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.
Protein film voltammetry (PFV) of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC) adsorbed at a graphite electrode reveals that the catalytic activity of this complex Mo-pterin/Fe-S enzyme is optimized within a narrow window of electrode potential. The upper and lower limits of this window are determined from the potential dependences of catalytic activity in reducing and oxidizing directions; i.e., for reduction of DMSO (or trimethylamine-N-oxide) and oxidation of trimethylphosphine (PMe(3)). At either limit, the catalytic activity drops despite the increase in driving force: as the potential is lowered below -200 mV (pH 7.0-8.9), the rate of reduction of DMSO decreases abruptly, while for PMe(3), an oxidative current is observed that vanishes as the potential is raised above +20 mV (pH 9.0). Analysis of the waveshapes reveals that both activity thresholds result from one-electron redox reactions that arise, most likely, from groups within the enzyme; if so, they represent "switches" that reflect the catalytic mechanism and may be of physiological relevance. The potential window of activity coincides approximately with the appearance of the Mo(V) EPR signal observed in potentiometric titrations, suggesting that crucial stages of catalysis are facilitated while the active site is in the intermediate Mo(V) oxidation state.
Factors governing the efficacy of long-range electron relays in enzymes have been examined using protein film voltammetry in conjunction with site-directed mutagenesis. Investigations of the fumarate reductase from Escherichia coli, in which three Fe-S clusters relay electrons over more than 30 A, lead to the conclusion that varying the medial [4Fe-4S] cluster potential over a 100 mV range does not have a significant effect on the inherent kinetics of electron transfer to and from the active-site flavin. The results support a proposal that the reduction potential of an individual electron relay site in a multicentered enzyme is not a strong determinant of activity; instead, as deduced from the potential dependence of catalytic electron transfer, electron flow through the intramolecular relay is rapid and reversible, and even uphill steps do not limit the catalytic rate.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.