gene encodes a mitochondrial copper shuttle necessary for metal delivery to complex IV (16). The ABC1 gene is essential for the correct assembly of complexes II-IV, and a loss of Abc1p impairs electron transport in complex III (8, 17). The BCS1 gene product may be necessary for the formation or insertion of the activesite iron-sulfur cluster of the Rieske...

“…However, to our knowledge, the only fumarateoxidizable cytochromes reported are associated with SDH. Furthermore, some mutations in the cytochrome b subunit of complex III, such as W142R, can severely impair SDH activity (50). Trp-142 is within 5 Å of the quinone-binding site to which myxothiazol binds, and we speculate that myxothiazol binding might similarly impair SDH functions.…”

Section: Discussionmentioning

confidence: 99%

Expression of Saccharomyces cerevisiae Sdh3p and Sdh4p Paralogs Results in Catalytically Active Succinate Dehydrogenase Isoenzymes

Szeto¹,

Reinke

Oyedotun

et al. 2012

Background: Succinate dehydrogenase is a tetrameric, mitochondrial membrane protein needed for respiratory energy generation. Results: Yeast succinate dehydrogenase containing Sdh3p and Sdh4p paralogs is catalytically active. Conclusion: Expression of paralogous subunits results in enzymes with novel kinetic properties. Significance: At least four functional succinate dehydrogenase isoenzymes containing zero, one, or two paralogs can be expressed in yeast mitochondria.