A LYSINE 73-]HISTIDINE VARIANT OF YEAST ISO-1-CYTOCHROME-C - EVIDENCEFOR A NATIVE-LIKE INTERMEDIATE IN THE UNFOLDING PATHWAY AND IMPLICATIONS FOR M-VALUE EFFECTS

In this paper we report thermodynamic studies on a variant of yeast is o-l-cytochrome c in which a surface lysine residue at position 73 has been replaced with a histidine (H73). Guanidine hydrochloride denatura tion studies monitored by circular dichroism spectroscopy indicated de creased thermodynamic stability (a lower Delta G degrees(u)(H2O)) and a smaller m value for the H73 protein as compared to the wild type (WT ) protein. Further investigations to probe the causes for the thermody namic stability differences between the two proteins involved guanidin e hydrochloride and urea denaturations monitored by tryptophan fluores cence. The stability of heme ligation in the denatured state in the pr esence of either guanidine hydrochloride or urea was monitored by the spin-state transition of the heme iron induced by pH. None of these st udies supported the hypothesis that the decreased m value was due to h eme-His73 ligation in the denatured state. Guanidine hydrochloride den aturations monitored by the change in the extinction coefficient at 69 5 nm, which is sensitive to the presence of heme-Met80 ligation, revea led a native-like intermediate for the H73 protein, probably caused by displacement of the Met80 heme ligand by histidine 73 at guanidine hy drochloride concentrations much lower than required for full cooperati ve unfolding. Presence of the native-like intermediate is most likely the cause of the smaller m value and decreased thermodynamic stability for the CD-monitored H73 protein unfolding as compared to the unfoldi ng of the WT protein. Guanidine hydrochloride denaturations in the pre sence of 200 mM imidazole provide further evidence in support of the p roposed mechanism.