An engineered two-iron superoxide reductase lacking the [Fe(SCys)4] site retains its catalytic properties in vitro and in vivo.
Superoxide reductases (SORs) 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 SORs, referred to as two-iron SORs (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-SORs 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-SORs are considered.[1]References
- An engineered two-iron superoxide reductase lacking the [Fe(SCys)4] site retains its catalytic properties in vitro and in vivo. Emerson, J.P., Cabelli, D.E., Kurtz, D.M. Proc. Natl. Acad. Sci. U.S.A. (2003) [Pubmed]
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