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Cited by 31Metalloenzymes and iron-sulfur proteinsEnzyme Structure and FunctionMetal-Catalyzed Oxygenation Mechanisms

Access to the Active Site of Periplasmic Nitrate Reductase: Insights from Site-Directed Mutagenesis and Zinc Inhibition Studies

Dementin, Sébastien, Arnoux, Pascal, Frangioni, Bettina, Grosse, Sandrine, Léger, Christophe, Burlat, Bénédicte, Guigliarelli, Bruno, Sabaty, Monique +1 more·2007·Biochemistry
DOI 10.1021/bi700928m
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Abstract

The periplasmic nitrate reductase (NapAB), a member of the DMSO reductase superfamily, catalyzes the first step of the denitrification process in bacteria. In this heterodimer, a di-heme NapB subunit is associated to the catalytic NapA subunit that binds a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) cofactor. Here, we report the kinetic characterization of purified mutated heterodimers from Rhodobacter sphaeroides. By combining site-directed mutagenesis, redox potentiometry, EPR spectroscopy, and enzymatic characterization, we investigate the catalytic role of two conserved residues (M153 and R392) located in the vicinity of the molybdenum active site. We demonstrate that M153 and R392 are involved in nitrate binding: the Vm measured on the M153A and R392A mutants are similar to that measured on the wild-type enzyme, whereas the Km for nitrate is increased 10-fold and 200-fold, respectively. The use of an alternative enzymatic assay led us to discover that NapAB is uncompetitively inhibited by Zn2+ ions (Ki' = 1 microM). We used this property to further probe the active site access in the mutant enzymes. It is proposed that R392 acts as a filter by preventing a direct reduction of the Mo atom by small reducing molecules and partially protecting the active site against zinc inhibition. In addition, we show that M153 is a key residue mediating this inhibition likely by coordinating Zn2+ ions via its sulfur atom. This residue is not conserved in the DMSO reductase superfamily while it is conserved in the periplasmic nitrate reductase family. Zinc inhibition is therefore likely to be specific and restricted to periplasmic nitrate reductases.

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  • Rhodobacter sphaeroides NapA is noncompetitively inhibited by Zn2+ ions with a Ki of ~1 μM.

    “Rhodobacter sphaeroides NapA is noncompetitively inhibited by Zn2+ ions with a Ki of ~1 µM.213”

    From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [213]

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Authors
Dementin, S., Arnoux, P., Frangioni, B., Grosse, S., Léger, C., Burlat, B., Guigliarelli, B., Sabaty, M., & Pignol, D.
Affiliations
Centre National de la Recherche Scientifique (FR)
Corresponding author
  • Dementin, Sébastien
  • Pignol, David
ORCID
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
2007-08-01
Volume
46
Issue
34
Pages
9713-9721
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi700928m
OpenAlex
W1978436100
Cited by
31
References
15
Citations by year
2024: 1 · 2023: 1 · 2022: 1 · 2021: 2 · 2019: 1 · 2018: 1 · 2017: 1 · 2016: 2 · 2015: 2 · 2014: 1 · 2013: 5 · 2012: 3
FWCI
1.32
Type
journal article
Language
en
Open access
closed
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
  • HAL AMU (submittedVersion)
Funders
Chongqing University of Arts and Sciences
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, active site access, zinc inhibition, nitrate binding, molybdenum active site, Rhodobacter sphaeroides, denitrification, [4Fe-4S] cluster, molybdenum cofactor, uncompetitive inhibition, site-directed mutagenesis
MeSH terms
Binding Sites, Molybdenum, Mutation, Nitrates, Oxidation-Reduction, Protein Binding, Rhodobacter sphaeroides, Zinc, Mutagenesis, Site-Directed, Periplasm, Nitrate Reductase

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