Identification of Quinone-binding and Heme-ligating Residues of the Smallest Membrane-anchoring Subunit (QPs3) of Bovine Heart Mitochondrial Succinate:Ubiquinone Reductase

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...

supporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

The Saccharomyces cerevisiae Succinate-ubiquinone Oxidoreductase

Oyedotun

Lemire

1999

“…This conclusion is also supported by the site-directed mutagenesis studies of E. coli SQR, where substitution of histidyl residues in the membrane binding domain suggests that SdhC His 84 and SdhD His 71 are the axial ligands to the heme (16,27). An example of the swapping of the heme axial ligands has been observed in both beef (43) and E. coli SQR (26), when the small membrane anchor subunit QPs3 or SdhD respectively, is expressed alone. In these examples, it appears that the histidyl ligands come from two different molecules of the respective small subunit (26,43).…”

Section: Discussionmentioning

confidence: 99%

Retention of Heme in Axial Ligand Mutants of Succinate-Ubiquinone Oxidoreductase (Complex II) from Escherichia coli

Maklashina

Rothery

Weiner

et al. 2001

Succinate-ubiquinone oxidoreductase (SdhCDAB, complex II) from Escherichia coli is a four-subunit membrane-bound respiratory complex that catalyzes ubiquinone reduction by succinate. In the E. coli enzyme, heme b 556 is ligated between SdhC His 84 and SdhD His 71 . Contrary to a previous report (Vibat, C. R. T., Cecchini, G., Nakamura, K., Kita, K., and Gennis, R. B. (1998) Biochemistry 37, 4148 -4159), we demonstrate the presence of heme in both SdhC H84L and SdhD H71Q mutants of SdhCDAB. EPR spectroscopy reveals the presence of low spin heme in the SdhC H84L (g z ‫؍‬ 2.92) mutant and high spin heme in the SdhD H71Q mutant (g ‫؍‬ 6.0). The presence of low spin heme in the SdhC H84L mutant suggests that the heme b 556 is able to pick up another ligand from the protein. CO binds to the reduced form of the mutants, indicating that it is able to displace one of the ligands to the low spin heme of the SdhC H84L mutant. The g ‫؍‬ 2.92 signal of the SdhC H84L mutant titrates with a redox potential at pH 7.0 (E m,7 ) of approximately ؉15 mV, whereas the g ‫؍‬ 6.0 signal of the SdhD H71Q mutant titrates with an E m,7 of approximately ؊100 mV. The quinone site inhibitor pentachlorophenol perturbs the heme optical spectrum of the wild-type and SdhD H71Q mutant enzymes but not the SdhC H84L mutant. This finding suggests that the latter residue also plays an important role in defining the quinone binding site of the enzyme. The SdhC H84L mutation also results in a significant increase in the K m and a decrease in the k cat for ubiquinone-1, whereas the SdhD H71Q mutant has little effect on these parameters. Overall, these data indicate that SdhC His 84 has an important role in defining the interaction of SdhCDAB with both quinones and heme b 556 .Succinate-ubiquinone oxidoreductases (SQR 1 ; succinate dehydrogenase, complex II) and menaquinol-fumarate reductase (QFR; fumarate reductase) are membrane-bound complexes that play critical roles in cellular metabolism in prokaryotic and eukaryotic organisms. The enzymes catalyze the reversible transfer of two electrons and two protons between succinatefumarate and the quinol-quinone couples (1); however, they normally are only expressed in aerobic (SQR) or anaerobic (QFR) environments (2, 3). SQR directly connects the Krebs cycle with the aerobic respiratory chain by transferring reducing equivalents via quinone, whereas QFR is a terminal reductase in anaerobic respiration, where it oxidizes low potential quinols and reduces fumarate as the final electron acceptor (2, 4 -6).Two distinct operons encode the subunits of SQR (sdhCDAB) (7, 8) and QFR (frdABCD) (9, 10) in Escherichia coli. X-ray structures for membrane-bound QFR from E. coli (11) 1ϩ,0 ). The soluble dehydrogenase fragment (FpIp) binds to the hydrophobic anchor domain to form a membrane-bound complex (complex II) that is able to carry out electron transfer with quinone-quinol. The hydrophobic membrane anchor subunit pairs (SdhC, FrdC and SdhD, FrdD) are essential for forming the quinone binding sites and assembly of t...

“…The experiments on many heme-containing members of the QFR/SQR family have yielded evidence for the participation of a site positioned similarly to Q D during electron transfer (21,40,43,53); however the molecular characteristics of these functional sites resemble those observed in Q P in the E. coli QFR-respiratory complex. Quinol oxidation at a site positioned similarly to Q D indicates that transmembrane electron transfer occurs and would be obligatory for the generation of a transmembrane electrochemical potential gradient during turnover (54,55).…”

Section: Discussionmentioning

confidence: 99%

Crystallographic Studies of the Escherichia coli Quinol-Fumarate Reductase with Inhibitors Bound to the Quinol-binding Site

Iverson

Luna-Chavez

Croal

et al. 2002

101

119

The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integralmembrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH 2 ) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH 2 , bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q P and Q D , indicating their positions proximal (Q P ) or distal (Q D ) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH 2 at the Q P site. In the structures with the inhibitor bound at Q P , no density is observed at Q D , which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q D . A comparison of the Q P site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover.The Escherichia coli quinol-fumarate reductase (QFR) 1 respiratory complex catalyzes the terminal step of anaerobic respiration when fumarate acts as the terminal electron acceptor (1). During this type of anaerobic respiration, electrons are donated to QFR by menaquinol (MQH 2 ) molecules in the membrane. The electrons are transferred to a covalently-bound flavin adenine nucleotide at the active site through three distinct iron-sulfur clusters and ultimately are used to reduce fumarate to succinate (2, 3). The QFR respiratory complex is composed of four polypeptide chains. Two of these chains, the flavoprotein (FrdA) and the iron protein (FrdB), comprise the soluble domain, which is involved in fumarate reduction, whereas the remaining two subunits (FrdC and FrdD) are membrane-spanning polypeptides involved in the electron transfer with quinones. High sequence identity between the soluble domain of the E. coli QFR and the soluble domain of succinate-quinone oxidoreductase (SQR, complex II) of the aerobic respiratory chain places these two complexes in the same family (4). Consistent with the sequence relationship, both enzymes from E. coli can bidirectionally catalyze the interconversion of succinate and fumarate (5), and each can functionally replace the other to support growth (6, 7). In contrast to the soluble domain, the transmembrane anchor subunits of the complex II family have little sequence identity and exhibit variable polypeptide and cofactor composition. Nevertheless...