Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c.
One unique aspect of cytochrome c folding concerns the involvement of the covalently attached heme group and its axial ligands. To elucidate the role of the ligands in stabilizing the native and molten globule states, we studied the conformational and thermodynamic features of the iron-free derivative of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV circular dichroism suggested that porphyrin-cytochrome c has native-like alpha-helices, whereas near-UV CD suggested that the side-chains are flexible. 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 holoprotein. These results indicate that, at neutral pH, the ligation of His18 of the iron is important for the maintenance of the native structure whereas the Met80 ligation is not essential, and that porphyrin-cytochrome c assumes a molten globule-like state. Porphyrin-cytochrome c was largely unfolded at pH 2.0 in the absence of salt, but assumed another molten globule-like structure in the presence of anions. The salt-induced stabilization of the molten globule-like state was the same as that of apocytochrome c, requiring a much higher salt concentration than holocytochrome c. These results indicate that, at acidic pH, the His18 ligation is important, although not essential, for stabilizing the molten globule state. Taken together, both specific (i.e. the His18 axial ligand, as 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.
Hamada
D
D
Department of Biology Faculty of Science Osaka University, Japan.
Kuroda
Y
Y
Kataoka
M
M
Aimoto
S
S
Yoshimura
T
T
Goto
Y
Y
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D011166:Porphyrins / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014965:X-Rays
D003574:Cytochrome c Group; D008024:Ligands; D011166:Porphyrins; D006418:Heme