FAST FOLDING OF CYTOCHROME-C

Native iso-2 cytochrome c contains two residues (His 18, Met 80) coord inated to the covalently attached heme. On unfolding of iso-2, the His 18 ligand remains coordinated to the heme iron, whereas Met 80 is dis placed 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 doub le mutant protein in which the non-native ligands are replaced by aspa ragine 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: tau = 14-2 6 ms for H33N,H39K iso-2 versus tau = 200-1,100 ms for iso-2. These re sults 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 s earch is about 10(11) s(-1), the results imply that the direct folding pathway of iso-2 involves passage through on the order of 10(9) or fe wer partially folded conformers.