FIGURE 6.

Extended exposure to H₂O₂ converts PDF and TDH to nonreactivatible forms in vivo and in vitro. A, TCEP did not restore PDF and TDH activities in the extracts of Hpx⁻ cells that had been grown in aerobic medium. Both enzymes were assayed in the presence of metal (500 μm Ni²⁺ and 500 μm Fe²⁺ respectively) to ensure full activity of undamaged enzymes. B, PDF polypeptide was not degraded in vivo. Anaerobic cells expressing PDF-FLAG were treated with chloramphenicol and aerated starting at time 0. Polypeptide content was monitored by Western blot. The strains used were MG1655/pPDF-FLAG, LC106/pPDF-FLAG (Hpx⁻) and AA30/pPDF-FLAG (Hpx⁻ ΔmntH). C, iron-PDF and apo-PDF can be overoxidized in vitro. Iron-charged PDF, apo-PDF, and nickel-charged PDF were exposed to H₂O₂ (133, 100, and 500 μm, respectively) for the indicated times. They were then treated ± TCEP and reconstituted with nickel prior to assay.