Thiocyanate binding to the molybdenum centre of the periplasmic nitrate reductase from Paracoccus pantotrophus

The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two haem groups, a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) (MGD) cofactor that catalyses the reduction of nitrate to nitrite. In the present study the effect of KSCN (potassium thiocyanate) as an inhibitor and Mo ligand has been investigated. Results are presented that show NAP is sensitive to SCN^- (thiocyanate) inhibition, with SCN^- acting as a competitive inhibitor of nitrate (K_i ≈ 4.0 mM). The formation of a novel EPR Mo(V) species with an elevated g_av value (g_av ∼ 1.994) compared to the Mo(V) High-g (resting) species was observed upon redox cycling in the presence of SCN^-. Mo K-edge EXAFS analysis of the dithionite-reduced NAP was best fitted as a mono-oxo Mo(IV) species with three Mo-S ligands at 2.35 Å (1 Å = 0.1 nm) and a Mo-O ligand at 2.14 Å. The addition of SCN^- to the reduced Mo(IV) NAP generated a sample that was best fitted as a mono-oxo (1.70 Å) Mo(IV) species with four Mo-S ligands at 2.34 Å. Taken together, the competitive nature of SCN^- inhibition of periplasmic nitrate reductase activity, the elevated Mo(V) EPR g_av value following redox cycling in the presence of SCN^- and the increase in sulphur co-ordination of Mo(IV) upon SCN^- binding, provide strong evidence for the direct binding of SCN^- via a sulphur atom to Mo.