Cited by 32Metalloenzymes and iron-sulfur proteinsEnzyme Structure and FunctionMetal-Catalyzed Oxygenation Mechanisms

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

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BUTLER, Clive S., CHARNOCK, John M., GARNER, C. David, THOMSON, Andrew J., FERGUSON, Stuart J., BERKS, Ben C., RICHARDSON, David J.·2000·Biochemical Journal

Abstract

The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two haem groups, a [4FeŐ4S] 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 (Ki ≈ 4.0mM). The formation of a novel EPR Mo(V) species with an elevated gav value (gav ∼ 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 MoŐS ligands at 2.35Å (1 Å = 0.1nm) and a MoŐO ligand at 2.14 Å. The addition of SCN- to the reduced Mo(IV) NAP generated a sample that was best fitted as a mono-oxo (1.70 Å) Mo(IV) species with four MoŐS ligands at 2.34 Å. Taken together, the competitive nature of SCN- inhibition of periplasmic nitrate reductase activity, the elevated Mo(V) EPR gav 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.

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  • NapA from Paracoccus pantotrophus is competitively inhibited by azide and thiocyanate with a Ki of 11.0 mM and 4.0 mM, respectively.

    “NapA from Paracoccus pantotrophus is competitively inhibited by azide212 and thiocyanate206 with a Ki 11.0 mM and 4.0 mM, respectively.”

    From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [206]

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Authors
BUTLER, C. S., CHARNOCK, J. M., GARNER, C. D., THOMSON, A. J., FERGUSON, S. J., BERKS, B. C., & RICHARDSON, D. J.
Affiliations
  • Newcastle University (GB)
  • Daresbury Laboratory (GB)
  • University of Nottingham (GB)
  • University of East Anglia (GB)
Venue
Biochemical Journal
Published
2000-12-08
Volume
352
Issue
3
Pages
859-864
ISSN
  • 0264-6021 (ISSN-L)
  • 1470-8728
OpenAlex
W2013692958
Cited by
32
References
26
Citations by year
2026: 1 · 2025: 1 · 2024: 1 · 2023: 1 · 2022: 1 · 2021: 1 · 2019: 1 · 2017: 1 · 2014: 2 · 2013: 3 · 2012: 2
FWCI
1.78
Type
journal article
Language
en
Open access
Locations
  • Biochemical Journal (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Science and Technology Facilities Council (submittedVersion)
  • UEA Digital Repository (University of East Anglia) (submittedVersion)
  • Newcastle University ePrints (Newcastle Univesity)
Funders
  • University of East Anglia
  • Directorate for Biological Sciences
  • Engineering and Physical Sciences Research Council
Topics
  • Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
  • Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
Keywords
periplasmic nitrate reductase, Paracoccus pantotrophus, thiocyanate inhibition, competitive inhibition, [4Fe-4S] cluster, heme, EXAFS, sulfur coordination, nitrate reduction, enzyme inhibition
MeSH terms
Dithionite, Electron Spin Resonance Spectroscopy, Fourier Analysis, Kinetics, Models, Chemical, Molybdenum, Nitrate Reductases, Oxidation-Reduction, Paracoccus, Protein Binding, Thiocyanates, Periplasm, Nitrate Reductase

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