FIGURE 4.

MtMrx1 thiol pK_a determinations and kinetics of oxidation by H₂O₂. A, pK_a titration curves for wild type (circles) (15) and the CXXA mutant (triangles). The specific absorption of the thiolate ion at 240 nm as a function of the pH is shown. A_exp is determined as described (31). Data were fitted with the Henderson-Hasselbach equation. B, time-dependent decrease in the total intrinsic fluorescence intensity (λ_ex = 295 nm, λ_em = 335 nm) of MtMrx1wt (10 μm) upon oxidation by H₂O₂ in 100 mm sodium phosphate buffer plus 0.1 mm DTPA, at pH 7.4 and room temperature. The first arrow indicates the addition of excess H₂O₂ (1.5 mm) and the second, the addition of DTT (1.5 mm). C, effect of H₂O₂ concentration on the observed rate constants of wtMtMrx1 intrinsic fluorescence change.