An engineered two-iron superoxide reductase lacking the [Fe(SCys)4] site retains its catalytic properties in vitro and invivo

doi:10.1073/pnas.0537177100

An engineered two-iron superoxide reductase lacking the [Fe(SCys)₄] site retains its catalytic properties in vitro and invivo

^*Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30605; and ^†Chemistry Department, Brookhaven National Laboratory, Upton, NY 11972

Edited by Jack Halpern, University of Chicago, Chicago, IL, and approved December 23, 2002 (received for review November 25, 2002)

Abstract

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.

oxidative stress‖2Fe-SOR‖1Fe-SOR‖pulse-radiolysis

Footnotes

↵ ‡ To whom correspondence should be addressed. E-mail: kurtz{at}chem.uga.edu.
This paper was submitted directly (Track II) to the PNAS office.
↵ § The absorption spectrum of the ferric [Fe(SCys)₄] site in a genetically engineered protein fragment corresponding to the N-terminal [Fe(SCys)₄]-containing domain of D. vulgaris 2Fe-SOR (residues 1–38) is provided as supporting information. The metal content and spectroscopic properties of this protein, which we call N-terminal 2Fe-SOR, were found to be very similar to those previously described for the same recombinant protein fragment by Ascenso et al. (27).
↵ ¶ During isolation and purification, a small quantity of C13S 2Fe-SOR was eluted as a separate fraction that, based on its absorption spectrum, may have contained a small portion of ferric [Fe(SCys)₃(OSer)] sites. However, the visible absorption attributed to these sites bleached irreversibly after several minutes at room temperature, whereas a much more intense blue color caused by the ferric [Fe(NHis)₄(SCys)] sites could be regenerated by addition of oxidants.
Abbreviations:

SOR,

superoxide reductase;

SOD,

superoxide dismutase;

2Fe-SOR,

two-iron SOR;

NHE,

normal hydrogen electrode