Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism

Abstract

Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in wt NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The S_Cys is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle.

Figure 1.

Top: Selected structures that have been previously proposed for sulfido-based nitrate reductase enzyme forms. Middle: Selected structures proposed for nonsulfido-based NapA enzyme forms. Bottom generalized structure of the reduced PDT ligand.

Figure 2.

Mo K-edge XANES spectra for nitrate-treated wt-Cj NapA and its as isolated NapA-C176A variant. The rising edge energies, defined as the inflection point of the first derivative, are observed at 20014.0 eV for oxidized wt-NapA and 20011.4 eV for NapA-C176A variant.

Figure 3.

Mo K-edge EXAFS data for oxidized wt-NapA (A and C) and the NapA-C176A variant (B and D). The experimental data are shown in black, and the best fits to the data are in red for wt-NapA and blue for NapA-C176A. Four Mo-C scatters from the dithiolene C=C carbon atoms of the two PDT ligands have been included in the fit for wt-NapA, yielding Mo-C vectors at an average distance of 3.339 Å. The observed oscillations arise from terminal oxo, S, C (second coordination sphere), and OH scatterers, and are colored in purple, green, orange, and brown, respectively. The computational models used as initial guesses in the fitting procedure are given in C and F. The XAS data for the wt protein was obtained by incubating the enzyme with nitrate.

Figure 4.

Top: X-band EPR spectra of the as-isolated NapA C176A variant in H₂O (Black) and D₂O (Blue) (50 mM HEPES pH 7.4) with simulations of the spectra in red. Enzyme concentrations are approximately 200 μM. The spectra in H₂O and D₂O buffer are virtually identical (A) and show no evidence for strongly coupled protons. EPR spin quantitation yields ~ 10–20% of the variant being in the Mo⁵⁺ oxidation state (see SI). The g-values determined from spectral simulations are g_1,2,3 = 2.0170, 1.9876, 1.9644 in H₂O sample (B) and g_1,2,3 = 2.0178, 1.9888, 1.9645 in D₂O (C). Bottom: Bond line drawings for small molecule analogs of the Mo⁵⁺ forms of C176A NapA (1-2) and wt-NapA (3).

Figure 5.

Proposed NapA oxygen atom transfer mechanism based on the results of this study.