Structural investigation of the molybdenum site of the periplasmic nitrate reductase from Thiosphaera pantotropha by X-ray absorption spectroscopy.
The molybdenum centre of the periplasmic respiratory nitrate reductase from the denitrifying bacterium Thiosphaera pantotropha has been probed using molybdenum K-edge X-ray absorption spectroscopy. The optimum fit of the Mo(VI) EXAFS suggests two ==O, three -S- and either a fourth -S- or an -O-/-N- as molybdenum ligands in the ferricyanide-oxidized enzyme. Three of the -S- ligands are proposed to be the two sulphur atoms of the molybdopterin dithiolene group and Cys-181. Comparison of the EXAFS of the ferricyanide-oxidized enzyme with that of a nitrate-treated sample containing 30% Mo(V) suggests that the Mo(VI)-->Mo(V) reduction is accompanied by conversion of one ==O to -O-. The best fit to the Mo(IV) EXAFS of dithionite-reduced enzyme was obtained using one ==O, one -O- and four -S-/-Cl ligands. The periplasmic nitrate reductase molybdenum co-ordination environment in both the Mo(VI) and Mo(IV) oxidation states is distinct from that found in the membrane-bound respiratory nitrate reductase.