The effect of His-heme misligation on folding has been investigated for a triple mutant of yeast iso-2 cytochrome c (N26H,H33N,H39K iso-2). The variant contains a single misligating His residue at position 26, a location at which His residues are found in several cytochrome c homologues, including horse, tuna, and yeast iso-1. The amplitude for fast phase folding exhibits a strong initial pH dependence. For GdnHCl unfolded protein at an initial pH < 5, the observed refolding at final pH 6 is dominated by a fast phase (tau(2f) = 20 ms, alpha(2f) = 90 %) that represents folding in the absence of misligation. For unfolded protein at initial pH 6, folding at final pH 6 occurs in a fast phase of reduced amplitude (alpha(2f) similar to 20%) but the same rate (tau(2f) = 20 ms), and in two slower phases (tau(m) = 6-8 seconds, alpha(m) similar to 45%; and tau(1b) = 16-20 seconds, alpha(1b) similar to 35%). Double jump experiments show that the initial pH dependence of the folding amplitudes results from a slow pH-dependent equilibrium between fast and slow folding species present in the unfolded protein. The slow equilibrium arises from coupling of the His protonation equilibrium to His-heme misligation and proline isomerization. Specifically, Pro25 is predominantly in trans in the unligated low-pH unfolded protein, but is constrained in a non-native cis isomerization state by His26-heme misligation near neutral pH. Refolding from the misligated unfolded form proceeds slowly due to the large energetic barrier required for proline isomerization and displacement of the misligated His26-heme ligand. (C) 2000 Academic Press.
