Spectroscopic studies on the conformation of cytochrome c and apocytochrome c

The appearance of the downfield region of the PMR spectrum of apocytochrome c is consistent with that of an extensively disordered protein. The resonances of the 3 histidine C 2 protons are almost equivalent and have a pKa of 6.2. In contrast, this region of the ferricytochrome c PMR spectrum shows many sharp resonances, due to the tertiary structure of the protein and contact shifts from the heme group. The complete titration of one histidine residue, with a pKa of 6.41, can be determined in the holoprotein. At low pH, the resonance of a second histidine residue can be observed. The acid induced conformational transition of ferricytochrome c can be resolved into several component transitions by the use of different spectral parameters. Measurements of absorption at 395 nm and fluorescence emission at 340 nm show a single cooperative transition with midpoint of about pH 2.5. These indices are closely related to the geometry of the heme group. The resonances of the 2 observable histidine residues change in a way suggesting denaturation only below pH 1.8 but indicate conformational changes at about pH 3. The resonances assigned to heme methyl groups, however, undergo significant changes between pH 3 and 4. The reduced viscosity of acid denatured ferricytochrome c and the fluorescence emission of its tryptophan residue increase markedly as salt concentration is lowered, indicating that the conformation of acid denatured protein is sensitive to ionic strength.