Nanosecond-Regime Correlation Time Scales for Equilibrium Protein Structural Fluctuations of Metal-Free Cytochrome c from Picosecond Time-Resolved Fluorescence Spectroscopy and the Dynamic Stokes Shift

We used picosecond time-resolved fluorescence spectroscopy to characterize the fluorescence Stokes shift (FSS) response function of metal-free (or free-base, fbCytc) cytochrome c under the solution conditions that favor the native states of ferricytochrome c (FeCytc) and Zn (II)-substituted cytochrome c (ZnCytc) The intrinsic porphyrin chromophore serves in these experiments as a fluorescent probe of the structural fluctuations of the surrounding protein and solvent Demetalation of the porphyrin destabilizes the folded structure of cytochrome c owing to the loss of the axial metal-histidine and metal-methionine bonds Thus, these experiments examine how the time scales detected in a dynamic solvation experiment in a chromoprotein report changes in the character of motion The FSS response function in fbCytc in water and pH 7 is well described by a biexponential response over the 100 Ps to 50 ns regime with time constants of 1 4 and 9 1 ns under similar conditions ZnCytc exhibits a biexponential FSS response with time constants of 250 Ps and 1 5 ns [Lampa-Pastirk and Beck, J Phys Chem B 2004, 108, 16288] These time constants correspond respectively to the correlation time scales for motions of the hydrophobic core and the solvent-contact layer of the protein Both of the time constants observed in fbCytc are further lengthened upon addition of glycerol to the external solvent so that a significant fraction of the protein dynamics is rendered effectively static on the fluorescence time scale The solvation reorganization energy the time-integrated Stokes shift of the fluorescence spectrum, is reduced by about a third to 33 cm(-1) in 50% glycerol from 43 cm(-1) in water These results are interpreted structurally using a model for Brownian diffusive motion with thermally activated barrier crossings on the protein-folding energy landscape The results suggest that the mean-squared deviations of the structural fluctuations exhibited by fbCytc are nearly a factor of 10 larger than those of ZnCytc This conclusion is consistent with the suggestion that fbCytc assumes a dynamic partially unfolded structure with some of the characteristics of a molten globule