Models for Molybdenum Coordination during the Catalytic Cycle of Periplasmic Nitrate Reductase from Paracoccus denitrificans Derived from EPR and EXAFS Spectroscopy

Abstract

The periplasmic nitrate reductase from Paracoccus denitrificans is a soluble two-subunit enzyme which binds two hemes (c-type), a [4Fe-4S] center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD). A catalytic cycle for this enzyme is presented based on a study of these redox centers using electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) spectroscopies. The Mo(V) EPR signal of resting NAP (High g [resting]) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, and is split by two weakly interacting protons which are not solvent-exchangeable. Addition of exogenous ligands to this resting state (e.g., nitrate, nitrite, azide) did not change the form of the signal. A distinct form of the High g Mo(V) signal, which has slightly lower anisotropy and higher rhombicity, was trapped during turnover of nitrate and may represent a catalytically relevant Mo(V) intermediate (High g [nitrate]). Mo K-edge EXAFS analysis was undertaken on the ferricyanide oxidized enzyme, a reduced sample frozen within 10 min of dithionite addition, and a nitrate-reoxidized form of the enzyme. The oxidized enzyme was fitted best as a di-oxo Mo(VI) species with 5 sulfur ligands (4 at 2. 43 A and 1 at 2.82 A), and the reduced form was fitted best as a mono-oxo Mo(IV) species with 3 sulfur ligands at 2.35 A. The addition of nitrate to the reduced enzyme resulted in reoxidation to a di-oxo Mo(VI) species similar to the resting enzyme. Prolonged incubation of NAP with dithionite in the absence of nitrate (i.e., nonturnover conditions) resulted in the formation of a species with a Mo(V) EPR signal that is quite distinct from the High g family and which has a g(av) = 1.973 (Low g [unsplit]). This signal resembles those of the mono-MGD xanthine oxidase family and is proposed to arise from an inactive form of the nitrate reductase in which the Mo(V) form is only coordinated by the dithiolene of one MGD. In samples of NAP that had been reduced with dithionite, treated with azide or cyanide, and then reoxidized with ferricyanide, two Mo(V) signals were detected with g(av) elevated compared to the High g signals. Kinetic analysis demonstrated that azide and cyanide displayed competitive and noncompetitive inhibition, respectively. EXAFS analysis of azide-treated samples show improvement to the fit when two nitrogens are included in the molybdenum coordination sphere at 2.52 A, suggesting that azide binds directly to Mo(IV). Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.

EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

“…As seen in other Naps [23,25], Dd NapA shows different paramagnetic species: resting (Fig. 1, spectrum a), lowpotential (Fig.…”

“…Attempts to develop a Mo(V) EPR signal by treating both as-prepared and dithionite-reduced samples with 100-fold molar excess of either sodium azide or potassium perchlorate followed by air oxidation failed to show an EPR signal associated with Mo(V) species. In contrast to Pp NapAB [25], Dd NapA has no activity when incubated in the presence of dithionite as the sole electron donor and the enzyme needs reduced MV to be catalytically competent. In a typical kinetics experiment, the enzyme is reacted with MV reduced either with dithionite or with Zn(0) and then the reaction is started by adding nitrate.…”

“…A least-squares fit to the data of the FeS signal with a Nernstian function (n=1) yielded E=À390 mV (vs. NHE). This value is unusually low for Naps, which usually have FeS centers with redox potentials around À200 mV [23,25,29]. The redox potentials of the low-potential species could not be precisely determined owing to the lack of data below À500 mV (Fig.…”

“…Particularly, nitrate addition to a dithionite-reduced sample yields an EPR signal called high g (nitrate), which was postulated to be produced by a catalytic intermediate of the reaction [25]. Previous EPR characterization of Dd NapA also detected a similar EPR signal [26] that develops upon nitrate addition to a dithionite-reduced sample.…”

“…The active site in the oxidized forms of Dd NapA and Rs NapAB is formed by a distorted hexa-coordinated Mo(VI) ion, in which the metal atom is coordinated by four sulfur atoms from two dithioline ligands, one hydroxo/water group, and S c from Cys 140 (in Dd NapA numbering) [20]. In contrast, on the basis of electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) studies, a hepta-coordinated molybdenum site was postulated for Pp NapAB [25], in which an additional oxo group constitutes the main difference from the oxidized active sites of Dd NapA and Rs NapAB [20,23], whereas a negatively charged hepta-coordinated complex was proposed for the reduced form. The structure of the reduced active site in Dd NapA and Rs NapAB has not been reported.…”