Ferricytochrome c. Refolding and the methionine 80-sulfur-iron linkage.

The refolding of urea-denatured horse heart ferricytochrome c in the presence of imidazole, 0.5 M, pH 7.0, has been examined using stopped-flow and equilibrium measurements at 407.5 nm. Thermodynamically, imidazole-cytochrome c folds and unfolds via a single transition with [urea]1/2 of 5.9 M. Kinetically, the refolding is a triphasic process: (i) a slow, urea-independent phase, time constant of 22 +/- 6 s, and an amplitude of 10-13%; (ii) an intermediate reaction, with a slightly positive urea-dependent rate constant, average time constant of 150 ms; and (iii) a fast phase with negative urea dependence of the rate constant from 4-6 M urea and positive dependence above the 6 M concentration, with the largest time constant, 25 +/- 6 ms, at 5.8 M urea, the midpoint of the transition. The amplitudes of the intermediate and the fast phases exhibit inverse dependence on the final urea concentrations, favoring the intermediate form at higher concentrations, while maintaining an almost constant sum of the two amplitudes throughout the range. The temperature dependence of the three apparent rate constants for the refolding from denatured base-line to midpoint of the transition, 9 to 6.03 M urea, yields linear Arrhenius plots with activation energies of 14, 19, and 23 +/- 3 kcal/mol for the slow, intermediate, and rapid reactions, respectively. These findings show that the slow reaction, time constant in decaseconds , does not require, directly or indirectly, the coordination of Met-80-S to heme iron. The formation of this linkage during the folding of the urea-denatured protein in the absence of extrinsic ligand, however, does alter the course of the refolding process. From a comparison of the proposed mechanisms and of the kinetic parameters for the folding of urea-denatured and of guanidine hydrochloride-denatured ferricytochrome c, it has been suggested that the two systems are distinct in detail, although both systems exhibit the slow, decasecond process.