Protein Film Voltammetry Reveals Distinctive Fingerprints of Nitrite and Hydroxylamine Reduction by a Cytochrome c Nitrite Reductase

Abstract

The cytochrome c nitrite reductases perform a key step in the biological nitrogen cycle by catalyzing the six-electron reduction of nitrite to ammonium. Graphite electrodes painted with Escherichia coli cytochrome c nitrite reductase and placed in solutions containing nitrite (pH 7) exhibit large catalytic reduction currents during cyclic voltammetry at potentials below 0 V. These catalytic currents were not observed in the absence of cytochrome c nitrite reductase and were shown to originate from an enzyme film engaged in direct electron exchange with the electrode. The catalytic current-potential profiles observed on progression from substrate-limited to enzyme-limited nitrite reduction revealed a fingerprint of catalytic behavior distinct from that observed during hydroxylamine reduction, the latter being an alternative substrate for the enzyme that is reduced to ammonium in a two electron process. Cytochrome c nitrite reductase clearly interacts differently with these two substrates. However, similar features underlie the development of the voltammetric response with increasing nitrite or hydroxylamine concentration. These features are consistent with coordinated two-electron reduction of the active site and suggest that the mechanisms for reduction of both substrates are underpinned by common rate-defining processes.

A needle in a haystack: The active site of the membrane‐bound complex cytochrome c nitrite reductase

“…In the presence of substrate, the catalytic process results in a steady-state electron flow whose detection allows instantaneous measurement of turnover rates. Catalytic electrochemical signals have been reported independently for the ccNiRs from D. desulfuricans [13] and E. coli [14,15]. Here we shall report on the former enzyme.…”

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

“…Certainly, activity from the E. coli penta-heme nitrite reductase, NrfA, a MQH 2 -dependent system is only detected below ca. À0.07 V. 41,42 In this light it is striking that potentials below ca. À0.30 V are required to see any appreciable activity from the assimilatory nitrate reductase (Nas) of Synechococcus elongatus.…”

On the Possibility of Uphill Intramolecular Electron Transfer in Multicopper Oxidases: Electrochemical and Quantum Chemical Study of Bilirubin Oxidase

“…Finally, the stability test of MvBOx-modified CPGCE at different pH values is a strong evidence for a single form of electrochemically active enzyme on the electrode surface [82]. Taking also into account the different pH dependences obtained for low-and high-potential processes, when both non-turnover and turnover signals were evaluated (Fig.…”

“…However, no detectable changes in shape or feature positions in the catalytic waveform were noted, suggesting the presence of a single form of electrocatalytically active BOx on the electrode surface (SI1 , Fig. S7); the population of which decreases over the time of the experiment, without formation of new species that contribute to the voltammetric response [82].…”