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

The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a s oluble two-subunit enzyme (NapAB) that binds two haem groups, a [4Fe-4S] cl uster and a bis(molybdoptelin guanine dinucleotide) (MGD) cofactor that cat alyses the reduction of nitrate to nitrite. In the present study the effect of KSCN (potassium thiocyanate) as an inhibitor and Mo ligand has been inv estigated. Results are presented that show NAP is sensitive to SCN- (thiocy anate) inhibition, with SCN- acting as a competitive inhibitor of nitrate ( K-i approximate to 4.0 mM). The formation of a novel EPR Mo(V) species with an elevated g(av) value (g(av) similar to 1.994) compared to the Mo(V) Hig h-g (resting) species was observed upon redox cycling in the presence of SC N-. 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 Angstrom (1 An gstrom = 0.1 nm) and a Mo-O ligand at 2.14 Angstrom. The addition of SCN- t o the reduced Mo(IV) NAP generated a sample that was best fitted as a mono- ore (1.70 Angstrom) Mo(IV) species with four Mo-S ligands at 2.34 Angstrom. Taken together, the competitive nature of SCN- inhibition of periplasmic n itrate reductase activity, the elevated Mo(V) EPR g(av) value following red ox cycling in the presence of SCN- and the increase in sulphur co-ordinatio n of Mo(IV) upon SCN- binding, provide strong evidence for the direct bindi ng of SCN- via a sulphur atom to Mo.