A Threonine on the Active Site Loop Controls Transition State Formation in Escherichia coli Respiratory Complex II

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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

Section: Discussionsupporting

confidence: 57%

Section: Discussionmentioning

confidence: 99%

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

Tomasiak¹,

Archuleta²,

Andréll

et al. 2011

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“…These domains have been proposed to move with respect to each other during cofactor insertion and catalysis. Investigation of the residue corresponding to Ala-524 has not been investigated in bacterial systems, although mutations within E. coli QFR that influence the interaction between these domains lower enzyme turnover to ϳ13% of wild-type levels (17).…”

Section: Aberrant Complex II Activity Associated With Neurodegenerationmentioning

confidence: 99%

Structural Basis for Malfunction in Complex II

Iverson

Maklashina

Cecchini

2012

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Complex II couples oxidoreduction of succinate and fumarate at one active site with that of quinol/quinone at a second distinct active site over 40 Å away. This process links the Krebs cycle to oxidative phosphorylation and ATP synthesis. The pathogenic mutation or inhibition of human complex II or its assembly factors is often associated with neurodegeneration or tumor formation in tissues derived from the neural crest. This brief overview of complex II correlates the clinical presentations of a large number of symptom-associated alterations in human complex II activity and assembly with the biochemical manifestations of similar alterations in the complex II homologs from Escherichia coli. These analyses provide clues to the molecular basis for diseases associated with aberrant complex II function.Over the past 6 decades, complex II (succinate dehydrogenase, succinate:quinone oxidoreductase (SQR) 3 ) has been at the forefront in the discovery of redox cofactors involved in bioenergetics. Helmut Beinert noted that in the 1950s-1960s, complex II was instrumental in the discovery of covalently bound flavin, tightly bound iron, and acid-labile sulfur associated with Fe-S clusters and bound ubiquinone of the mitochondrial respiratory chain (1). In 1995, a mutation of human complex II was the first report of a nuclear gene mutation shown to cause a mitochondrial respiratory chain deficiency (2). More recently, germ-line mutations in complex II have been found to be associated with hereditary tumors, suggesting that complex II genes may act as tumor suppressors (3).Complex II enzymes ( Fig. 1A) are heterotetramers containing two soluble subunits (SdhA (flavoprotein) and SdhB (Fe-S protein)) and two integral membrane subunits (SdhC and SdhD). This architecture houses two distinct active sites, and the coordinated catalysis at these sites links two key biological pathways, i.e. succinate oxidation to the Krebs cycle and quinone reduction to the electron transport chain. The SdhA subunit harbors a covalently attached FAD redox moiety and the dicarboxylate-binding site (Fig. 1B), where succinate is oxidized to fumarate. The product protons of this oxidation are transferred to bulk solvent, and fumarate acts as the next substrate in the Krebs cycle. The product electrons are transferred over 40 Å via three Fe-S clusters in the SdhB protein. These electrons act as co-substrates at the second active site, located at the interface of the integral membrane subunits. At this quinonereducing site (Fig. 1C), 2H ϩ and 2eϪ reduce ubiquinone to ubiquinol. The resultant quinol pool supports ATP synthesis by oxidative phosphorylation.The complex II superfamily contains both SQRs and quinol: fumarate reductases (QFRs). QFRs are enzymes that are kinetically poised to catalyze quinol oxidation and fumarate reduction as part of anaerobic electron transport chains. Both enzymes have evolved to function optimally in the physiological niche in which they are expressed. SQR and QFR are capable of functionally replacing each other in vivo in E...

“…This covalent bond increases the FAD redox potential by ϳ60 mV to permit succinate oxidation (6). SDH is the major mitochondrial protein containing a covalent bound flavin (7).…”

mentioning

confidence: 99%

Flavinylation and Assembly of Succinate Dehydrogenase Are Dependent on the C-terminal Tail of the Flavoprotein Subunit

Kim¹,

Jeong²,

et al. 2012

Background: Succinate dehydrogenase (SDH) requires a covalent addition of FAD for catalytic function. Results: Mutational analyses of Sdh1 implicate C-terminal region Arg residues involvement in covalent flavinylation and SDH assembly. Conclusion: SDH assembly is dependent on FAD binding to Sdh1 but not covalent binding. Significance: These results document the basis for the SDH deficiency and pathology seen with mutations in human Sdh1.