A flash photolysis method to characterize hexacoordinate hemoglobin kinetics.

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Dynamics

Background:

• Hexacoordinate hemoglobins, prevalent in plants and microbes, possess a unique ferrous state with six coordination sites.
• Understanding ligand binding kinetics is crucial for elucidating hemoglobin function and evolutionary adaptations.
• Previous methods were insufficient for characterizing the dynamic interactions in these hexacoordinate systems.

Purpose of the Study:

• To develop and validate a flash photolysis method for quantifying ligand binding kinetics in hexacoordinate hemoglobins.
• To determine the rate constants for hexacoordinate complex formation and dissociation, as well as CO binding.
• To investigate the role of the hexacoordinating residue, specifically His(73), in ligand binding dynamics.

Main Methods:

• Flash photolysis coupled with time-resolved spectroscopy to monitor CO rebinding kinetics.
• Analysis of CO rebinding time courses at varying CO concentrations using a two-exponential fit.
• Application of the method to a nonsymbiotic plant hemoglobin from rice.

Main Results:

• The developed method successfully yielded rate constants for CO binding, hexacoordinate complex formation, and dissociation.
• For rice hemoglobin, the dissociation and rebinding of the hexacoordinating His(73) side chain were found to be rapid.
• These dynamics were comparable to the rate of CO binding at high CO concentrations.

Conclusions:

• Hexacoordination significantly influences ligand binding affinity in these hemoglobins.
• The His(73) side chain plays a dynamic role, rapidly dissociating and rebinding during ligand exchange.
• The developed flash photolysis method provides a valuable tool for studying hexacoordinate hemoglobin kinetics.