Raman and kinetic studies of myoglobin structure and dynamics

Resonance Raman spectroscopy is used to analyze the absorption line shapes of myoglobin (Mb), its CO ligated complex (MbCO) and the low‐temperature photoproduct (Mb*, CO). Energetically significant inhomogeneous broadening is detected in the line shapes of the unligated species and a strong coupling to low‐frequency (CO torsional) modes is found for MbCO. These observations lead naturally to a simple model of the protein–heme system and its thermally induced conformational fluctuations. The model is used to analyze the non‐exponential ligand binding, optical line shifts and kinetic holeburning exhibited by (Mb*, CO) at low temperatures. The model makes a specific prediction for the high‐temperature Arrhenius barrier at the heme that is found to be in good agreement with kinetic measurements as a function of temperature near 293 K. It is also found that the geminate phase of ligand rebinding remains non‐exponential even at these higher temperatures and it is shown that this is due to a homogeneous relaxation process (probably involving changes in the distal pocket protein conformation following photolysis) rather than to an inhomogeneous distribution of heme rebinding barriers. Slow distal pocket fluctuation time scales are also revealed by using a two‐pulse photolysis protocol to select kinetically a rapidly rebinding sub‐population of the ensemble. These experiments allow the direct monitoring of the time scales of the large‐scale distal pocket interconversions that average the ‘open’ (A0) and ‘closed’ (A1) states of myoglobin.