Structural investigation of the molybdenum site of the periplasmic nitrate reductase from Thiosphaera pantotropha by X-ray absorption spectroscopy

• Published in: Biochemical journal 1996-07, Vol.317 ( Pt 2) (2), p.557-563
• Main Authors: Bennett, B, Charnock, J M, Sears, H J, Berks, B C, Thomson, A J, Ferguson, S J, Garner, C D, Richardson, D J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Ferricyanides - pharmacology; Fourier Analysis; Gram-Negative Chemolithotrophic Bacteria - enzymology; Ligands; Metalloproteins - chemistry; Metalloproteins - drug effects; Models, Chemical; Molecular Sequence Data; Molybdenum - chemistry; Nitrate Reductase; Nitrate Reductases - chemistry; Nitrate Reductases - drug effects; Nitrates - pharmacology; Oxidation-Reduction; Sequence Homology, Amino Acid; Spectrum Analysis; X-Rays
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3170557

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.