Electronic and vibrational interactions of porphyrins with proteins: A fluorescence line narrowing study

We have used Fluorescence Line Narrowing (FLN) spectroscopy to study electronic and vibrational interactions of porphyrins with proteins. Zn-substituted cytochrome c (Zn cyt c) was chosen as the model porphyrin-protein system, along with a number of Zn porphyrins embedded in organic and aqueous solvents. For Zn cyt c and the individual Zn porphyrins FLN spectra show characteristics of relaxed fluorescence from an inhomogeneously broadened sample. Zero phonon lines and phonon wings can be clearly distinguished, and vibrational frequencies of the ground and excited states of Zn cyt c have been identified. Ground-state vibrational levels have also been measured for each of the Zn porphyrins. Zn cyt c is characterized by a continuous inhomogeneous energy distribution. When fitted by a gaussian function, we have found the width of the energy distribution function to be approximately 65 cm$\sp{-1}$, indicating that the porphyrin cavity of the protein is relatively rigid. Upon denaturation the width of the distribution increases almost six times to approximately 361 cm$\sp{-1}$, reflecting a very significant increase in the conformational heterogeneity of the protein. The spectra of the denatured protein showed increased broad background and decreased peak resolution when compared to the native protein, indicating that denaturation results in increased phonon coupling. In general, we have found that the phonon coupling in the energy selected spectra and the Stokes shift in conventional optical spectra increase with increased polarity of the solvent matrix. Ligation of guanidine to Zn coproporphyrin in 5 M GuHCl is reflected in the zero phonon lines and the phonon contribution of the corresponding spectra. By comparing FLN spectra of Zn cyt c before and after long-term irradiation we have also observed photochemical changes in the sample. When studying the temperature dependence of the FLN spectra of Zn cyt c we have noted a discontinuous broadening pattern with zero phonon lines rapidly losing resolution within a narrow temperature range. We have interpreted this observation in terms of a low activation energy change in the protein, which is uniquely resolved by using the FLN technique. (Abstract shortened with permission of author.