Fumarate Reductase and Succinate Oxidase Activity of Escherichia coli Complex II Homologs Are Perturbed Differently by Mutation of the Flavin Binding Domain

Abstract

The Escherichia coli complex II homologues succinate:ubiquinone oxidoreductase (SQR, SdhCDAB) and menaquinol:fumarate oxidoreductase (QFR, FrdABCD) have remarkable structural homology at their dicarboxylate binding sites. Although both SQR and QFR can catalyze the interconversion of fumarate and succinate, QFR is a much better fumarate reductase, and SQR is a better succinate oxidase. An exception to the conservation of amino acids near the dicarboxylate binding sites of the two enzymes is that there is a Glu (FrdA Glu-49) near the covalently bound FAD cofactor in most QFRs, which is replaced with a Gln (SdhA Gln-50) in SQRs. The role of the amino acid side chain in enzymes with Glu/Gln/Ala substitutions at FrdA Glu-49 and SdhA Gln-50 has been investigated in this study. The data demonstrate that the mutant enzymes with Ala substitutions in either QFR or SQR remain functionally similar to their wild type counterparts. There were, however, dramatic changes in the catalytic properties when Glu and Gln were exchanged for each other in QFR and SQR. The data show that QFR and SQR enzymes are more efficient succinate oxidases when Gln is in the target position and a better fumarate reductase when Glu is present. Overall, structural and catalytic analyses of the FrdA E49Q and SdhA Q50E mutants suggest that coulombic effects and the electronic state of the FAD are critical in dictating the preferred directionality of the succinate/fumarate interconversions catalyzed by the complex II superfamily. . The membrane-extrinsic domain is bound to the membrane through interactions with the hydrophobic subunits of the complex. These subunits comprise two membrane anchor polypeptides, each containing three transmembrane helices and providing a binding site(s) for quinone (for reviews, see Refs. 5-8). In addition, the E. coli SQR hydrophobic peptides bind one b-type heme, whereas the E. coli QFR lacks heme.Comparison of the structures of complex II is possible due to the availability of x-ray crystallographic structures for both SQR and QFR of E. coli (9, 10), the porcine SQR (11), the QFR from Wolinella succinogenes (12), and soluble homologs of the flavoprotein subunit that function as periplasmically localized fumarate reductases (13-16). The flavoprotein subunits from the E. coli SQR and QFR are highly homologous, with 64% similarity and 44% identity of amino acid residues (3). The sequence similarity within the SQR/QFR superfamily is reflected in structural alignments of members whose structures are known (17)(18)(19). Within this group, the backbone C ␣ atoms can be superimposed with a maximal root mean square deviation of 1.5 Å (10). A detailed hydride transfer mechanism for fumarate reduction has been proposed based on structural data and enzyme assays of wild type and mutant enzymes (15, 18 -20). QFRs and SQRs all contain covalently bound FAD and are bidirectional (i.e. they will catalyze both succinate oxidation and fumarate reduction). There are, however, significant differences for the kinetics of fumarate reduction by...

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

“…CT amplitude does not change at pH 7 or 9 and does not correlate with the pK a of the reaction (36,49). This suggests that microscopic pK a values of amino acid residues involved in substrate activation are below pH 7, and the observed pK a of the reaction is influenced by other active site or proton shuttle residues.…”

“…Potassium ferricyanide and phenazine ethosulfate/2,6-dichlorophenol indophenol were used as electron acceptors for reactions of succinate oxidation for QFR and SQR, respectively (35). Fumarate reduction was determined with reduced methyl viologen or by menaquinol oxidation, as described previously (36). Optical spectra were recorded with an Agilent 8453 diode array spectrophotometer 1 min after the addition of ligand to an isolated enzyme in 30 mM BTP, 0.1 mM EDTA, 0.01% Anapoe C 12 E 9 .…”

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…314 The reduction potentials of the four centers were also determined using PFV ( Figure 26B) and found to match independent measurements. 36,38,66,278 The assumption that the centers are redox-independent was explicitly made and questioned, 36 but it should be recognized that the relatively low intensity of the peak currents observed for large multicenter enzymes often precludes the use of noncatalytic PFV to investigate the possible redox cooperativity between the different redox centers (section 2.1.1.2). Should redox interactions be suspected or detected 76 (as in multiheme cytochromes for example), it is important to think of the nontrivial relation that exists between the "macroscopic" reduction potentials measured in PFV (or UV-vis if the centers are indistinguishable for example) and their "microscopic" counterparts, which can sometimes be determined from titrations followed by NMR or EPR.…”

“…This includes nanoparticles (graphite, 20 silica, 264,265 gold, [266][267][268] Pt,etc. )andnanotubes, 235,269 quantum dots, and mesoporous materials (electrodeposited oxides, 278,271 ITO, carbon, etc). The reader will find many examples in recent reviews.…”

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

“…Previous analyses have suggested a role of the reduction potentials of the Fe-S clusters, the properties of the covalently bound FAD cofactor, and quinone substrates in catalytic proficiency (10,14,18,32). Because electrons are substrates and products, the tuning of the potentials can control substrate availability at each active site.…”