Picosecond time-resolved fluorescence spectroscopy was employed to characterize the equilibrium and non-equilibrium protein structural fluctuations in ZnII substituted (ZnCytc) and metal-free (fbCytc) cytochromes c using dynamic fluorescence Stokes shift (FSS) and fluorescence anisotropy (FA) measurements. The intrinsic porphyrin chromophore is used as the probe for the structural fluctuations of thesurrounding protein and solvent. The FSS experiments examine how the time scales detected from the dynamic solvation of a chromoprotein report changes in the character of motion. ZnCytc and fbCytc serve as limited, single-chromophore models for photosynthetic reaction center and light-harvesting proteins. The dynamic solvation of redox and light-harvesting chromophores in photosynthesis plays an important role in the quantum efficiency of electron transfer and energy transfer performed by these systems, respectively. The FSS response function of fbCytc in water is biexponential over the 100-ps&mdash 50-ns regime and the two time constants are 1.4 ns and 9.1 ns. ZnCytc under similarsolution conditions shows a biexponential FSS response but with time constants of 0.2 ns and 1.5 ns. The two correlation times from the FSS response function correspond to motions of the hydrophobic core and the solvent-contact layer, respectively. Both FSS correlation times were lengthened and the solvation reorganization energy was reduced from 43 cm-1 to 33 cm-1 in the presence of 50 % (v/v) glycerol. A Brownian diffusion model with thermally activated barrier crossings on the protein folding energy landscape is used to interpret these results. The conclusion is that the mean-squared deviations of the fluctuations exhibited by fbCytc are perhaps a factor of ten larger than those in ZnCytc, which is consistent with the suggestion that fbCytcassumes a dynamic, partially unfolded structure with some of the characteristics of a molten globule.The nature of the motion associated with the unfolding reaction coordinate was then studied in ZnCytc using the FA response of the ZnII-porphyrin probe as guanidinium ion (Gdm+ ) is added to the solution. An unfolding transition-state-like intermediate is formed at &sim 1.0 M Gdm+ , well below the unfolding transition concentration at &sim 2.0 M. The intermediate is characterized by an enhanced angular fluctuation ofthe porphyrin compared to the native and the denatured structures. The correlation time for internal angular motion returned by the FA response for the 1.0 M intermediate is similar to those observed for the FSS response of fbCytc, which suggests that the axial ligands for the ZnII porphyrin have been lost.The FSS response was then obtained as ZnCytc is titrated with Gdm+ using the same data sets as the FA studies. The FSS results show that even at the lowest concentrations of Gdm+ , the native fold is destabilized so that the optical excitation of the ZnII porphyrin chromophore perturbs the motions of the surrounding protein and solvent so that they depart from the linear-response regime. This conclusion is supported by the observation of an unusual bidirectional FSS response. This action of Gdm+ appears to saturate well prior to the formation of the 1-M intermediate structure noted in the FA studies. The main implication of these results is that structural changes in the hydration layer surrounding the folded protein are the origin of the dynamical changes reported in the FSS response in the presence of Gdm+ .
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