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Cited by 65Metal-Catalyzed Oxygenation MechanismsMetalloenzymes and iron-sulfur proteinsRedox biology and oxidative stress

Superoxide Reductase from Desulfoarculus baarsii: Identification of Protonation Steps in the Enzymatic Mechanism

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Nivière, Vincent, Asso, Marcel, Weill, Claire O., Lombard, Murielle, Guigliarelli, Bruno, Favaudon, Vincent, Houée-Levin, Chantal·2004·Biochemistry
DOI 10.1021/bi035698i
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Abstract

Superoxide reductase (SOR) is a metalloenzyme that catalyzes the reduction of O2*- to H2O2 and provides an antioxidant mechanism in some anaerobic and microaerophilic bacteria. Its active site contains an unusual mononuclear ferrous center (center II). Protonation processes are essential for the reaction catalyzed by SOR, since two protons are required for the formation of H2O2. We have investigated the acido-basic and pH dependence of the redox properties of the active site of SOR from Desulfoarculus baarsii, both in the absence and in the presence of O2*-. In the absence of O2*-, the reduction potential and the absorption spectrum of the iron center II exhibit a pH transition. This is consistent with the presence of a base (BH) in close proximity to the iron center which modulates its reduction properties. Studies of mutants of the closest charged residues to the iron center II (E47A and K48I) show that neither of these residues are the base responsible for the pH transitions. However, they both interact with this base and modulate its pKa value. By pulse radiolysis, we confirm that the reaction of SOR with O2*- involves two reaction intermediates that were characterized by their absorption spectra. The precise step of the catalytic cycle in which one protonation takes place was identified. The formation of the first reaction intermediate, from a bimolecular reaction of SOR with O2*-, does not involve proton transfer as a rate-limiting step, since the rate constant k1 does not vary between pH 5 and pH 9.5. On the other hand, the rate constant k2 for the formation of the second reaction intermediate is proportional to the H+ concentration in solution, suggesting that the proton arises directly from the solvent. In fact, BH, E47, and K48 have no role in this step. This is consistent with the first intermediate being an iron(III)-peroxo species and the second one being an iron(III)-hydroperoxo species. We propose that BH may be involved in the second protonation process corresponding to the release of H2O2 from the iron(III)-hydroperoxo species.

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Authors
Nivière, V., Asso, M., Weill, C. O., Lombard, M., Guigliarelli, B., Favaudon, V., & Houée-Levin, C.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, DRDC-CEA/CNRS/Université Joseph Fourier, 17 Avenue des Martyrs, 38054 Grenoble Cedex 9, France, Unité de Bioénergétique et Ingénierie des Protéines, UPR 9036, CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France, INSERM U 350, Institut Curie-Recherche, Bâtiment 112, Centre Universitaire 91405 Orsay Cedex, France, Laboratoire de Chimie Physique, CNRS/Université Paris-Sud, Bâtiment 350, Centre Universitaire 91405 Orsay Cedex, France
Corresponding author
Nivière, Vincent
ORCID
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
2003-12-24
Volume
43
Issue
3
Pages
808-818
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi035698i
OpenAlex
W1989118744
Cited by
65
References
13
Citations by year
2023: 1 · 2021: 1 · 2020: 1 · 2019: 3 · 2017: 2 · 2016: 2 · 2015: 1 · 2014: 5 · 2013: 2 · 2012: 5
FWCI
4.15
Type
journal article
Language
en
Open access
Licence
other-oa
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, other-oa)
Topics
  • Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
  • Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
Keywords
superoxide reductase, catalytic mechanism, hydrogen peroxide production, pH dependence, redox properties, pulse radiolysis
MeSH terms
Alanine, Enzyme Stability, Hydrogen-Ion Concentration, Isoleucine, Lysine, Oxidation-Reduction, Oxidoreductases, Protons, Pulse Radiolysis, Recombinant Proteins, Spectrum Analysis, Superoxides, Desulfovibrio vulgaris, Glutamic Acid, Amino Acid Substitution, Deltaproteobacteria

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