Investigations of spectral broadening mechanisms in biomolecules: Cytochrome-c

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Abstract
We have studied the optical line shape of the intense π–π* ‘‘Soret’’ transition of ferrocytochrome‐c as a function of temperature. Use of the linear electron–nuclear coupling strengths found from the transform analysis of the resonance Raman excitation profiles allows us to simulate the measured line shape changes using a multimode time correlator model that is exact at all temperatures. Four major sources of line broadening are considered: (1) Multimode broadening due to the observed Raman (Franck–Condon) active modes; (2) broadening due to a low frequency Franck–Condon active bath that is not observed directly in the resonance Raman spectrum; (3) inhomogeneous broadening, σ, modeled as a Gaussian distribution of 0–0 transition frequencies; (4) homogeneous broadening, Γ, due to exponential population decay of the excited electronic state. We find conclusive evidence that mechanism (2) is not important in determining the Soret band line shape and that mechanism (3) is constrained to an upper limit of σmax=200 cm−1. The results giving the best fits over the entire temperature range yield σ=100 cm−1, Γ=370 cm−1 and show that the thermal properties of the line shape are well described by the observed low frequency Raman modes. The resulting large value of Γ corresponds to ultrafast population decay times (10–20 fs) that are difficult to measure directly. As a result, we have expanded upon a light scattering sum rule that allows direct determination of Γ even in the presence of inhomogeneous broadening. Recent tests of this independent approach are also discussed. Finally, the analysis uncovers strong evidence for a set of weak temperature dependent transitions underlying the Soret band. These transitions are quantified via fitting to Gaussian line shapes and their temperature dependence is explicitly presented.