Catalytic Protein Film Voltammetry from a Respiratory Nitrate Reductase Provides Evidence for Complex Electrochemical Modulation of Enzyme Activity

Abstract

The first step in the respiratory reduction of nitrate to dinitrogen in Paracoccus pantotrophus is catalyzed by the quinol-nitrate oxidoreductase NarGHI. This membrane-anchored protein directs electrons from quinol oxidation at the membrane anchor, NarI, to the site of nitrate reduction in the membrane extrinsic [Fe-S] cluster and Mo-bis-MGD containing dimer, NarGH. Liberated from the membrane, NarGH retains its nitrate reductase activity and forms films on graphite and gold electrodes within which direct and facile exchange of electrons between the electrode and the enzyme occurs. Protein film voltammetry has been used to define the catalytic behavior of NarGH in the potential domain and a complex pattern of reversible, nitrate concentration dependent modulation of activity has been resolved. At low nitrate concentrations the local maximum observed in the catalytic current-potential profile reveals how NarGH can catalyze nitrate reduction via two pathways having distinct specificity constants, k(obs)(cat)/K(obs)(M). Catalysis is directed to occur via one of the pathways by an electrochemical event within NarGH. On increasing the nitrate concentration, the local maximum in the catalytic current becomes less distinct, and the catalytic waveform adopts an increasingly sigmoidal form. A pattern of voltammetry similar to that observed during nitrate reduction is observed during reduction of the stereochemically distinct substrate chlorate. Centers whose change of oxidation state may define the novel catalytic voltammetry of NarGH have been identified by EPR-monitored potentiometric titrations and mechanisms by which the electrochemistry of Mo-bis-MGD or [Fe-S] clusters can account for the observed behavior are discussed.

Tuning a Nitrate Reductase for Function

“…By contrast, in the periplasmic enzyme from P. pantotrophus the high g Mo 6ϩ/5ϩ couple has a midpoint potential above 400 mV, with the E m of the Mo 5ϩ/4ϩ couple lying closer to that of the NarB Mo 6ϩ/5ϩ couple at around Ϫ120 mV. The E m of the NarB Mo 6ϩ/5ϩ couple is also much lower that of the membrane-bound nitrate reductase, which lies in the range of 250 to Ͼ450 mV depending on the pH (32,36).…”

“…In terms of the catalytic properties NarB appears highly specific for nitrate, the k cat /K m for nitrate being 800-fold higher than for chlorate. This is also a property of periplasmic nitrate reductases but not of membrane-bound nitrate reductase, where the k cat /K m for nitrate and chlorate are comparable (32,33). Likewise both the Synechococcus sp.…”

The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

“…2. However, in the absence of information on the catalytically relevant reduction potentials of the Mo 15,39,59 it cannot be ruled out that this behaviour arises from Mo-based redox chemistry in a situation analogous to that proposed for the active site redox chemistry of succinate dehydrogenase and NarGH. 39,60 An opportunity to resolve contributions of the various cofactors to the activity-potential relationship is provided by the ability of P. pantotrophus to insert W into NapAB in place of Mo.…”

“…However, their approach is readily extended to include steps that describe redox events and so a dependence of the catalytic rate on electrochemical potential. 12,39,49 In the simplest case the activity of a reductase would be expected to be negligible at more positive potentials since the fully oxidised enzyme lacks reducing equivalents that can be passed to substrate. Lowering the potential into a critical domain related to the reduction potentials of centres in that enzyme will significantly increase the population of reduced, catalytically-competent enzyme.…”

“…As yet there is no consensus on the molecular mechanism that gives rise to the activity-potential relationship displayed by NapAB. Details of the specific mechanisms that have been proposed can be found in a number of papers 13,15,21,39 and we present the discussion above to stimulate further interest in accounting for activity-potential relationships at the molecular level. Experimental approaches that resolve enzyme states structuro-spectroscopically across the potential domain will be valuable.…”