FIGURE 7.

Mia40 catalyzes the rapid oxidation of Cox19 into a single disulfide species. A, Mia40 (100 μm) was added to reduced Cox19 (10 μm), and aliquots were removed at the indicated time points and analyzed by RP-HPLC. The retention times of the detected species coincide exactly with those detected in spontaneous Cox19 oxidative folding. B, different peaks were integrated, and their area plotted as a function of time; reduced Cox19 (▴), Native Cox19 (□), Peak I (♢), and Peak III (○). For clarity the first 5 min of reactions are shown, with a rapid conversion of reduced Cox19 into single disulfide species, mainly peak III that slowly converts into native protein. C, Mia40 (100 μm) was added to reduced Cox19 single disulfide mutants (10 μm); aliquots were removed at the indicated time points and analyzed. Peak areas were integrated and plotted as a function of time. At early stages, Mia40 is only able to catalyze formation of the Cys40-Cys52 disulfide. D, TFE was added to reduced Cox19 at different final percentages, and the protein CD spectrum analyzed: 0% (black), 5% (−), 10% (○), 20% (×), and 40% (♢). E, TFE was added to reduced Cox19, and 1 h folding mixtures were analyzed by RP-HPLC. The percentage of native protein formed was plotted as a function of TFE %.