Enzyme Electrokinetics:  Energetics of Succinate Oxidation by Fumarate Reductase and Succinate Dehydrogenase

Abstract

Protein film voltammetry is used to probe the energetics of electron transfer and substrate binding at the active site of a respiratory flavoenzyme--the membrane-extrinsic catalytic domain of Escherichia coli fumarate reductase (FrdAB). The activity as a function of the electrochemical driving force is revealed in catalytic voltammograms, the shapes of which are interpreted using a Michaelis-Menten model that incorporates the potential dimension. Voltammetric experiments carried out at room temperature under turnover conditions reveal the reduction potentials of the FAD, the stability of the semiquinone, relevant protonation states, and pH-dependent succinate--enzyme binding constants for all three redox states of the FAD. Fast-scan experiments in the presence of substrate confirm the value of the two-electron reduction potential of the FAD and show that product release is not rate limiting. The sequence of binding and protonation events over the whole catalytic cycle is deduced. Importantly, comparisons are made with the electrocatalytic properties of SDH, the membrane-extrinsic catalytic domain of mitochondrial complex II.

Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

“…Several models for electrocatalysis by adsorbed enzymes have been developed and summarized (11), although they do not implicitly address the simple but special case of reversible catalysis. As examples, specific models have accounted for MichaelisMenten concentration and rotation-rate dependencies (12,13), diode-like behavior (14), irreversible electrocatalytic voltammetry (15), dispersion of electronic couplings between electrode and enzyme (16,17), and the influence of intramolecular electron transfer rates (18). These models are all variations on the classical procedure of treating electrocatalytic reactions as a special type of coupled electrochemical (EC) process (19,20 (8).…”

Campylobacter jejuni Gene Expression in the Chick Cecum: Evidence for Adaptation to a Low-Oxygen Environment

“…Both enzymes can function to interconvert succinate and fumarate (5,34). In E. coli, Sdh can function as an effective fumarate reductase in vitro (21,28), even supporting its anaerobic growth (22). However, it is also possible that succinate dehydrogenase is highly activated in C. jejuni in vivo to maintain the oxidative function of the citricacid cycle under the low oxygen tensions found in the gut.…”

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

“…In the membrane-bound forms of complex II, covalent binding of FAD raises its potential (E m7 ϭ ϳϪ55 to Ϫ70 mV) and allows membrane-bound enzymes to proficiently oxidize succinate as well as reduce fumarate (3)(4)(5)(6). In contrast, noncovalently bound FAD in the soluble bacterial homologues, such as flavocytochrome c 3 (Fcc 3 ) and L-aspartate oxidase, has a redox potential ϳ100 mV lower (ϳϪ150 mV) (7).…”