Geminate carbon monoxide rebinding to a c-type haem

Area of Science:

• Biochemistry
• Photochemistry
• Protein Dynamics

Background:

• Carboxymethyl cytochrome c (cm cyt. c) is a modified form of cytochrome c with a unique sixth coordination site available for ligand binding.
• Understanding ligand binding and dissociation kinetics in heme proteins is crucial for elucidating their biological functions and reaction mechanisms.

Purpose of the Study:

• To investigate the rapid flash photolysis of carbon monoxide (CO) from the ferrous heme iron of cm cyt. c.
• To characterize the kinetics and spectral transitions associated with CO recombination.
• To compare the ligand escape and recombination dynamics of cm cyt. c with those of myoglobin.

Main Methods:

• Utilized 30-femtosecond laser pulses and a white light continuum for time-resolved spectral monitoring.
• Employed global analysis to fit kinetic data with a minimal number of exponential components.
• Investigated both the neutral and high pH ferrous forms of cm cyt. c.

Main Results:

• Observed a high photo-dissociation quantum yield for CO from cm cyt. c, but a small yield of CO escape due to rapid geminate recombination.
• Identified multiphasic CO recombination with time constants of 16 ps, 120 ps, and 1 ns, indicating three spectrally distinct components.
• The high pH ferrous form of cm cyt. c showed rapid recombination (approx. 7 ps) after photo-dissociation of a bound lysine ligand.

Conclusions:

• The efficient CO-heme geminate recombination in cm cyt. c underscores the sterically hindered nature of its distal heme pocket.
• The dynamics observed in cm cyt. c contrast with the more flexible heme pocket of myoglobin, suggesting structural rigidity in cyt. c is optimized for electron transfer.
• The rapid recombination of the high pH ferrous form of cm cyt. c with lysine is comparable to native cytochrome c's Met80 residue rebinding.