Porphyrin electronic transitions in heme proteins provide a useful tool for probing the protein environment, since the surrounding protein affects the porphyrin pi-electron cloud. Perturbations can arise from structural distortions of the porphyrin ring, from the internal electric field generated by charged and polar groups, or from axial ligation to the heme iron. In this work, cytochrome c in aqueous solution or in glasses of trehalose or glycerol/water was examined as a function of temperature to evaluate the effect of fluctuations on the heme. The amide I band of cytochrome c in trehalose remains constant over a wide temperature excursion, indicating that interactions between the protein and the matrix do not change with temperature. The width of the Q(0,0) optical transition measured at low temperature (i.e., < 100 K) reflects the temperature at which the glass was formed, while the temperature profiles of the widths for the protein in different solvents and glasses are similar at high temperature. The results were interpreted in terms of contributions from solvent-coupled and solvent-uncoupled motions. Molecular dynamics simulations of cytochrome c in explicit solvent were performed to investigate the structural distortions in the protein, and semiempirical quantum mechanics (Zindo/ S) was used to calculate the resultant changes in the spectroscopic transitions. A correlation between the calculated transition energies and the structural distortions in both the heme and the surrounding protein environment was observed and was invoked to characterize the origins of the temperature-dependent broadening of the electronic transitions seen in the visible spectra.