Spectroelectrochemical Characterization of a Pentaheme Cytochrome in Solution and as Electrocatalytically Active Films on Nanocrystalline Metal-Oxide Electrodes

Abstract

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.

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

“…A nitrite concentration of 1.7 mM is well below the K M values for all potentials where NrfA has significant nitrite reductase activity so that the reduction potentials of the enzyme cofactors may inform on the redox transformations that modulate the activity. 20 The value of E cat correlates with E m of the active site heme while E switch has a value close to the reduction potential of the lowest potential relay, Fig. 8B.…”

Electron Accepting Units of the Diheme Cytochrome c TsdA, a Bifunctional Thiosulfate Dehydrogenase/Tetrathionate Reductase

“…The enzyme-coated electrode was rinsed with 2 mM neomycin, 50 mM NaCl, 50 mM HEPES, pH 7, to remove unbound protein, taken into a N 2 -filled chamber (atmospheric O 2 Ͻ 2 ppm) and immersed in an anaerobic solution of the same composition within a previously described spectroelectrochemical cell (42). The cell was sealed, removed from the anaerobic chamber, and inserted into a Jasco V650 UV-visible spectrophotometer thermostated at 4°C and flushed with argon to maintain anaerobic status.…”

Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

“…Potentiometric titrations monitored by electronic absorbance were performed with a working electrode composed of mesoporous nanocrystalline SnO 2 in a previously described cell configuration (23,24). SoxAX was adsorbed onto SnO 2 electrodes during 2 h of cyclic voltammetry (ϩ60 mV to Ϫ540 mV, 20 mV s Ϫ1 ) in 35 M SoxAX, 50 mM Hepes, 100 mM NaCl, pH 7, 20°C.…”