Determination of an Optimal Potential Window for Catalysis by E. coli Dimethyl Sulfoxide Reductase and Hypothesis on the Role of Mo(V) in the Reaction Pathway

Abstract

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.

Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme

“…4B). The reduction potential for DMSO is +160 mV at pH 7, and work by Heffron et al (25) shows that the enzyme E. coli DMSO reductase is capable of reducing DMSO at more positive voltages than YedY. In Fig.…”

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…Figure 44 shows steady state voltammograms for DMSO reduction by DmsABC. 39 The voltammogram recorded at high pH illustrates a typical electrochemical signature for this family of enzymes. 41 When the electrode potential is taken down, the reductive activity first increases to a maximum (i decreases to a minimum), before it drops and eventually plateaus off at high driving force; since this occurs at steady state, the same profile of activity is observed on the return scan.…”

“…This was first described in the case of the membrane bound DMSO reductase from E. coli (DmsABC, 90 kDa) 39 and then with several related enzymes, namely periplasmic 156,160,186,343 and membrane bound 42,159 nitrate reductases. Since these enzymes share a common catalytic subunit but have very little overall homology, it is likely that the electrochemical data relate to the mechanism at the active site.…”

“…In the case of E. coli DmsABC, 39 experimental limitations precluded the substrate concentration dependence to be studied, but the fact that the shape of the signal is strongly dependent on pH ( Figure 44) suggested a catalytic mechanism involving a crucial protonation step when the Mo is in its intermediate redox state (Mo V ); at low potential, the Mo is quickly reduced to Mo IV before the protonation can proceed, forcing the mechanism into a less-active route (hence the decrease in activity). 39 This mechanism explains that the low potential attenuation of activity is no longer seen under acidic conditions, when the rate of proton uptake may be high enough that taking the high or low potential reaction pathway makes no difference. In short, according to this model, the fact that the sequence of events in the catalytic cycle depends on the driving force is the reason the turnover rate need not vary in a monotonic fashion with the electrode potential.…”

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

“…DmsA, the RR-leader containing subunit of the enzyme, functions as the catalytic subunit that coordinates the MobisPGD catalytic cofactor in addition to one [4Fe-4S] (40,41). DmsB serves as the electron conduit subunit coordinating four [4Fe-4S] clusters through conserved Cys residues, and is essential for anchoring to the cytoplasmic membrane via the integral membrane protein (42).…”