The pentaheme containing cytochrome, NrfA, from Escherichia coli catalyzes the six-electron reduction of nitrite and the five-electron reduction of nitric oxide. Crystallographic and spectroscopic studies have provided a structural framework for these mechanisms. The active site includes a high-spin heme, and four low-spin, bis-his coordinated hemes are positioned to facilitate intra- and intermolecular electron exchange. However, despite the use of protein film voltammetry to provide kinetic descriptions of NrfA catalysis at graphite and gold electrodes, the thermodynamic descriptions of heme redox activity remain incomplete. Here we rectify this situation with the observation of nonturnover signals from NrfA adsorbed on mesoporous SnO2 electrodes. Simultaneous cyclic voltammetry and electronic absorption spectroscopy define reduction potentials for the high- and low-spin hemes. These reduction potentials are shown to be similar to those exhibited by the enzyme in solution and defined by electrodic reduction monitored by magnetic circular dichroism. Thus, NrfA is shown to undergo minimal perturbation of its electronic and thermodynamic properties on adsorption giving confidence to correlations of properties deduced from various methods and in approaches that may well facilitate studies of other oxidoreductases where catalytic protein film voltammetry is well-defined but nonturnover signals elusive.

In protein film electrochemistry a redox protein of interest is studied as an electroactive film adsorbed on an electrode surface. For redox enzymes this configuration allows quantification of the relationship between catalytic activity and electrochemical potential. Considered as a function of enzyme environment, i.e., pH, substrate concentration etc., the activity-potential relationship provides a fingerprint of activity unique to a given enzyme. Here we consider the nature of the activity-potential relationship in terms of both its cellular impact and its origin in the structure and catalytic mechanism of the enzyme. We propose that the activity-potential relationship of a redox enzyme is tuned to facilitate cellular function and highlight opportunities to test this hypothesis through computational, structural, biochemical and cellular studies.