Structure of Superoxide Reductase Bound to Ferrocyanide and Active Site Expansion upon X-Ray-Induced Photo-Reduction

Abstract

Some sulfate-reducing and microaerophilic bacteria rely on the enzyme superoxide reductase (SOR) to eliminate the toxic superoxide anion radical (O2*-). SOR catalyses the one-electron reduction of O2*- to hydrogen peroxide at a nonheme ferrous iron center. The structures of Desulfoarculus baarsii SOR (mutant E47A) alone and in complex with ferrocyanide were solved to 1.15 and 1.7 A resolution, respectively. The latter structure, the first ever reported of a complex between ferrocyanide and a protein, reveals that this organo-metallic compound entirely plugs the SOR active site, coordinating the active iron through a bent cyano bridge. The subtle structural differences between the mixed-valence and the fully reduced SOR-ferrocyanide adducts were investigated by taking advantage of the photoelectrons induced by X-rays. The results reveal that photo-reduction from Fe(III) to Fe(II) of the iron center, a very rapid process under a powerful synchrotron beam, induces an expansion of the SOR active site.

Detoxification of superoxide without production of H 2 O 2 : Antioxidant activity of superoxide reductase complexed with ferrocyanide

“…The recent crystal structure of SOR from D. baarsii revealed that the organometallic compound ferrocyanide makes a complex with the enzyme active site (8). This structure is the only example of a complex between ferrocyanide and an iron site of a metalloenzyme.…”

“…1). The complex was observed for both the reduced and oxidized forms of the SOR iron active site (8). The almost perfect steric and electrostatic complementarities of ferrocyanide with the active site suggested that it could act as a potent inhibitor of SOR activity by preventing O 2 .…”

“…by SOD or SOR appears to be an essential cellular process to allow organisms to survive in the presence of O 2 (1,2,6). The SOR active site consists of a mononuclear ferrous ion in an unusual [Fe 2ϩ (N-His) 4 (S-Cys)] square pyramidal pentacoordination (7,8). The free, solvent-exposed, sixth coordination position is the site of O 2 .…”

“…This intermediate undergoes two sequential protonation processes, first yielding a second intermediate, possibly a Fe 3ϩ -hydroperoxo species and then the final reaction products, H 2 O 2 and the ferric active site (10). Recently, spectroscopic (11,12) and crystallographic studies (8) have shown that the SOR active site binds ferrocyanide [also referred as hexacyanoferrate (II) or K 4 Fe(CN) 6 ] at its sixth coordination position through a cyano bridge between the iron and the ferrocyanide molecule (Fig. 1).…”

Temperature-dependent macromolecular X-ray crystallography

“…In another example, reduction of the haem iron by synchrotron radiation followed by transient warming to room temperature was used to generate and trap intermediate states in crystalline myoglobin (Hersleth et al, 2008). Research on other crystalline redox-sensitive proteins for which X-rayinduced reduction has been reported at temperatures close to 100 K (Berglund et al, 2002;Adam et al, 2004;Baxter et al, 2004;Mees et al, 2004;Roberts et al, 2005;Echalier et al, 2006;Beitlich et al, 2007;Kuhnel et al, 2007;Pearson et al, 2007;Hough et al, 2008) could benefit from temperaturecontrolled crystallography if structural information on intermediate states were of interest.…”