Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence of interactions across the dimeric interface

Abstract

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, conducts the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by "boosts" and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to the properties of ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed; e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA [Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) J. Biol. Chem. 277, 23374-23381]. Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which has previously been observed in any cytochrome c nitrite reductase. Collectively, these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer.

Figure 1

Crystal structure of soNrfA. One monomer of the complete homodimer is transparent to highlight the positions of c-type hemes. Image rendered in PyMol from PDB file 3UBR.pdb.

Figure 2

Current-potential profile of soNrfA nitrite reduction. (A) Cyclic voltammograms of soNrfA in the presence of increasing concentrations of nitrite. Reactions were carried out at 20°C, pH ~8.3, scan rate 10 mV/s, electrode rotation rate 3000 rpm. Bold arrow indicates direction of increasing nitrite concentration (1.4, 5.5, 17, 54, 160, 490, and 1460 μM). Full scans are shown in top panel. Filled horizontal double-headed arrows indicate the potential range where the boost is observed, −300 to −525 mV. Open horizontal double-headed arrows indicate the potential range where the switch is observed, −300 to −470 mV. First derivative of reductive scans of catalytic waves are shown in bottom panel. (B) Baseline-subtracted reductive catalytic wave for soNrfA at 1.37 μM nitrite showing the decrease in activity at potentials below ~ −300 mV. (C) First derivative of reductive catalytic wave for soNrfA at 54.1 μM nitrite showing that three distinct features can be simultaneously observed.

Figure 3

Variation in limiting current as a function of the concentration of nitrite. Experiment carried out at pH 8.3, 20°C, 20 mV/s. Data is fit to a substrate inhibition model (Equation 1). Limiting current was measured at −550 mV.

Figure 4

Catalytic current-potential profile for hydroxylamine turnover by soNrfA. Cyclic voltammograms of soNrfA in the presence of increasing concentrations of hydroxylamine. Reactions were carried out at 20°C, pH ~8.3, scan rate 20 mV/s, electrode rotation rate 3000 rpm. Nitrite concentrations are listed to the left of each voltammogram. Full scans are shown in the top panel and first derivatives of reductive scans of the catalytic waves are shown in bottom panel. The dotted line shows soNrfA on an electrode in the absence of hydroxylamine.

Figure 5

Variation in limiting current with hydroxylamine concentration Experiment carried out at pH 8.3, 20°C, 20 mV/s. Data is fitted with a two site model (Eq 2), and a simple model for Michaelis-Menten kinetics Limiting current measured at −550 mV.

Figure 6

Variation of the position of E_cat1 (squares) and E_cat2 (diamonds) with the concentration of nitrite (filled symbols) and hydroxylamine (open symbols). Data were collected at pH 8.3, 20°C, 3000 rpm, 20 mV/s, 2 mM CaCl₂. Nitrite concentration is plotted on the lower x-axis and hydroxylamine concentration is plotted on the upper x-axis.