Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system

Abstract

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "small tetraheme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-Å-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

Figure 1

Arrangement of the 10 hemes within the ccNiR dimer. The yellow and green hemes are 6-coordinate and used for electron transport only, while the two orange hemes are the active sites. The red arrows show likely paths of electron flow. Electrons are believed to enter via the green hemes, but can move between subunits as shown (the dotted line separates the monomeric subunits).

Figure 2

(a) SDS-Page gel documenting the ccNiR purification procedure; (b) CcNiR activity recovered after each purification step; 1 enzyme unit is the amount required to reduce 1 µmol nitrite per minute under the assay conditons. HIC: hydrophobic interaction chromatography; IEx: ion exchange chromatography; IP: isopropyl.

Figure 3

(a) Spectra obtained at applied potentials of 0.034V, −0.106V, −0.196V, −0.256V, −0.316V and −0.506V vs SHE. Solid blue lines show the experimentally obtained data, while the dashed red lines show the fit obtained using the matrix Eq. 6 (b) The least-squares best fit of the data by Eq. 6 at λ=426 nm.

Figure 4

(a) Extinction coefficient difference spectra corresponding to each of the reduced ccNiR species C₁ – C₅ (Scheme 2), as calculated by fitting the experimental spectropotentiometric titration data using Eq. 6. The vertical line shows the point at which a high-spin ferroheme should have an absorbance maximum. (b) Similar to (a), but here the calculated absolute extinction coefficient spectra of C₁ – C₅, together with the spectrum of C_ox, are shown. (c) Concentrations of the various ccNiR species present in solution at a given applied potential (vs SHE), obtained by fitting the data to Eq. 6.

Figure 5

Protein Film Voltammetry of S. oneidensis ccNiR. (a) Typical signal on a graphite electrode. Experiment was carried out at pH 5.15, 0°C, scan rate 45 mV/s. Both the full cyclic voltammogram and background-subtracted data are shown. (b) Baseline-subtracted non-turnover voltammogram recorded at 0°C, pH 6, 250 mV/s. Black trace is the baseline-subtracted voltammogram, red trace and dotted lines are from the resulting fit; equation for fitting derived from the Nernst equation. Sub-plots show the residuals for fits of the oxidative (Ox) and reductive (Red) scans. Calculated E_m values are −0.295 V, −0.230 V, −0.166 V, −0.105 V, and −0.036 V vs. SHE.

Figure 6

Comparison between the heme arrangement within a monomer of S. oneidensis ccNiR (lighter shade) and that within a monomer of E. coli (darker shade). Subunit A is shown. Irons are shown in yellow, while the heme color scheme matches that of Fig. 1.

Figure 7

Coordination environment of the conserved Ca²⁺ site within the S. oneidensis ccNiR. Subunit B is shown here; in subunit A the difference electron density feature that corresponds to the Ca-bound water is only 3.5 sigma. This is extremely close to the noise level, and consequently the water was left out of the structure. The E. coli structure revealed two waters bound to the Ca²⁺.

Figure 8

Overlay comparing the protein structures of S. oneidensis and E. coli ccNiRs. The rectangle in the lower right-hand side highlights the region near Hemes 2 that shows the greatest sequence divergence.

Scheme 1

Scheme 2

Fully oxidized ccNiR is referred to as Ox, while C₁ – C₅ refer to the 1 – 5 electron reduced species, respectively.