Fast folding of cytochrome c

Native iso‐2 cytochrome c contains two residues (His 18, Met 80) coordinated to the covalently attached heme. On unfolding of iso‐2, the His 18 ligand remains coordinated to the heme iron, whereas Met 80 is displaced by a non‐native heme ligand, His 33 or His 39. To test whether non‐native His‐heme ligation slows folding, we have constructed a double mutant protein in which the non‐native ligands are replaced by asparagine and lysine, respectively (H33N,H39K iso‐2). The double mutant protein, which cannot form non‐native histidine‐heme coordinate bonds, folds significantly faster than normal iso‐2 cytochrome c: t = 14‐26 ms for H33N,H39K iso‐2 versus r = 200‐1,100 ms for iso‐2. These results with iso‐2 cytochrome c strongly support the hypothesis that non‐native His‐heme ligation results in a kinetic barrier to fast folding of cytochrome c. Assuming that the maximum rate of a conformational search is about 10″ s1, the results imply that the direct folding pathway of iso‐2 involves passage through on the order of 109 or fewer partially folded conformers.