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

Abstract

The Complex II family of enzymes, comprising the respiratory succinate dehydrogenases and fumarate reductases, catalyze reversible interconversion of succinate and fumarate. In contrast to the covalent flavin adenine dinucleotide (FAD) cofactor assembled in these enzymes, the soluble fumarate reductases (e.g. that from Shewanella frigidimarina) that assemble a noncovalent FAD cannot catalyze succinate oxidation but retain the ability to reduce fumarate. In this study, an SdhA-H45A variant that eliminates the site of the 8α-N3-histidyl covalent linkage between the protein and the FAD was examined. The variants SdhA-R286A/K/Y and -H242A/Y, that target residues thought to be important for substrate binding and catalysis were also studied. The variants SdhA-H45A and -R286A/K/Y resulted in assembly of a noncovalent FAD cofactor, which led to a significant decrease (−87 mV or more) in its reduction potential. The variant enzymes were studied by electron paramagnetic resonance spectroscopy following stand-alone reduction and potentiometric titrations. The “free” and “occupied” states of the active site were linked to the reduced and oxidized states of the FAD, respectively. Our data allows for a proposed model of succinate oxidation that is consistent with tunnel diode effects observed in the succinate dehydrogenase enzyme and a preference for fumarate reduction catalysis in fumarate reductase homologues that assemble a noncovalent FAD.

Structural and biochemical analyses reveal insights into covalent flavinylation of the Escherichia coli Complex II homolog quinol:fumarate reductase

“…Wild-type QFR, the QFR-FrdA R114 variant, and the QFR-FrdA H44S variant were used as comparators. Five of our designed variants (FrdA E245Q , FrdA R287K , FrdA H355S , FrdA R390K , and FrdA R390Q ) had almost a complete absence of covalent flavinylation, consistent with studies of the SQR protein (14). The FrdA D288N variant showed minimal residual fluorescence, suggesting covalent flavinylation was severely compromised.…”

“…From this, it was proposed that one reason for the inability of the variants to oxidize succinate was because of a lowered redox potential of the non-covalent FAD (3). This was later supported by findings where similar sitedirected variants of E. coli SQR result in an ϳ90 mV reduction in the E m,7 of the non-covalent flavin compared with wild-type enzyme (14) and in E. coli QFR where there was a Ϫ100 mV drop in redox potential of the FAD compared with the wildtype enzyme (15).…”

The unassembled flavoprotein subunits of human and bacterial complex II have impaired catalytic activity and generate only minor amounts of ROS

“…FAD in the range of Ϫ51 to Ϫ55 mV. This is in good agreement with the potential of the FAD found by protein film voltammetry for the soluble E. coli FrdAB (Ϫ50 mV) form (34) or by electron paramagnetic spectroscopy in the mammalian or bacterial SQR complex (Ϫ79 to Ϫ100 mV, respectively) (39,40). The FAD reduction achieved with succinate ( Fig.…”

“…4B). The iron-sulfur protein may also have an electronic interaction with the FAD in the flavoprotein via the proximal redox center [2Fe-2S] in the ironsulfur subunit (40).…”

The Fumarate Reductase of Bacteroides thetaiotaomicron , unlike That of Escherichia coli , Is Configured so that It Does Not Generate Reactive Oxygen Species

“…Subunit A of fumarate reductase was detected after electrophoresis of membrane samples containing 275 μg protein on SDS-polyacrylamide gels (62). The gels were exposed to UV transillumination, and the UV fluorescence of the bound flavin was quantified by a Quantity One system (Bio-Rad).…”