Oxidation of malate by the mitochondrial succinate-ubiquinone reductase

Succinate:quinone oxidoreductase (SQR, 4 succinate dehydrogenase) and menaquinol:fumarate oxidoreductase (QFR, fumarate reductase), members of the Complex II family, are homologous integral membrane proteins which couple the interconversion of succinate and fumarate with quinone and quinol (1-4). SQR is a key enzyme in the Krebs cycle, oxidizing succinate to fumarate during aerobic growth and reducing quinone to quinol and, thus, acts as a direct link between the Krebs cycle and the respiratory chain. QFR is found in anaerobic or facultative bacteria and lower eukaryotes, where it couples the oxidation of reduced quinones to the reduction of fumarate (1, 4). Escherichia coli SQR has four subunits, two hydrophilic subunits exposed to the cytoplasm (SdhA and SdhB), which interact with two hydrophobic membrane-intrinsic subunits (SdhC and SdhD) (5). SdhA contains the dicarboxylate-binding site and a covalently bound FAD cofactor which cycles between FAD and FADH 2 redox states during succinate oxidation (6). The electrons from succinate oxidation are sequentially transferred via a [2Fe-2S], a [4Fe-4S], and a [3Fe-4S] iron-sulfur cluster relay system in SdhB to a quinone-binding site (Q P ) located at the interface of the SdhB, SdhC, and SdhD subunits. SdhC and SdhD are both composed of three transmembrane helices and coordinate a low spin b-type heme via His residues contributed by each subunit (7,8).The first structural information about members of the Complex II family came from x-ray structures of the QFR enzymes from E. coli at 3.3 Å resolution (9) and Wolinella succinogenes at 2.2 Å resolution (10). These structures revealed details of the overall architecture of the subunits, the position of key redox cofactors, the electron transfer pathway, and the quinone-binding sites. At around the same time, the structures of soluble fumarate reductases found in anaerobic and microaerophilic bacteria and structurally homologous to the flavoprotein subunit of Complex II were solved by x-ray crystallography (1). Analysis of these soluble fumarate reductases has proven particularly informative in describing the mechanism of fumarate reduction and succinate oxidation at the dicarboxylate-binding site (11)(12)(13)(14).Structures of SQRs lagged behind those of the QFRs until the structure of the E. coli enzyme was solved at 2.6 Å (15). This structure, solved in space group R32, revealed that the E. coli enzyme is packed as a trimer. The structures of the SdhA and SdhB subunits were highly similar to those of E. coli and W. succinogenes QFRs, but the transmembrane SdhC and SdhD subunits showed differences compared with their QFR counterparts. The structure revealed the position of the redox sites and the dicarboxylate-and quinone-binding (Q) sites. The heme b molecule was shown to lie away from the electron transfer pathway, suggesting electrons are preferentially transferred from * This work was supported, in whole or in part, by National Institutes of Health Grant GM61606. This work was also supported by the Department of Ve...

Section: Comparison Of E Coli and Eukaryotic Sqr Structures-thesupporting

confidence: 81%

Structure of Escherichia coli Succinate:Quinone Oxidoreductase with an Occupied and Empty Quinone-binding Site

Ruprecht

Yankovskaya

Maklashina

et al. 2009

“…It is known that malate can be oxidized to OAA by FRD or SQR (40,41), and there is evidence that OAA is bound as the enol tautomer (42), with the double bond between C-2 and C-3, rather than the keto form, which is more stable in aqueous solution. The geometry about C-2 (the oxygen-bearing central carbon) is nearly planar rather than tetrahedral, which suggests sp 2 hybridization.…”

Section: Architecture Of the Succinate-binding Site And Identificatiomentioning

confidence: 99%

3-Nitropropionic Acid Is a Suicide Inhibitor of Mitochondrial Respiration That, upon Oxidation by Complex II, Forms a Covalent Adduct with a Catalytic Base Arginine in the Active Site of the Enzyme

Huang¹,

Sun²,

Cobessi³

et al. 2006

274

256

The toxin 3-nitropropionic acid 5 is produced by certain plants and fungi. It is a specific inhibitor of mitochondrial respiratory complex II. Fatalities after eating moldy sugarcane have been linked to 3-NP toxicity (1, 2). Ruminants grazing in regions with 3-NP-producing plants acquire resistance because of reduction of the nitro group to an amine by ruminal bacteria (3).The effectiveness of 3-NP in vivo after injection or oral administration has made it useful in studies involving tissues or whole animals. Ingestion of 3-NP results in neurodegeneration with symptoms resembling those of Huntington disease (4), and conversely Huntington disease results in a loss of complex II activity (5); thus 3-NP has been used to produce an animal model for the study of Huntington disease (6, 7). Symptoms also include convulsions, and 3-NP is being looked at for inducing a model of epilepsy (8). Prior subacute 3-NP poisoning seems to provide resistance to ischemic damage to nervous tissue by a preconditioning effect (9) similar to that resulting from mild ischemia.The target of 3-NP is Complex II, which is both a member of the Krebs tricarboxylic acid cycle (oxidizing succinate to fumarate) and an entry point for electrons into the respiratory chain at the level of ubiquinol. It consists of a large flavoprotein subunit containing covalently bound FAD, an iron-sulfur protein (IP) with three different iron-sulfur clusters, and two small membrane anchor subunits (chains C and D) ligating a single low spin heme of type B. Human genetic defects in the IP subunits or chains C or D lead to development of paragangliomas (10, 11). A mutation in chain C leads to premature aging in nematodes, presumably through excessive production of free radicals (12). Bacterial homologs succinate:quinone oxidoreductase (SQR) and menaquinol: fumarate oxidoreductase (FRD) in Escherichia coli have been studied as genetically manipulable models for the mitochondrial protein. Recent reviews cover this family of enzymes (13-18). X-ray crystallographic structures are available for a number of members of the family. The available mitochondrial structures and representative bacterial examples are listed in Table 1.The toxin 3-NP, structurally similar to and isoelectronic with the substrate succinate, is believed to be a suicide inactivator of Complex II. Alston et al. (19) proposed, based on previous observations and on their own experience with another flavoprotein, that the normal reaction pathway involves a temporary adduct with the N-5 nitrogen of flavin, which in the case of 3-NP collapses to a stable adduct resulting in permanent inactivation. Irreversible covalent modification of the flavin was ruled out by later work (20) examining the spectral change induced and showing that unmodified flavin peptide could be isolated from the inactivated complex by mild proteolysis. It was proposed that 3-NP is oxidized to 3-nitroacrylate, an unstable molecule that then reacts with some residue in the active site. A cysteine that was believed to be in the active ...

“…It has additionally been proposed that oxaloacetate can be formed through off-pathway catalysis by complex II when fumarate hydration to malate is followed by oxidation to oxaloacetate (3,4). Oxaloacetate binding to the complex II active site results in a charge transfer complex between the oxaloacetate carbonyl and the FAD hydride that can be monitored spectroscopically (4,5).…”

mentioning

confidence: 99%

Geometric Restraint Drives On- and Off-pathway Catalysis by the Escherichia coli Menaquinol:Fumarate Reductase

Tomasiak¹,

Archuleta²,

Andréll

et al. 2011

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.Complex II superfamily enzymes provide a crucial link between oxidoreduction reactions in the membrane bilayer and in the soluble milieu (1). During aerobic respiration in Escherichia coli, the complex II enzyme succinate:ubiquinone oxidoreductase oxidizes succinate and reduces ubiquinone. In bacterial anaerobic respiration with fumarate as the terminal electron acceptor, the reaction proceeds in the opposite direction, and the complex II homolog menaquinol:fumarate oxidoreductase (QFR) 4 oxidizes menaquinol and transfers the electrons to fumarate. Complex II superfamily members contain either three or four polypeptide chains (Fig. 1A): two soluble subunits (the flavoprotein, FrdA, and the iron protein, FrdB, in E. coli QFR) and either one or two integral membrane subunits (FrdC and FrdD in the E. coli QFR). Although there are significant differences in the integral membrane subunits across the family, complex II enzymes all share a high percentage of sequence identity in the soluble subunits, including the flavoprotein, where the kinetically challenging oxidoreduction of fumarate and succinate takes place (1).Numerous molecules, including metabolic intermediates and ingested toxins, structurally resemble succinate and fumarate. Some of these are excluded from the active site, whereas others act as inhibitors (Fig. 1B). Several of these come from normal respiratory processes. For example, oxaloacetate is a Krebs cycle intermediate and acts as a tight, slow