Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 100, Issue 7, Pages 3802-3807Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0537177100
Keywords
oxidative stress; 2Fe-SOR; 1Fe-SOR; pulse-radiolysis
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Funding
- NIGMS NIH HHS [R01 GM040388, GM 40388] Funding Source: Medline
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Superoxide reductases (SORB) contain a characteristic square-pyramidal [Fe(NHis)(4)(SCys)] active site that catalyzes reduction of superoxide to hydrogen peroxide in several anaerobic bacteria and archaea. Some SORB, referred to as two-iron SORB (2Fe-SORs), also contain a lower-potential [Fe(SCys)(4)] site that is presumed to have an electron transfer function. However, the intra- and inter-subunit distances between [Fe(SCys)(4)] and [Fe(NHis)(4)(SCys)] iron centers within the 2Fe-SOR homodimer seem too long for efficient electron transfer between these sites. The possible role of the [Fe(SCys)(4)] site in 2Fe-SORB was addressed in this work by examination of an engineered Desulfovibrio vulgaris 2Fe-SOR variant, C13S, in which one ligand residue of the [Fe(SCys)(4)] site, cysteine 13, was changed to serine. This single amino acid residue change destroyed the native [Fe(SCys)(4)] site with complete loss of its iron, but left the [Fe(NHis)(4)(SCys)] site and the protein homodimer intact. The spectroscopic, redox and superoxide reactivity properties of the [Fe(NHis)(4)(SCys)] site in the C13S variant were nearly indistinguishable from those of the wild-type 2Fe-SOR. Aerobic growth complementation of a superoxide dismutase (SOD)-deficient Escherichia coli strain showed that the presence of the [Fe(NHis)(4)(SCys)] site in C13S 2Fe-SOR was apparently sufficient to catalyze reduction of the intracellular superoxide to nonlethal levels. As is the case for the wild-type protein, C13S 2Fe-SOR did not show any detectable SOD activity, i.e., destruction of the [Fe(SCys)(4)] site did not unmask latent SOD activity of the [Fe(NHis)(4)(SCys)] site. Possible alternative roles for the [Fe(SCys)(4)] site in 2Fe-SORB are considered.
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