FIGURE 3.

Oxidation and repair of the P. putida ANR protein. A, UV-visible spectrum of reconstituted ANR containing ∼15 μm [4Fe-4S] cluster under anaerobic conditions (thick line). The changes in the ANR spectrum upon successive additions of aerobic buffer (25 mm Tris-HCl containing 500 mm NaCl, pH 7.5) (thin lines) are presented along with the final spectrum ([2Fe-2S] form) shown in gray. B, CD spectra of [4Fe-4S] ANR (29.8 μm) before (solid line) and after (dashed line) exposure to O₂ (∼2-fold molar excess). The arrow indicates the movement of spectral features in response to O₂. The buffer was 9 mm Tris, 17 mm HEPES, 1.7 mm CaCl₂, 236 mm NaCl, 66 mm NaNO₃, pH 7.5. C, detection of persulfide forms of apo-ANR after exposure of [4Fe-4S] ANR to O₂. Mixtures of ANR reconstituted under anaerobic conditions (initially 80 μm [4Fe-4S]²⁺ cluster) were analyzed by LC-MS after incubation with anaerobic buffer for 15 min (gray line) and after treatment with 2 molar equivalents of O₂ for 15 min (black line). The peak at 28,343 Da corresponds to the peak ANR monomer (mass, 28,347 Da) with two disulfide bonds. The peaks labeled S⁰–5S⁰ correspond to successive S⁰ additions (+32 Da). D, restoration of the ANR [4Fe-4S] cluster by treatment of purified [2Fe-2S] ANR (∼40 μm cluster) with ferrous ions (4-fold molar excess) and DTT (3 mm). The gray line shows the initial spectrum of [2Fe-2S] ANR, the dashed and solid black lines show the spectra obtained 50 and 160 min after the addition of ferrous ions and DTT.