KINETIC MECHANISM OF CYTOCHROME-C FOLDING - INVOLVEMENT OF THE HEME AND ITS LIGANDS

The covalently attached heme and its axial ligands not only are essential for the structure and function Of cytochrome c but they also play an important role in the folding process. Under typical denaturing conditions (concentrated guanidine hydrochloride or urea near pH 7), one of the axial ligands, His 18, remains bound to the oxidized heme iron, but the second ligand, Met 80, is replaced by a non-native histidine ligand (His 26 or His 33 in horse cytochrome c). Using quenched-flow and NMR methods, hydrogen exchange rates were measured for several individual amide protons in guanidine-denatured horse cytochrome c. The observation of a single highly protected (140-fold) backbone amide, that of His 18, suggests the presence of a persistent H-bond consistent with heme ligation of the His 18 side chain in the unfolded state. Heme absorbance changes induced by rapid acidification of oxidized cytochrome c in 4.5 M guanidine hydrochloride from pH 7.8 to 4.6 or below exhibit two kinetic phases with rates of 110 and 25 s(-1), attributed to the dissociation of non-native histidine ligands from the heme in the unfolded state. The kinetics of folding from guanidine-denatured cytochrome c under a variety of initial and final conditions was investigated by stopped-flow methods, using tryptophan fluorescence as a conformational probe and Soret absorbance as a probe for the ligation state of the heme. A fast kinetic phase (80 s(-1)) accompanied by a major decrease in fluorescence and a minor absorbance change coincides with the formation of a partially folded intermediate with interacting chain termini detected in earlier pulsed NH exchange measurements [Roder, H., Elove, G. A., and Englander, S. W. (1988) Nature 335 700]. At neutral pH, an intermediate kinetic phase (1.8 s(-1)) accounts for 78% of the absorbance change and 47% of the fluorescence change. In contrast, the folding kinetics at pH 5 is dominated by the fast phase, and the amplitude of the intermediate phase is reduced to similar to 10%. The pH-dependent amplitude changes show titration behavior with an apparent pK of similar to 5.7, consistent with the protonation of a single histidine residue. The intermediate phase can also be suppressed by the addition of 200 mM imidazole. Since both of these conditions interfere with histidine ligation, the intermediate kinetic phase is attributed to the presence of a non-native histidine ligand (His 26 or His 33) that can become trapped in a partially folded intermediate. In order to investigate the formation of H-bonded structure without interference from heme ligation events, quenched-flow and two-dimensional NMR methods were used to measure the time course of protection against NH exchange during the folding reaction at pH 5. In contrast to earlier results at higher pH, all amide protons had already acquired extensive protection during the fast folding phase, indicating a more cooperative structural transition, All except the N- and C-terminal amide protons exhibit a minor protection phase on the 100-ms time scale, suggesting that some preferential interaction of N- and C-terminal helices is also found at lower pH. A kinetic mechanism is presented that accounts for most of the observed structural and kinetic data on the cytochrome c folding process under various conditions. The model predicts that distinct populations of unfolded molecules with alternative axial ligands give rise to multiple parallel folding pathways, as previously observed [Elove, G. A., and Roder, H. (1991) ACS Symp. Ser. 470, 50].