The Quaternary Structure of the Saccharomyces cerevisiae Succinate Dehydrogenase

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. 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. Succinate dehydrogenase (SDH), 1The abbreviations used are: SDH, succinate dehydrogenase; FRD, fumarate reductase; s-BDNP, 2-sec-butyl-4,6-dinitrophenol; UQ, ubiquinone; QP, QD, proximal and distal quinone-binding sites, respectively; DPPC, dipalmitoylphosphatidylcholine; LGA, Lamarckian genetic algorithm; ΔGBIND, free energy of binding; PDB, Protein Data Bank; SPC, simple point charge; RMSD, root mean square deviation; MD, molecular dynamics.1The abbreviations used are: SDH, succinate dehydrogenase; FRD, fumarate reductase; s-BDNP, 2-sec-butyl-4,6-dinitrophenol; UQ, ubiquinone; QP, QD, proximal and distal quinone-binding sites, respectively; DPPC, dipalmitoylphosphatidylcholine; LGA, Lamarckian genetic algorithm; ΔGBIND, free energy of binding; PDB, Protein Data Bank; SPC, simple point charge; RMSD, root mean square deviation; MD, molecular dynamics. also known as Complex II or succinate:quinone oxidoreductase, is a key enzyme in intermediary metabolism and aerobic energy production in both prokaryotic and eukaryotic cells. SDH couples the oxidation of succinate in the Krebs cycle to the reduction of ubiquinone (UQ) in the electron transport chain (1Ackrell B.A.C. Johnson M.K. Gunsalus P.R. Cecchini G. Muller F. Chemistry and Biochemistry of Flavoenzymes. Vol. III. CRC Press, Boca Raton, FL1992: 229-297Google Scholar, 2Cecchini G. Schröder I. Gunsalus R.P. Maklashina E. Biochim. Biophys. Acta. 2002; 1553: 140-157Crossref PubMed Scopus (203) Google Scholar, 3Hägerhäll C. Biochim. Biophys. Acta. 1997; 1320: 107-141Crossref PubMed Scopus (371) Google Scholar, 4Hederstedt L. Ohnishi T. Ernster L. Molecular Mechanisms in Bioenergetics. Elsevier Science Publishers, New York1992: 163-197Google Scholar, 5Lemire B.D. Oyedotun K.S. Biochim. Biophys. Acta. 2002; 1553: 102-116Crossref PubMed Scopus (96) Google Scholar). Membrane-bound fumarate reductases (FRD) or menaquinol:fumarate oxidoreductases, which are found in anaerobic organisms respiring with fumarate as terminal electron acceptor, are functionally and structurally related to SDH (2Cecchini G. Schröder I. Gunsalus R.P. Maklashina E. Biochim. Biophys. Acta. 2002; 1553: 140-157Crossref PubMed Scopus (203) Google Scholar, 6Cole S.T. Condon C. Lemire B.D. Weiner J.H. Biochim. Biophys. Acta. 1985; 811: 381-403Crossref PubMed Scopus (133) Google Scholar, 7Hägerhäll C. Hederstedt L. FEBS Lett. 1996; 389: 25-31Crossref PubMed Scopus (120) Google Scholar, 8Hederstedt L. Science. 1999; 284: 1941-1942Crossref PubMed Scopus (54) Google Scholar). FRD catalyzes the reduction of fumarate to succinate, the reverse of the SDH reaction. The structures of the Escherichia coli and the Wollinella succinogenes FRDs have been solved to resolutions of 2.7 and 2.2 Å, respectively (9Lancaster C.R. Kröger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (310) Google Scholar, 10Iverson T.M. Luna-Chavez C. Cecchini G. Rees D.C. Science. 1999; 284: 1961-1966Crossref PubMed Scopus (365) Google Scholar). More recently, the structure of the E. coli SDH was solved to 2.6 Å resolution (11Yankovskaya V. Horsefield R. Törnroth S. Luna-Chavez C. Miyoshi H. Léger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (676) Google Scholar). SDH and FRD enzymes consist of a hydrophilic catalytic dimer that protrudes into the mitochondrial matrix or the bacterial cytoplasm and either one or two integral membrane subunits. Table I lists the subunit and cofactor compositions of the relevant enzymes. The catalytic dimers are comprised of two subunits that exhibit a high degree of sequence conservation across species and as expected, the structures of the catalytic subunits are very similar. The larger subunit carries a covalently bound flavin adenine dinucleotide (FAD), while the smaller subunit contains three iron-sulfur clusters. In contrast, the membrane portions of SDH and FRD enzymes show considerable variability in the primary structures of their subunits and in cofactor composition (3Hägerhäll C. Biochim. Biophys. Acta. 1997; 1320: 107-141Crossref PubMed Scopus (371) Google Scholar, 7Hägerhäll C. Hederstedt L. FEBS Lett. 1996; 389: 25-31Crossref PubMed Scopus (120) Google Scholar). In the E. coli SDH and FRD enzymes, two hydrophobic subunits are present. The E. coli FRD was crystallized without heme and with two bound menaquinone molecules, while the E. coli SDH was crystallized with one heme and one ubiquinone. The W. succinogenes FRD has a single membrane subunit, which contains two hemes, but the crystals did not contain any bound quinone. Although, this latter enzyme is known to oxidize quinone, the positions of any quinone-binding sites have yet to be determined. The structures of the membrane subunits of each enzyme are considerably different, although in all three x-ray structures, four antiparallel helices form a central helix bundle. It is not possible to superimpose the membrane subunits without changing the position, orientation, and tilt of the transmembrane helices (12Iverson T.M. Luna-Chavez C. Schröder I. Cecchini G. Rees D.C. Curr. Opin. Struct. Biol. 2000; 10: 448-455Crossref PubMed Scopus (30) Google Scholar). The variability among membrane subunits and the cofactors they harbor are major determinants of the distinct properties of SDH and FRD enzymes in different biological systems.Table IThe subunit and cofactor compositions of SDH and FRD enzymes from E. coli, S. cerevisiae, and W. enzyme is by the in subunit and subunit and and cofactors in the are coli SDH ubiquinone coli FRD succinogenes FRD cerevisiae SDH Sdh4p ubiquinone The enzyme is by the in cofactors in the are in a SDH not in mitochondrial energy but also has a in and in the in mitochondrial or while in the or genes are with in the and A. A. B. Science. 2000; PubMed Scopus Google Scholar, T. M. M. E. A. A. PubMed Scopus Google Scholar, A. Biochim. Biophys. Acta. 2002; 1553: PubMed Scopus Google Scholar). The in the in and the production of M. M. S. Nature. PubMed Scopus Google Scholar). The variability of with SDH and a possible for the enzyme in have in the structure and the molecular that The Saccharomyces cerevisiae SDH is a tetrameric enzyme that has been as a model for the mitochondrial enzymes B.D. Oyedotun K.S. Biochim. Biophys. Acta. 2002; 1553: 102-116Crossref PubMed Scopus (96) Google Scholar, K.S. Lemire B.D. FEBS Lett. 1999; PubMed Scopus Google Scholar). on the of by the and on mutagenesis the enzyme is proposed to harbor two quinone-binding sites K.S. Lemire B.D. Biol. 1999; PubMed Scopus Google Scholar, K.S. Lemire B.D. Biol. PubMed Scopus Google Scholar). In of the Sdh4p is for the of reduction and for the of a K.S. Lemire B.D. Biol. 1997; PubMed Scopus Google Scholar, K.S. Lemire B.D. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). In this a model for the quaternary structure of the S. cerevisiae iron-sulfur heme and the analog s-BDNP, were docked into the In used molecular dynamics on the membrane dimer embedded in a phospholipid to Sdh4p which may as heme axial We discuss the possible of heme and the of two quinone-binding sites in electron transport. structure of the SDH was constructed by using the A. A. Protein 2000; PubMed Scopus Google Scholar, A. R. F. A. Biophys. Struct. 2000; PubMed Scopus Google Scholar, A. Biol. PubMed Scopus Google Scholar). used for were the E. coli SDH, the E. coli FRD, and the W. succinogenes FRD Data and and were using T. PubMed Scopus Google and in the 1997; PubMed Scopus Google Scholar, A. 1999; PubMed Google Scholar, T. A. 2000; PubMed Scopus Google by the mean energy F. E. Biol. 1997; PubMed Scopus Google Scholar, Biol. PubMed Scopus Google of the and the iron-sulfur subunits have high sequence all three and were used for the catalytic dimer. is not possible to model the membrane subunits in this to the low primary sequence conservation (3Hägerhäll C. Biochim. Biophys. Acta. 1997; 1320: 107-141Crossref PubMed Scopus (371) Google Scholar, 7Hägerhäll C. Hederstedt L. FEBS Lett. 1996; 389: 25-31Crossref PubMed Scopus (120) Google Scholar). the three structures, (11Yankovskaya V. Horsefield R. Törnroth S. Luna-Chavez C. Miyoshi H. Léger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (676) Google the for the membrane dimer. of the membrane subunits were by the positions of the three transmembrane for each subunit K.S. Lemire B.D. Biol. 1999; PubMed Scopus Google Scholar, K.S. Lemire B.D. Biol. PubMed Scopus Google the heme and residues that may with ubiquinone the Sdh3p with the E. coli that three was in the of the second transmembrane of the three that be identified in a sequence of Sdh3p related of structures, of which were was and the one with the was for sequence among and FRDs from E. coli, S. cerevisiae, and W. cerevisiae cerevisiae cerevisiae cerevisiae coli sequence coli succinogenes coli coli succinogenes coli coli sequence in a of the three used for any for the Sdh4p structure that residues are with a hydrophobic of We for to in using the of the The with the energy and sequence was and to the Sdh4p with the A. PubMed Scopus Google using as a The model was of using R. Scopus Google Scholar, E. B. Google Scholar). We the of the with the of mutagenesis which that may with K.S. Lemire B.D. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). The for the model have been in the Protein Data for New The and Sdh4p subunits to in the and for and heme were from the PubMed Scopus Google Scholar). The of the analog inhibitor were with the C. Scopus Google and with R. Scopus Google Scholar, E. B. Google Scholar). of the three iron-sulfur and heme were determined by with using the of and inhibitor s-BDNP, into the SDH model subunit was not to the of the or heme binding was with the 1996; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, R. 1996; 10: PubMed Scopus Google Scholar, R. Scopus Google using the The Lamarckian genetic was used to molecular R. Scopus Google Scholar). for the of energy and were to and The docked were on the of energy with of the with residues the site. and for SDH residues and cofactors were in using the and in the cofactors were with and all were to In were all single in a ligand were using the into the proximal quinone-binding the was the of SDH, while for into the distal quinone-binding the was the of the The of the was by the of mutagenesis of the quinone-binding sites K.S. Lemire B.D. Biol. 1999; PubMed Scopus Google Scholar, K.S. Lemire B.D. Biol. PubMed Scopus Google Scholar). the was a Å with separated by the was a Å with separated by The cofactors were a from the site. energies of binding were using the in R. Scopus Google Scholar). The the as a the as a and the The was by and and from of structures are known high and energy was used to the degree of of the were with the Google Scholar, PubMed Scopus Google Scholar). of the model was by the F. E. Biol. 1997; PubMed Scopus Google which on single residues and of residues with the was the of or the of a of the Molecular dynamics simulation was in R. Scopus Google Scholar, E. B. Google with the The subunit was not used in the simulation not the Sdh3p and Sdh4p subunits. The were in a with and simple point The model for the is a of molecules 1996; Scopus Google Scholar). Å Å with the in the was using the of The was with using the The simulation molecules molecules in each on both with The were to the simulation was with of the to as the structures for in which the was with of of the and while the of the The and were to the and and were and using the A. Scopus Google with and of and The were using a with the and second and were the T. L. Scopus Google Scholar, L. T. H. Scopus Google with a of for the and a for the were using the to a of Molecular dynamics were on a of with A. Google Scholar). for with the for were and using W. A. 1996; Scopus Google Scholar). A of the S. cerevisiae the model of the S. cerevisiae SDH constructed by which structures by the of A. A. Protein 2000; PubMed Scopus Google Scholar, A. R. F. A. Biophys. Struct. 2000; PubMed Scopus Google Scholar, A. Biol. PubMed Scopus Google Scholar). It and on the sequence from with the in with the were used to a of the with and molecular dynamics to a model that all the In the the structures of the Sdh1p and Sdh2p subunits the structures of the The model of the catalytic dimer is structure based on from the three E. coli SDH (11Yankovskaya V. Horsefield R. Törnroth S. Luna-Chavez C. Miyoshi H. Léger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (676) Google E. coli FRD T.M. Luna-Chavez C. Cecchini G. Rees D.C. Science. 1999; 284: 1961-1966Crossref PubMed Scopus (365) Google and W. succinogenes FRD (9Lancaster C.R. Kröger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (310) Google Scholar). were to model the subunits using the three as this to the in the position, orientation, and the of W. succinogenes of transmembrane The crystal structure of the E. coli SDH that is Å of UQ, while is to the residues are in the S. cerevisiae SDH subunits A and the heme and the transmembrane as sequence of the membrane subunits and K.S. Lemire B.D. Biol. 1999; PubMed Scopus Google and K.S. Lemire B.D. Biol. PubMed Scopus Google Scholar). The is a model that is to the E. coli SDH mean square of Å for as determined with the The of the model is as as of the for the S. cerevisiae and the E. coli SDH cerevisiae coli Protein Protein in a FAD cofactor and the three iron-sulfur clusters be docked by the catalytic dimer of the E. coli SDH that of the S. cerevisiae SDH cofactor are among the catalytic dimers from the three crystal structures and heme be docked into the S. cerevisiae SDH by The of and inhibitor s-BDNP, were determined with the A and The in excellent agreement with determined structures of 1996; PubMed Scopus Google Scholar, R. Scopus Google Scholar). in the heme was docked into the crystal structure of the E. coli SDH all cofactors from the the docked heme the heme in the crystal the positions of the two are in In for the S. cerevisiae SDH, each of with each a single docked The of structures was in of their A of structures a root mean square in positions of than Å was a A of clusters and while a of clusters a or of binding as the into different binding conformations or The cofactor structure was identified as the with the energy and was for for and into the S. cerevisiae of of in the energy in a FAD and the from sequence the of FAD and the three iron-sulfur clusters in the S. cerevisiae SDH model and are similar to for the E. coli SDH (11Yankovskaya V. Horsefield R. Törnroth S. Luna-Chavez C. Miyoshi H. Léger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (676) Google the E. coli FRD T.M. Luna-Chavez C. Cecchini G. Rees D.C. Science. 1999; 284: 1961-1966Crossref PubMed Scopus (365) Google and the W. succinogenes FRD (9Lancaster C.R. Kröger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (310) Google Scholar). In the three crystal structures, FAD is with the subunit a the flavin and the of a histidine residue. In the S. cerevisiae SDH Sdh1p is in to the flavin FRDs from E. coli and W. succinogenes and SDH from E. coli contain three iron-sulfur which are by residues in the or the subunits. The is the E. coli SDH is a In the S. cerevisiae SDH, the residues are A and show the docked heme in the S. cerevisiae In this the of Sdh3p and the of Sdh4p Cys-78 are to form with the central of the heme. The the and the is Å, which is the of in the E. coli SDH (11Yankovskaya V. Horsefield R. Törnroth S. Luna-Chavez C. Miyoshi H. Léger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (676) Google and W. succinogenes FRD crystal structures (9Lancaster C.R. Kröger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (310) Google Scholar). the of the second heme axial ligand in the SDH, is Å from the heme than in crystal is not the model is based on from the E. coli SDH crystal structure is the Molecular dynamics were to low energy conformations for the Cys-78 model be docked into two separated sites with of Å A and similar to the sites in the E. coli The free energies of binding the and sites are and respectively residues near each of the two quinone-binding sites are in binding free energies of of the UQ, and and the experimental inhibitory are in are in and were from Oyedotun and Lemire are in are in and were from Oyedotun and Lemire K.S. Lemire B.D. Biol. PubMed Scopus Google Scholar). in a The of are known analog of the respiratory chain I. Miyoshi H. R. H. PubMed Scopus (30) Google Scholar, V. Cecchini G. Miyoshi H. Biol. 1996; PubMed Scopus Google Scholar). the S. cerevisiae SDH with K.S. Lemire B.D. Biol. 1999; PubMed Scopus Google Scholar, K.S. Lemire B.D. Biol. PubMed Scopus Google Scholar). The with the of mutagenesis have to a two quinone-binding model for the molecules of were docked into the and sites The for the two inhibitor binding sites by one of with the determined K.S. Lemire B.D. Biol. 1999; PubMed Scopus Google Scholar, K.S. Lemire B.D. Biol. PubMed Scopus Google Scholar). Molecular the membrane subunits of the three structures a similar of four the and of helices We used molecular dynamics to energy conformations of the membrane dimer for the for were with the membrane dimer embedded in a with a The from the of simulation the the structure was to the and the conformations this were the structure of this by the simulation on the The is Å of the and Sdh4p subunits. The are transmembrane helix II and the of The of the helix the of Sdh4p Cys-78 to Å of the of heme The in the from the complex and the are Å and Å, respectively S. T. C. 2002; PubMed Scopus Google Scholar, H. H. S. A. 1999; PubMed Scopus Google mean square of simulation of SDH from the was with the SDH model with a to the and the root mean square was calculated the using the of structure of the calculated simulation and the of electron of the SDH structure molecular dynamics with the Sdh4p residues that are molecular dynamics simulation are The structure was calculated from the of the using the of structure of the a molecular dynamics cofactor and of electron the cofactors are of the structure and of SDH and FRD enzymes has the of the E. coli SDH, the E. coli FRD, and the W. succinogenes FRD crystal structures (9Lancaster C.R. Kröger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (310) Google Scholar, 10Iverson T.M. Luna-Chavez C. Cecchini G. Rees D.C. Science. 1999; 284: 1961-1966Crossref PubMed Scopus (365) Google Scholar, V. Horsefield R. Törnroth S. Luna-Chavez C. Miyoshi H. Léger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (676) Google Scholar). the variability in heme and the of electron the membrane dimer (3Hägerhäll C. Biochim. Biophys. Acta. 1997; 1320: 107-141Crossref PubMed Scopus (371) Google Scholar). is also considerable in SDH is with The structure and of the membrane dimer is in of the roles for SDH as a of and as a A. A. B. Science. 2000; PubMed Scopus Google Scholar, M. M. S. Nature. PubMed Scopus Google Scholar, Lemire B.D. Biol. 2003; PubMed Scopus Google Scholar, M. T. S. H. Biol. PubMed Scopus Google Scholar). and in the E. coli FRD and the S. cerevisiae SDH have the of the site, in the has yet to be B.D. Oyedotun K.S. Biochim. Biophys. Acta. 2002; 1553: 102-116Crossref PubMed Scopus (96) Google Scholar, Gunsalus R.P. Cecchini G. Biol. PubMed Google Scholar, Gunsalus R.P. H. Cecchini G. Biol. PubMed Google Scholar). The in this have considerable insight into the structure of the and heme binding sites of a mitochondrial In proposed that the S. cerevisiae SDH has two quinone-binding sites B.D. Oyedotun K.S. Biochim. Biophys. Acta. 2002; 1553: 102-116Crossref PubMed Scopus (96) Google Scholar). A and show the residues in the of the site. Sdh4p and Sdh2p are in with of residues are to and of the E. coli In that the of Sdh3p is of the of the and may with the bound the site. is polar chain in the of the in the E. coli in that to the Sdh3p The by the chain may that the is more bound in the SDH than is in the E. coli by the C. M. M. S. Nature. PubMed Scopus Google SDH be a of may be a eukaryotic to that the S. cerevisiae SDH free We are this mutagenesis that the S. cerevisiae SDH residues near the are that the free may from binding or reduction Lemire B.D. Biol. 2003; PubMed Scopus Google Scholar). Sdh3p and are residues proposed to be in the of the site, of residues K.S. Lemire B.D. Biol. 1999; PubMed Scopus Google Scholar). the of two residues in model that they not with the but that they are to residues that be docked into the membrane dimer this to the A and In the E. coli SDH crystal this is with a (11Yankovskaya V. Horsefield R. Törnroth S. Luna-Chavez C. Miyoshi H. Léger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (676) Google Scholar). Sdh4p is of the A and in a in for the K.S. Lemire B.D. Biol. PubMed Scopus Google Scholar). polar in the of the in model is Sdh4p which may form a with the A of Sdh3p and is in the and hydrophobic We have identified Sdh3p as important for reduction and binding K.S. Lemire B.D. Biol. 1999; PubMed Scopus Google Scholar). the environment of the is more hydrophobic than that of the A and In are residues with the site. for the of the for and The the and the sites by one of K.S. Lemire B.D. Biol. 1999; PubMed Scopus Google Scholar, K.S. Lemire B.D. Biol. PubMed Scopus Google Scholar). The free energies of binding calculated using the are in agreement with the experimental The of the and the of polar with bound this the of bound with the Sdh3p and Sdh4p Cys-78 are in to the in the heme and as are the for the axial and this is the axial heme ligand to be proposed for the SDH and FRD of enzymes. The E. coli, and subunits also have a this position, but enzymes not contain heme (3Hägerhäll C. Biochim. Biophys. Acta. 1997; 1320: 107-141Crossref PubMed Scopus (371) Google Scholar). It is not possible to Cys-78 to a histidine the larger chain to with heme in to the E. coli SDH and the W. succinogenes FRD, the of the SDH is in may for why with smaller chain is the axial ligand in the S. cerevisiae enzyme instead of We have used mutagenesis and to this S. Oyedotun and B. The that Sdh3p and Sdh4p Cys-78 are the heme axial of Sdh4p Cys-78 for a enzyme and The of the in the membrane dimer that the heme and the two quinone-binding sites are in electron from the catalytic dimer to the the proposed of all in the S. cerevisiae SDH the are the of electron Nature. 1999; 402: PubMed Scopus Google Scholar). the and the sites, which are Å the heme SDH identified to contain one but in electron The of the for ubiquinone may to the for which a while the of The of the that is from which to to the In this contain two binding sites to the for with the to the production of free the roles of the and sites and of heme be to this The quaternary structure model of the S. cerevisiae SDH in this has identified residues that may the or catalytic properties of each of the two binding The model has also insight into the of a as the second heme ligand instead of the histidine residues in all of the SDH and FRD from this model and to a of the catalytic properties of a mitochondrial SDH and their to We for of the

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