This dissertation describes two studies that provide new information on the nature of the protein and solvent dynamics that is probed in ZnII-substituted cytochrome c (ZnCytc) by the intrinsic ZnII porphyrin, which serves as a probe of the surrounding protein and solvent. In the first study, the nature of the partially unfolded structures that are generated in ZnCytc upon optical excitation significantly above the vibronic origin of the Q (S0→S1, &pi&rarr&pi* transition) band was investigated using continuous-wave fluorescence spectroscopy. The results show that step-like transitions of the fluorescence Stokes shift correspond to the activation threshold for changes in structure from the native state to a partially unfolded state associated with the &omega loop formed by residues 20-35, which is adjacent to the Cys14 and Cys17 thioether linkages from the porphyrin to the polypeptide backbone. The excitation energy for optical formation of the unfolded state is consistent with the previous determination by Englander and coworkers using hydrogen-exchange NMR spectroscopy in ferrocytochrome c in the presence of Gdm+.In the second study, the hydration shell of ZnCytc was probed using the indolecyanine dye Cy5 using femtosecond pump-continuum-probe spectroscopy. Cy5 was attached to a surface lysine residue by a flexible linker so that it senses the viscosity of the surrounding medium owing to its nonpolar solvation response. The main conclusion of this work is that the hydration shell is as much as 200 times as viscous as bulk water. A simple structural interpretation of this finding is that longer or more persistent chains of hydrogen-bonded water molecules are present than in the bulk.
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