Rebinding and relaxation in the myoglobin pocket

The infrared stretching bands of carboxymyoglobin (MbCO) and the rebinding of CO to Mb after photodissociation have been studied in the temperature range 10–300 K in a variety of solvents. Four stretching bands imply that MbCO can exist in four substates, A 0–A 3. The temperature dependences of the intensities of the four bands yield the relative binding enthalpies and entropies. The integrated absorbances and pH dependences of the bands permit identification of the substates with the conformations observed in the X-ray data (Kuriyan et al., J. Mol. Biol. 192 (1986) 133). At low pH, A 0 is hydrogen-bonded to His E7. The substates A 0–A 3 interconvert above about 180 K in a 75% glycerol/water solvent and above 270 K in buffered water. No major interconversion is seen at any temperature if MbCO is embedded in a solid polyvinyl alcohol matrix. The dependence of the transition on solvent characteristics is explained as a slaved glass transition. After photodissociation at low temperature the CO is in the heme pocket B. The resulting CO stretching bands which are identified as B substates are blue-shifted from those of the A substates. At 40 K, rebinding after flash photolysis has been studied in the Soret, the near- infrared, and the integrated A and B substates. All data lie on the same rebinding curve and demonstrate that rebinding is nonexponential in time from at least 100 ns to 100 ks. No evidence for discrete exponentials is found. Flash photolysis with monitoring in the infrared region shows four different pathways within the pocket B to the bound substates A i . Rebinding in each of the four pathways B → A is nonexponential in time to at least 10 ks and the four pathways have different kinetics below 180 K. From the time and temperature dependence of the rebinding, activation enthalpy distributions g( H BA) and preexponentials A BA are extracted. No pumping from one A substate to another, or one B substate to another, is observed below the transition temperature of about 180 K. If MbCO is exposed to intense white light for 10–10 3 s before being fully photolyzed by a laser flash, the amplitude of the long-lived states increases. The effect is explained in terms of a hierarchy of substates and substate symmetry breaking. The characteristics of the CO stretching bands and of the rebinding processes in the heme pocket depend strongly on the external parameters of solvent, pH and pressure. This sensitivity suggests possible control mechanisms for protein r