Abstract
We have used sol-gel encapsulation protocols to trap kinetically and spectroscopically distinct conformational populations of native horse carbonmonoxy myoglobin. The method allows for direct comparison of functional and spectroscopic properties of equilibrium and non-equilibrium populations under the same temperature and viscosity conditions. The results implicate tertiary structure changes that include the proximal heme environment in the mechanism for population-specific differences in the observed rebinding kinetics. Differences in the resonance Raman frequency of nu(Fe-His), the iron-proximal histidine stretching mode, are attributed to differences in the positioning of the F helix. For myoglobin, the degree of separation between the F helix and the heme is assigned as the conformational coordinate that modulates both this frequency and the innermost barrier controlling CO rebinding. A comparison with the behavior of encapsulated derivatives of human adult hemoglobin indicates that these CO binding-induced conformational changes are qualitatively similar to the tertiary changes that occur within both the R and T quaternary states. Protein-specific differences in the time scale for the proposed F helix relaxation are attributed to variations in the intra-helical hydrogen bonding patterns that help stabilize the position of the F helix.
Highlights
We have used sol-gel encapsulation protocols to trap kinetically and spectroscopically distinct conformational populations of native horse carbonmonoxy myoglobin
Protein-specific differences in the time scale for the proposed F helix relaxation are attributed to variations in the intra-helical hydrogen bonding patterns that help stabilize the position of the F helix
Raman (18 –23) and absorption studies (24 –30), in which the equilibrium deoxy derivative is compared with the non-equilibrium deoxy derivative derived from the photodissociation of COMb under conditions where there is either no or slowed relaxation of the photoproduct, indicate that the proximal heme environment is different in the two cases
Summary
We have used sol-gel encapsulation protocols to trap kinetically and spectroscopically distinct conformational populations of native horse carbonmonoxy myoglobin. In addition the presence of apolar intra-protein substrate docking sites (the so-called Xe cavities) [4, 5] have recently been linked both to these newly proposed functions and to the kinetic patterns associated with geminate and bimolecular ligand binding (3, 6 –13). These new developments highlight fundamental biophysical issues that have not been fully addressed despite the extensive research effort that has been directed toward myoglobin. We describe the use of sol-gel encapsulation as a method of trapping different tertiary conformations of COMb that allows for both spectroscopic and functional characterization at the same ambient conditions.
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