Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Dec 15;352(Pt 3):859–864.

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

C S Butler 1, J M Charnock 1, C D Garner 1, A J Thomson 1, S J Ferguson 1, B C Berks 1, D J Richardson 1
PMCID: PMC1221527  PMID: 11104696

Abstract

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) approximately 4.0 mM). The formation of a novel EPR Mo(V) species with an elevated g(av) value (g(av) approximately 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 A (1 A=0.1 nm) and a Mo-O ligand at 2.14 A. The addition of SCN(-) to the reduced Mo(IV) NAP generated a sample that was best fitted as a mono-oxo (1.70 A) Mo(IV) species with four Mo-S ligands at 2.34 A. 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.

Full Text

The Full Text of this article is available as a PDF (147.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bennett B., Benson N., McEwan A. G., Bray R. C. Multiple states of the molybdenum centre of dimethylsulphoxide reductase from Rhodobacter capsulatus revealed by EPR spectroscopy. Eur J Biochem. 1994 Oct 1;225(1):321–331. doi: 10.1111/j.1432-1033.1994.00321.x. [DOI] [PubMed] [Google Scholar]
  2. Bennett B., Berks B. C., Ferguson S. J., Thomson A. J., Richardson D. J. Mo(V) electron paramagnetic resonance signals from the periplasmic nitrate reductase of Thiosphaera pantotropha. Eur J Biochem. 1994 Dec 15;226(3):789–798. doi: 10.1111/j.1432-1033.1994.00789.x. [DOI] [PubMed] [Google Scholar]
  3. Berks B. C., Ferguson S. J., Moir J. W., Richardson D. J. Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim Biophys Acta. 1995 Dec 12;1232(3):97–173. doi: 10.1016/0005-2728(95)00092-5. [DOI] [PubMed] [Google Scholar]
  4. Berks B. C., Richardson D. J., Reilly A., Willis A. C., Ferguson S. J. The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha. Biochem J. 1995 Aug 1;309(Pt 3):983–992. doi: 10.1042/bj3090983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berks B. C., Richardson D. J., Robinson C., Reilly A., Aplin R. T., Ferguson S. J. Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha. Eur J Biochem. 1994 Feb 15;220(1):117–124. doi: 10.1111/j.1432-1033.1994.tb18605.x. [DOI] [PubMed] [Google Scholar]
  6. Boyington J. C., Gladyshev V. N., Khangulov S. V., Stadtman T. C., Sun P. D. Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster. Science. 1997 Feb 28;275(5304):1305–1308. doi: 10.1126/science.275.5304.1305. [DOI] [PubMed] [Google Scholar]
  7. Breton J., Berks B. C., Reilly A., Thomson A. J., Ferguson S. J., Richardson D. J. Characterization of the paramagnetic iron-containing redox centres of Thiosphaera pantotropha periplasmic nitrate reductase. FEBS Lett. 1994 May 23;345(1):76–80. doi: 10.1016/0014-5793(94)00445-5. [DOI] [PubMed] [Google Scholar]
  8. Butler C. S., Charnock J. M., Bennett B., Sears H. J., Reilly A. J., Ferguson S. J., Garner C. D., Lowe D. J., Thomson A. J., Berks B. C. Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy. Biochemistry. 1999 Jul 13;38(28):9000–9012. doi: 10.1021/bi990402n. [DOI] [PubMed] [Google Scholar]
  9. Craske A., Ferguson S. J. The respiratory nitrate reductase from Paracoccus denitrificans. Molecular characterisation and kinetic properties. Eur J Biochem. 1986 Jul 15;158(2):429–436. doi: 10.1111/j.1432-1033.1986.tb09771.x. [DOI] [PubMed] [Google Scholar]
  10. Czjzek M., Dos Santos J. P., Pommier J., Giordano G., Méjean V., Haser R. Crystal structure of oxidized trimethylamine N-oxide reductase from Shewanella massilia at 2.5 A resolution. J Mol Biol. 1998 Nov 27;284(2):435–447. doi: 10.1006/jmbi.1998.2156. [DOI] [PubMed] [Google Scholar]
  11. Dias J. M., Than M. E., Humm A., Huber R., Bourenkov G. P., Bartunik H. D., Bursakov S., Calvete J., Caldeira J., Carneiro C. Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods. Structure. 1999 Jan 15;7(1):65–79. doi: 10.1016/s0969-2126(99)80010-0. [DOI] [PubMed] [Google Scholar]
  12. Gangeswaran R., Lowe D. J., Eady R. R. Purification and characterization of the assimilatory nitrate reductase of Azotobacter vinelandii. Biochem J. 1993 Jan 15;289(Pt 2):335–342. doi: 10.1042/bj2890335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hensel M., Hinsley A. P., Nikolaus T., Sawers G., Berks B. C. The genetic basis of tetrathionate respiration in Salmonella typhimurium. Mol Microbiol. 1999 Apr;32(2):275–287. doi: 10.1046/j.1365-2958.1999.01345.x. [DOI] [PubMed] [Google Scholar]
  14. Ludwig W., Mittenhuber G., Friedrich C. G. Transfer of Thiosphaera pantotropha to Paracoccus denitrificans. Int J Syst Bacteriol. 1993 Apr;43(2):363–367. doi: 10.1099/00207713-43-2-363. [DOI] [PubMed] [Google Scholar]
  15. Schindelin H., Kisker C., Hilton J., Rajagopalan K. V., Rees D. C. Crystal structure of DMSO reductase: redox-linked changes in molybdopterin coordination. Science. 1996 Jun 14;272(5268):1615–1621. doi: 10.1126/science.272.5268.1615. [DOI] [PubMed] [Google Scholar]
  16. Schneider F., Löwe J., Huber R., Schindelin H., Kisker C., Knäblein J. Crystal structure of dimethyl sulfoxide reductase from Rhodobacter capsulatus at 1.88 A resolution. J Mol Biol. 1996 Oct 18;263(1):53–69. doi: 10.1006/jmbi.1996.0555. [DOI] [PubMed] [Google Scholar]
  17. Sears HJ, Little PJ, Richardson DJ, Berks BC, Spiro S, Ferguson SJ. Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient in nitrate respiration. Arch Microbiol. 1997 Jan 29;167(1):61–66. doi: 10.1007/s002030050417. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES