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.

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Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity

Journal of General Microbiology ◽

10.1099/00221287-139-12-3205 ◽

1993 ◽

Vol 139 (12) ◽

pp. 3205-3214 ◽

Cited By ~ 35

Author(s):

L. C. Bell ◽

M. D. Page ◽

B. C. Berks ◽

D. J. Richardson ◽

S. J. Ferguson

Keyword(s):

Nitrate Reductase ◽

Structural Gene ◽

Nitrate Reductase Activity ◽

Reductase Activity ◽

Transposon Tn5 ◽

Periplasmic Nitrate Reductase ◽

Thiosphaera Pantotropha ◽

Membrane Bound

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The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha

Biochemical Journal ◽

10.1042/bj3090983 ◽

1995 ◽

Vol 309 (3) ◽

pp. 983-992 ◽

Cited By ~ 90

Author(s):

B C Berks ◽

D J Richardson ◽

A Reilly ◽

A C Willis ◽

S J Ferguson

Keyword(s):

Nitrate Reductase ◽

Sequence Similarity ◽

Alcaligenes Eutrophus ◽

Integral Membrane Protein ◽

Periplasmic Nitrate Reductase ◽

Thiosphaera Pantotropha ◽

Cysteine Motif ◽

Membrane Bound ◽

K 12 ◽

Nitrate Reductases

The napEDABC locus coding for the periplasmic nitrate reductase of Thiosphaera pantotropha has been cloned and sequenced. The large and small subunits of the enzyme are coded by napA and napB. The sequence of NapA indicates that this protein binds the GMP-conjugated form of the molybdopterin cofactor. Cysteine-181 is proposed to ligate the molybdenum atom. It is inferred that the active site of the periplasmic nitrate reductase is structurally related to those of the molybdenum-dependent formate dehydrogenases and bacterial assimilatory nitrate reductases, but is distinct from that of the membrane-bound respiratory nitrate reductases. A four-cysteine motif at the N-terminus of NapA binds a [4Fe-4S] cluster. The DNA- and protein-derived primary sequence of NapB confirm that this protein is a dihaem c-type cytochrome and, together with spectroscopic data, indicate that both NapB haems have bis-histidine ligation. napC is predicted to code for a membrane-anchored tetrahaem c-type cytochrome that shows sequence similarity to the NirT cytochrome c family. NapC may be the direct electron donor to the NapAB complex. napD is predicted to encode a soluble cytoplasmic protein and napE a monotopic integral membrane protein, napDABC genes can be discerned at the aeg-46.5 locus of Escherichia coli K-12, suggesting that this operon encodes a periplasmic nitrate reductase system, while napD and napC are identified adjacent to the napAB genes of Alcaligenes eutrophus H16.

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