An investigation of processes that occur during the rebinding of carbon monoxide to myoglobin
Lamb, Don Carroll
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Permalink
https://hdl.handle.net/2142/23551
Description
Title
An investigation of processes that occur during the rebinding of carbon monoxide to myoglobin
Author(s)
Lamb, Don Carroll
Issue Date
1993
Doctoral Committee Chair(s)
Frauenfelder, Hans
Department of Study
Physics, Molecular
Biophysics, General
Discipline
Physics, Molecular
Biophysics, General
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Physics, Molecular
Biophysics, General
Language
eng
Abstract
The binding of small ligands to myoglobin at room temperature appears to be a simple, one-step process. However, experiments performed over a large range in time and temperature have shown that the situation is much more complex. Between 60 K and 160 K, the rebinding kinetics are non-exponential in time and are described by a distribution of enthalpy barriers, g(H), between a bound and pocket state. Above 160 K, the structure of the low-temperature photoproduct of myoglobin relaxes towards the deoxy structure. The relaxation increases the enthalpy barriers for rebinding at the heme, and the rebinding kinetics slow down. Above 200 K, ligands can escape to the solvent, and the rebinding process becomes more complicated. Four peaks can be seen in the distribution of lifetimes in the dissociated state, f(log $\tau$): peaks I, 2, 3, and S. Peaks I and S are well understood. Peak I arises from rebinding to the unrelaxed or relaxing g(H) distribution. Peak S is the only process with rates that depend on the concentration of CO in the solvent and, therefore, represents ligands that have rebound from the solvent. Peaks 2 and 3 are geminate processes, but their cause is not yet unambiguously determined.
To investigate the relaxation of the g(H) distribution, band III was monitored because of its sensitivity to conformational changes at the binding site. In the low-temperature photoproduct, the peak position of band III is red-shifted with respect to the deoxy value but relaxes to the deoxy value within 10 ns at room temperature. The rebinding kinetics of band III were monitored with an optical multichannel analyzer between 370 ns and 1 s from 100 K to 300 K. The relaxation of band III was observed and is in agreement with the model presented in a recent paper by Steinbach et al. The time at which band III reaches the deoxy position is measured as a function of temperature and corresponds with peak 2.
The CO stretch bands in MbCO reveal three taxonomic substates: A$\sb0$, A$\sb1$, and A$\sb3$. The rebinding kinetics of the individual A substates are different and provide valuable information about the processes that occur. The exchange between A$\sb1$ and A$\sb3$ is observable on the time scale of peak 3, consistent with the results from pressure release experiments. Since an exchange between A substates is not visible in the Soret kinetics, peak 3 must arise from another process. There is either an additional relaxation of the barrier for rebinding at the heme, an additional A substate contributing to A$\sb1$, or a trap state in myoglobin. These models are discussed along with the supporting experimental evidence for each.
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