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Cited by 89Metal-Catalyzed Oxygenation MechanismsMetalloenzymes and iron-sulfur proteinsRedox biology and oxidative stress

Kinetics and Mechanism of Superoxide Reduction by Two-Iron Superoxide Reductase from Desulfovibrio vulgaris

Emerson, Joseph P., Coulter, Eric D., Cabelli, Diane E., Phillips, Robert S., Kurtz, Donald M.·2002·Biochemistry
DOI 10.1021/bi0119159
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Abstract

Superoxide reductases (SORs) contain a novel square pyramidal ferrous [Fe(NHis)(4)(SCys)] site that rapidly reduces superoxide to hydrogen peroxide. Here we report extensive pulse radiolysis studies on recombinant two-iron SOR (2Fe-SOR) from Desulfovibrio vulgaris. The results support and elaborate on our originally proposed scheme for reaction of the [Fe(NHis)(4)(SCys)] site with superoxide [Coulter, E. D., Emerson, J. E., Kurtz, D. M., Jr., and Cabelli, D. E. (2000) J. Am. Chem. Soc. 122, 11555-11556]. This scheme consists of second-order diffusion-controlled formation of an intermediate absorbing at approximately 600 nm, formulated as a ferric-(hydro)peroxo species, and its decay to the carboxylate-ligated ferric [Fe(NHis)(4)(SCys)] site with loss of hydrogen peroxide. The second-order rate constant for formation of the 600-nm intermediate is essentially pH-independent (pH 5-9.5), shows no D(2)O solvent isotope effect at pH 7.7, and decreases with increasing ionic strength. These data indicate that formation of the intermediate does not involve a rate-determining protonation, and are consistent with interaction of the incoming superoxide anion with a positive charge at or near the ferrous [Fe(NHis)(4)(SCys)] site. The rate constant for decay of the 600-nm intermediate follows the pH-dependent rate law: k(2)(obs) = k(2)'[H(+)] + k(2)' ' and shows a significant D(2)O solvent isotope effect at pH 7.7. The values of k(2)' and k(2)' ' indicate that the 600-nm intermediate decays via diffusion-controlled protonation at acidic pHs and a first-order process involving either water or a water-exchangeable proton on the protein at basic pHs. The formation and decay rate constants for an E47A variant of 2Fe-SOR are not significantly perturbed from their wild-type values, indicating that the conserved glutamate carboxylate does not directly displace the (hydro)peroxo ligand of the intermediate at basic pHs. The kinetics of a K48A variant are consistent with participation of the lysyl side chain in directing the superoxide toward the active site and in directing the protonation pathway of the ferric-(hydro)peroxo intermediate toward release of hydrogen peroxide.

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Authors
Emerson, J. P., Coulter, E. D., Cabelli, D. E., Phillips, R. S., & Kurtz, D. M.
Affiliations
University of Georgia (US)
Corresponding author
Kurtz, Donald M.
ORCID
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
2002-03-06
Volume
41
Issue
13
Pages
4348-4357
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi0119159
OpenAlex
W2021326856
Cited by
89
References
15
Citations by year
2025: 1 · 2023: 1 · 2021: 2 · 2020: 1 · 2019: 2 · 2018: 1 · 2017: 2 · 2016: 4 · 2015: 3 · 2014: 4 · 2013: 1 · 2012: 8
FWCI
5.47
Type
journal article
Language
en
Open access
closed
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
Funders
National Institute of General Medical Sciences (GM 40388, R01GM040388)
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
Desulfovibrio vulgaris, superoxide reduction, hydrogen peroxide production, reaction mechanism, pulse radiolysis, pH dependence, solvent isotope effect, protonation, iron-sulfur clusters, superoxide reductase
MeSH terms
Bacterial Proteins, Electrons, Hydrogen-Ion Concentration, Iron, Kinetics, Models, Chemical, Oxidoreductases, Oxygen, Protein Binding, Recombinant Proteins, Rubredoxins, Spectrophotometry, Superoxides, Temperature, Time Factors, Desulfovibrio vulgaris, Crystallography, X-Ray

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