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

Abstract

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

Redox State of Flavin Adenine Dinucleotide Drives Substrate Binding and Product Release in Escherichia coli Succinate Dehydrogenase

“…In agreement with the T. thermophilus study, we also believe that the EPR lineshape changes are suggestive of two distinct conformations of the enzyme: an “occupied” state with substrate/inhibitor bound (g = 1.91 trough) and a “free” state without substrate/inhibitor bound (g = 1.92 trough) that are in rapid equilibrium. This is consistent with the hypothesis that structural changes occur, especially of the hinge region and the capping domain, to allow opening and closing of the active site during catalysis (5, 9, 26, 57). …”

“…Interestingly, all three substitutions of SdhA-R286 studied herein resulted in assembly of non-covalent FAD, including the Lys variant. It has been proposed that the guanidinium side chain of the conserved SdhA-R286 residue can: ( i ) H-bond to and stabilize the substrate in the dicarboxylate binding site, and ( ii ) act as a proton donor for fumarate reduction and a proton acceptor during succinate oxidation (9, 15, 35, 52). In comparison, the imidazole ring of SdhA-H242 plays a more limited role by H-bonding to the substrate and is not believed to be involved in proton shuttling during catalysis.…”

“…Alternatively, oxidation of FADH − by the [Fe-S] clusters in SdhB may also induce conformational change to the “free” form. In E. coli Sdh, movements of the capping and FAD binding domains are important for activating the C2-C3 bond of fumarate/succinate to attain the transition state (5, 9). In X-ray crystallographic studies of W. succinogenes fumarate reductase, the capping domain has been captured at different positions relative to the FAD binding domain, hinting at structural changes that occur during enzyme turnover (35, 58).…”

“…X-ray crystallographic structures of Sdh from Sus domesticus (1), Gallus gallus (2, 3) and Escherichia coli (4, 5), as well as those of the homologous fumarate reductase (Frd) from Wolinella succinogenes (6) and E. coli (7–9), show remarkable conservation of tertiary architecture in the Complex II family of enzymes. Succinate oxidation is initialized by a hydride transfer mechanism that donates 2 electrons to the covalent flavin adenine dinucleotide (FAD) cofactor in SdhA (10).…”

Plasticity of the Quinone-binding Site of the Complex II Homolog Quinol:Fumarate Reductase

“…Structure Determination and Refinement-Crystals were isomorphous with crystals of previously determined structures of QFR and were determined by rigid body refinement in CNS using the structure of QFR bound to fumarate (Protein Data Bank code 3P4P) (22) as the starting model. Model building was performed in Coot (23), and refinement was performed using CNS (24) and Refmac (25) with translation-liberation-screw (TLS) parameters determined with the TLSMD server (26).…”