Structures of the Superoxide Reductase from Pyrococcus furiosus in the Oxidized and Reduced States ^,

Abstract

Superoxide reductase (SOR) is a blue non-heme iron protein that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide [Jenney, F. E., Jr., Verhagen, M. F. J. M., Cui, X. , and Adams, M. W. W. (1999) Science 286, 306-309]. Crystal structures of SOR from the hyperthermophilic archaeon Pyrococcus furiosus have been determined in the oxidized and reduced forms to resolutions of 1.7 and 2.0 A, respectively. SOR forms a homotetramer, with each subunit adopting an immunoglobulin-like beta-barrel fold that coordinates a mononuclear, non-heme iron center. The protein fold and metal center are similar to those observed previously for the homologous protein desulfoferrodoxin from Desulfovibrio desulfuricans [Coelho, A. V., Matias, P., Fülöp, V., Thompson, A., Gonzalez, A., and Carrondo, M. A. (1997) J. Bioinorg. Chem. 2, 680-689]. Each iron is coordinated to imidazole nitrogens of four histidines in a planar arrangement, with a cysteine ligand occupying an axial position normal to this plane. In two of the subunits of the oxidized structure, a glutamate carboxylate serves as the sixth ligand to form an overall six-coordinate, octahedral coordinate environment. In the remaining two subunits, the sixth coordination site is either vacant or occupied by solvent molecules. The iron centers in all four subunits of the reduced structure exhibit pentacoordination. The structures of the oxidized and reduced forms of SOR suggest a mechanism by which superoxide accessibility may be controlled and define a possible binding site for rubredoxin, the likely physiological electron donor to SOR.

Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

“…The spectrum is essentially rhombic, with a pronounced feature at g=4.3, and a shoulder at g=9.4 (E/D=0.28), characteristic of a high-spin iron S=5/2, and very similar to previously published EPR spectra of rubredoxin [13,24,26,48,52,53].…”

“…Enzymes in this family share a conserved domain of 100 amino acids accommodating a single active site Fe ion coordinated by four equatorial histidine nitrogen atoms (three e and one d) and an axial cysteinyl sulfur atom [8,11,13,14,15]. Only the reduced colorless ferrous form of the active site iron is able to react with superoxide, with a virtually diffusion controlled rate of 10 9 M À1 s À1 , leading to the formation of the blue ferric state of the enzyme, which suggests the existence of an electron donor to regenerate the ferrous active form and complete the catalytic cycle of the enzyme [6,16,17,18].…”

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

“…The SOR active site is located at the surface of the protein and consists of a mononuclear iron center, named center II, pentacoordinated in its ferrous state by four nitrogen atoms from histidine residues in an equatorial plane and one sulfur atom from a cysteine residue in an axial position [11][12][13][14]. 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].…”

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

“…1) [12]. SORs from the three classes share a conserved C-terminal domain of approximately 100 amino acids that accommodates a single iron-containing active site coordinated by four equatorial histidine nitrogen atoms (three e and one d) and an axial cysteinyl sulfur atom, designated as center II [3,9,[13][14][15]. Class I SORs, also called desulfoferrodoxins, have been isolated from the sulfate-reducing bacteria Desulfovibrio vulgaris (Dv) [9,[16][17][18], Desulfovibrio desulfuricans [9,11], and Desulfoarculus barsii [19,20].…”

“…SORs from Pyrococcus furiosus [4,15], Archeoglobus fulgidus [1,25], or Methanococcus Janaschii [26] also belong to this family.…”