Different relaxations in myoglobin after photolysis.

To clarify the interplay of kinetic hole-burning (KHB), structural relaxation, and ligand migration in myoglobin (Mb), we measured time-resolved absorption spectra in the Soret region after photolysis of carbon monoxide Mb (MbCO) in the temperature interval 120-260 K and in the time window 350 ns to 200 ms. The spectral contributions of both photolyzed (Mb*) and liganded Mb (MbCO) have been analyzed by taking into account homogeneous bandwidth, coupling to vibrational modes, and static conformational heterogeneity. We succeeded in separating the "time-dependent" spectral changes, and this work provides possibilities to identify the events in the process of ligand rebinding. KHB is dominant at T <190 K in both the Mb* and the MbCO components. For MbCO, conformational substates interconversion at higher temperatures tends to average out the KHB effect. At 230-260 K, whereas almost no shift is observed in the MbCO spectrum, a shift of the order of approximately 80 cm(-1) is observed in Mb*. We attribute this shift to protein relaxation coupled to ligand migration. The time dependence of the Mb* spectral shift is interpreted with a model that enables us to calculate the highly nonexponential relaxation kinetics. Fits of stretched exponentials to this kinetics yield Kohlrausch parameter values of 0.25, confirming the analogy between proteins and glasses.