Role of Heme Axial Ligands in the Conformational Stability of the Native and Molten Globule States of Horse Cytochrome (original) (raw)

1996, Journal of Molecular Biology

One unique aspect of cytochrome c folding concerns the involvement of the 1 Department of Biology covalently attached heme group and its axial ligands. To elucidate the role Faculty of Science of the ligands in stabilizing the native and molten globule states, we studied Osaka University, Toyonaka the conformational and thermodynamic features of the iron-free derivative Osaka 560, Japan of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV 2 Protein Engineering Research circular dichroism suggested that porphyrin-cytochrome c has native-like Institute, Furuedai, Suita a-helices, whereas near-UV CD suggested that the side-chains are flexible. Osaka 565, Japan Its stability against heat or denaturants was much less than that of the intact protein, and similar to that of the acidic molten globule state of the 3 Department of Earth and holoprotein. These results indicate that, at neutral pH, the ligation of His18 Space Science, Faculty of to the iron is important for the maintenance of the native structure whereas Science, Osaka University the Met80 ligation is not essential, and that porphyrin-cytochrome c Toyonaka, Osaka 560, Japan assumes a molten globule-like state. Porphyrin-cytochrome c was largely 4 Institute for Protein unfolded at pH 2.0 in the absence of salt, but assumed another molten Research, Osaka University globule-like structure in the presence of anions. The salt-induced Suita, Osaka 565, Japan stabilization of the molten globule-like state was the same as that of apocytochrome c, requiring a much higher salt concentration than 5 Institute for Enzyme holocytochrome c. These results indicate that, at acidic pH, the His18 Research, University of ligation is important, although not essential, for stabilizing the molten Tokushima, Tokushima, 770 globule state. Taken together, both specific (i.e. the His18 axial ligand, as Japan observed at acidic pH) and nonspecific interactions (the hydrophobic effects of the heme, as observed at neutral pH) contribute to stabilizing the molten globule state.