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

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

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

“…These results agree with previously published data which demonstrate that Dv rubredoxin is a competent proximal electron donor to Dv SOR [33,34,46]. In addition, the existence of a rubredoxin/SOR redox partnership is consistent with the cotranscription of their genes in Dv [16].…”

“…Besides, on the basis of redox potential and the solvent accessibility, center II should be reduced more efficiently than center I (Scheme 1, pathway 2). It has also been demonstrated that an engineered SOR lacking center I retains its catalytic activity and that rubredoxin is an efficient electron donor to center II [34], and that removal of center I has no influence on the rubredoxin/center II electron transfer rates [28,34,46]. In fact, iron-iron distances in class I SORs seem to be too large to enable efficient electron transfer (monomer, intrasubunit Fe-Fe 22 Å ; dimer, intersubunit Fe-Fe 32 Å ) [14,[58][59][60].…”

“…Therefore, the presence of an electron donor is necessary to regenerate the ferrous active form and complete the catalytic cycle of the enzyme [10,19,[28][29][30][31]. Recently, several groups, including ours, have clearly established rubredoxin as the proximal electron donor to SORs in the case of Tp, Dv, P. furiosus, and A. fulgidus [4,[32][33][34][35][36].…”

Intermolecular electron transfer in two-iron superoxide reductase: a putative role for the desulforedoxin center as an electron donor to the iron active site

“…It displays a high redox potential of about + 300 mV (vs. NHE) at neutral pH and remains mainly in a reduced form in the presence of air [15][16][17][18]. Center II has an open coordination site, which represents the site where superoxide binds and is reduced by the ferrous iron to H 2 O 2 (eq.…”

Sulfur K-Edge X-ray Absorption Spectroscopy and Density Functional Theory Calculations on Superoxide Reductase: Role of the Axial Thiolate in Reactivity

“…Both 1Fe and 2Fe SOR's have been characterized and found to have similar active site structures and reactivities. 8,9 The second Fe in the 2Fe SOR is in a rubredoxin-like Fe(SCys) 4 site for which the function is unknown as eliminating it by mutation does not affect the reactivity of this enzyme. 9…”