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Fig. 6. Fluorimetric titration of superoxide reductase (SOR) with ferrocyanide (A) or ferricyanide (B). The SOR from Desulfoarculus baarsii was in 10 mM Tris·HCl (pH 7.6), and the fluorescence emission spectra were recorded at 18°C by using a 1-cm square cuvette. Inner filter effects due to the absorption of ferro/ferricyanide at 295 nm and between 300-420 nm were corrected on the data presented in the inset plots. (A) Top to bottom, SOR (2 mM) fluorescence spectra (excitation at 295 nm) after the addition of 0, 0.2, 3, 10, and 30 mM ferrocyanide. Inset shows a plot of bound SOR as a function of free ferrocyanide, determined from the fluorescence at 340 nm. A K_d value of 0.80 ±0.07 mM was determined. (B) Top to bottom, SOR (1 mM) fluorescence spectra (excitation at 295 nm) after the addition of 0, 0.1, 1, 5, and 15 mM ferricyanide. Inset shows a plot of bound SOR as a function of free ferricyanide, determined from the fluorescence at 340 nm. A K_d value of 0.48 ±0.05 mM was determined. A tryptophan residue (Trp 122), in the second coordination sphere of the iron active site of SOR (8), is most likely responsible for the fluorescence quenching upon ferro/ferricyanide binding.

Fig. 7. Dependence of k_{2 app} versus formate (A) and Tris (B) concentrations. The reaction of SOR (100 mM) with O₂^•- (3 mM), generated by pulse radiolysis, was followed at 580 nm, in the presence 5 molar equivalents of ferrocyanide with respect to SOR at pH 7.6. The ionic strength was kept constant by addition of sodium chloride.

Fig. 8. UV-visible difference spectra calculated from the absorption spectra of SOR-ferrocyanide solutions after minus before the reaction with 55 mM O₂^•-, generated by g-ray radiolysis (88 Gy at 17.6 Gy/min). The solution contained 100 mM of SOR and various concentration of ferrocyanide at pH 7.6. (●), absence of ferrocyanide. ■, presence of 2 molar equivalents of ferrocyanide with respect to SOR. ○, presence of 5 molar equivalents of ferrocyanide with respect to SOR.

Fig. 9. FTIR absorption spectra of SOR plus ferrocyanide solution before (thin line) and after (thick line) the reaction with 55 mM O₂^•-, generated by g-ray radiolysis (88 Gy at 17.6 Gy/min). The solutions contained 100 mM SOR and 500 mM ferrocyanide at pH 7.6. Spectra were recorded from 8-fold concentrated solutions by using a microconcentrator 10 Amicon. The spectra were corrected from the absorbance of a 10 mM Tris·HCl (pH 7.6) buffer solution. Before the reaction, the characteristic n(CN) IR modes are observed at 2,095, 2,047, and 2,024 cm^-1, together with a fraction of free ferrocyanide in solution with a maximum at 2,038 cm^-1. After irradiation, there are no significant modifications of the frequency and intensities of the bands associated with the ferrocyanide complexed with SOR, whereas a small band at 2,116 cm^-1 is due to a fraction of free ferricyanide in solution. A ferricyanide species bound to SOR would be expected to exhibit Fe³⁺-CN vibrations at higher frequencies centered around the n(CN) IR mode of free ferricyanide at 2,116 cm^-1.

1. Auchere F, Raleiras P, Benson L, Venyaminov SY, Tavares P, Moura JJG, Moura I, Rusnak F (2003) Inorg Chem 42:938-940.

Fig. 10. Formation of the SOR-Fe(CN)₆ complex within the cell. E. coli DH5a pMJ25 were grown in LB medium (3 liters) complemented with 1 mM IPTG and 0 or 1 mM ferrocyanide. At the mid-exponential phase, cells were harvested and washed three times with 10 mM Tris·HCl buffer (pH 7.6). The washed cell pellets were resuspended in 15 ml of 0.1 M Tris·HCl (pH 7.6) with 60 mM KCl and incubated 45 min at 4°C in the presence of lysozyme (0.2 mg/ml). Cells were lysed by three cycles of freeze-thawing treatment. The extracts were centrifuged 90 min at 250,000 ´ g. The supernatant (13 ml of 15 mg/ml protein) was kept as the soluble extract. (A) Oxidized-minus-reduced absorbance spectra of soluble extracts obtained from cell cultures grown in the presence of ferrocyanide 1 mM (solid line) or in the absence of ferrocyanide (dashed line). The soluble extracts were oxidized by using ammonium persulfate. The 645-nm band is characteristic of the presence of the oxidized SOR active site (52 mM in the soluble extracts, which represents »5% of the total soluble proteins). The line with filled squares is the difference spectrum calculated from the absorbance spectra of oxidized extracts of the culture with ferrocyanide minus culture in the absence of ferrocyanide. The 420-nm band is superimposable to a spectrum of a ferricyanide solution 70 mM (data not shown). (B) Superdex 75 chromatography of the soluble extracts. Soluble extracts (190 mg) were loaded onto a Superdex 75 column (Amersham Pharmacia Biosciences, Uppsala, Sweden; 124-ml bed volume) and eluted with 10 mM Tris·HCl buffer, pH 7.6. B shows the difference spectra (oxidized with ammonium persulfate minus nonoxidized) of a fraction eluted with 96 ml of buffer. Solid line, fraction from a soluble extract of cells grown in the presence of ferrocyanide. Dashed line, fraction from a soluble extract of cells grown in the absence of ferrocyanide. The 645-nm band is characteristic of the oxidized SOR active site at »12 mM. The line with squares is the difference spectrum of solid minus dashed lines. The 420-nm band is surimposable to a spectrum of a ferricyanide solution at »8 mM (data not shown).