Reductive activation of E. coli respiratory nitrate reductase

Abstract

Over the past decades, a number of authors have reported the presence of inactive species in as-prepared samples of members of the Mo/W-bisPGD enzyme family. This greatly complicated the spectroscopic studies of these enzymes, since it is impossible to discriminate between active and inactive species on the basis of the spectroscopic signatures alone. Escherichia coli nitrate reductase A (NarGHI) is a member of the Mo/W-bisPGD family that allows anaerobic respiration using nitrate as terminal electron acceptor. Here, using protein film voltammetry on NarGH films, we show that the enzyme is purified in a functionally heterogeneous form that contains between 20 and 40% of inactive species that activate the first time they are reduced. This activation proceeds in two steps: a non-redox reversible reaction followed by an irreversible reduction. By carefully correlating electrochemical and EPR spectroscopic data, we show that neither the two major Mo(V) signals nor those of the two FeS clusters that are the closest to the Mo center are associated with the two inactive species. We also conclusively exclude the possibility that the major "low-pH" and "high-pH" Mo(V) EPR signatures correspond to species in acid-base equilibrium.

Redox (In)activations of Metalloenzymes: A Protein Film Voltammetry Approach

“…For some enzyme preparations, it was observed that the first voltammogram recorded with a film display a very pronounced hysteresis that is absent from subsequent ones (Figure g), reflecting an activation that occurs only the first time the enzyme is reduced . This phenomenon is in fact common for molybdenum enzymes, having been observed on other nitrate reductases like Synechococcus elongatus NarB, Rhodobacter sphaeroides NapAB, E. coli NarGH, but also in the case of formate dehydrogenase …”

“…A similar process also occurs with the respiratory nitrate reductases, as was first evidenced with Paracoccus pantotrophus NarGH (Figure g). This behaviour was later observed with E. coli NarGH samples, and studied using chronoamperometry . It was shown that the activation is biphasic.…”

“…(15)], since the amplitudes of the two phases strongly depends on potential. A global fitting strategy was used to model the experimental data at different potentials, by assuming that in Equation (15), the first step is potential‐independent, and thus its rate constants are common to all the activations at different potentials, while that of the second step is assumed to depend on potential; this approach provided very satisfactory fits, showing that this scheme is enough to reproduce the experimental data …”

“…The study went on to look for spectroscopic signatures of the initially inactive states, but no correlations between the fractions of initially inactive enzymes and any of the spectroscopic signatures observed on the enzyme could be found, unlike in the case of NapAB …”

Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

“…3C). The quasi-axial component that forms about 33% of the composite FS1 EPR signature in holoNarGH2425 was not detected in the substituted enzyme and the EPR lines associated to the remaining rhombic component were broadened (Fig. 3C).…”