Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy

The periplasmic nitrate reductase from Paracoccus denitrificans is a solubl e two subunit enzyme which binds two hemes (c-type), a [4Fe-4S] center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD). A catalytic c ycle for this enzyme is presented based on a study of these redox centers u sing electron paramagnetic resonance (EPR) and extended X-ray absorption fi ne structure (EXAFS) spectroscopies. The Mo(V) EPR signal of resting NAP (H igh g [resting]) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, an d is split by two weakly interacting protons which are not solvent-exchange able. Addition of exogenous Ligands to this resting state (e.g., nitrate, n itrite, azide) did not change the form of the signal. A distinct form of th e High g Mo(V) signal, which has slightly lower anisotropy and higher rhomb icity, was trapped during turnover of nitrate and may represent a catalytic ally relevant Mo(V) intermediate (High g [nitrate]). Mo K-edge EXAFS analys is was undertaken on the ferricyanide oxidized enzyme, a reduced sample fro zen 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 Angstrom and 1 at 2.82 Angstrom), and the reduced form was fitted best as a mono-ore Mo(IV) species with 3 sulfur lig ands at 2.35 Angstrom. The addition of nitrate to the reduced enzyme result ed 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 th e dithiolene of one MGD. In samples of NAP that had been reduced with dithi onite, 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 c ompetitive and noncompetitive inhibition, respectively. EXAFS analysis of a zide-treated samples show improvement to the fit when two nitrogens are inc luded in the molybdenum coordination sphere at 2.52 Angstrom, suggesting th at azide binds directly to Mo(IV). Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.