Rebinding and relaxation in the myoglobin pocket

Area of Science:

• Biophysics
• Protein dynamics
• Spectroscopy

Background:

• Carboxymyoglobin (MbCO) is a key model for studying protein dynamics and ligand binding.
• Understanding the substates of MbCO is crucial for elucidating reaction mechanisms and protein function.

Purpose of the Study:

• To investigate the infrared stretching bands of MbCO and CO rebinding kinetics after photodissociation.
• To characterize the temperature and solvent dependence of MbCO substates and their interconversions.
• To explore the pathways and kinetics of CO rebinding within the heme pocket.

Main Methods:

• Infrared spectroscopy to study MbCO stretching bands across a temperature range (10-300 K) in various solvents.
• Flash photolysis to induce CO photodissociation and monitor rebinding kinetics.
• Analysis of temperature and time dependence to extract thermodynamic and kinetic parameters.

Main Results:

• Four distinct MbCO substates (A0-A3) were identified, with temperature-dependent enthalpies and entropies.
• Substate interconversion is solvent-dependent, occurring above ~180 K in glycerol/water and ~270 K in buffered water, but not in a solid matrix.
• CO rebinding after photodissociation is nonexponential across a wide time scale (100 ns to 100 ks) and occurs via multiple pathways within the heme pocket.
• Activation enthalpy distributions and pre-exponentials were extracted, indicating distinct kinetic pathways below 180 K.

Conclusions:

• MbCO substates and their dynamics are sensitive to external parameters like solvent, pH, and pressure.
• The observed slaved glass transition explains the solvent dependence of substate interconversion.
• CO rebinding is a complex, nonexponential process involving multiple kinetic pathways and substates.
• The findings suggest potential mechanisms for controlling protein reactions through external parameters.