Binding of carbon monoxide to isolated hemoglobin chains

Area of Science:

• Biochemistry
• Physical Chemistry
• Molecular Biophysics

Background:

• Hemoglobin's function involves reversible binding of oxygen and other small molecules like carbon monoxide (CO).
• Understanding the kinetics and thermodynamics of CO binding to hemoglobin is crucial for elucidating its biological mechanisms and potential toxicological effects.
• Previous studies have explored CO-hemoglobin interactions, but detailed kinetic analyses across a wide temperature range, especially at cryogenic temperatures, are limited.

Purpose of the Study:

• To investigate the kinetics of carbon monoxide rebinding to separated alpha and beta chains of hemoglobin.
• To characterize the multiple kinetic barriers involved in CO binding and their temperature dependence.
• To explore the role of quantum mechanical tunneling in CO binding at low temperatures and correlate kinetic parameters with protein structure.

Main Methods:

• Flash photolysis was employed to initiate CO dissociation from hemoglobin variants.
• CO rebinding kinetics were measured over a broad temperature range (5–340 K) and time scale (2 μs–1 ks).
• Kinetic data were analyzed using a multi-barrier model, and activation parameters (enthalpies and entropies) were determined.

Main Results:

• All four hemoglobin proteins exhibited three distinct CO rebinding processes.
• Temperature dependence revealed activation enthalpies and entropies for each barrier.
• Non-exponential binding below 200 K indicated a distribution of activation enthalpies for the innermost barrier.
• At temperatures below 30 K, CO binding approached finite limits, consistent with quantum mechanical tunneling.
• Barrier widths were extracted from tunneling rates, and a correlation between the innermost barrier height and equilibrium CO pressure was found.

Conclusions:

• CO binding to hemoglobin involves at least three sequential kinetic barriers.
• Quantum mechanical tunneling significantly contributes to CO binding kinetics at cryogenic temperatures.
• The structural features of hemoglobin chains influence the kinetic barriers, affecting CO binding affinity and dynamics.