pH effects and rebinding pathways in CO adducts of heme proteins
Braunstein, David Phillip
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https://hdl.handle.net/2142/22777
Description
Title
pH effects and rebinding pathways in CO adducts of heme proteins
Author(s)
Braunstein, David Phillip
Issue Date
1991
Doctoral Committee Chair(s)
Frauenfelder, Hans
Department of Study
Physics
Discipline
Physics
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Physics, Molecular
Language
eng
Abstract
CO-adducts of heme proteins have IR absorption bands over the range 2200-1900 cm$\sp{-1}$. Flash photolysis can break the Fe-C bond, freeing the ligand. Below the glass-transition temperature of the protein-solvent system, $T\sb{g} \approx$ 185K, heme proteins are frozen into static conformations, and the photolyzed ligand, trapped within the protein, is restricted to the local environment of the binding site. The IR spectra of the bound and photolyzed ligands display several bands that are sensitive to changes in the local structure of the binding site and the pH of the solvent surrounding the protein. Each band represents a distinct conformation substate (CS) of the protein. The kinetics of the geminate rebinding of CO in heme proteins following flash photolysis, below $T\sb{g}$, is well described by a time- and temperature-independent distribution of enthalpic barriers, $g(H)$.
The IR spectra of several heme proteins and a model compound are studied statically and kinetically to better understand the connections between the bound ($A$) and photolyzed ($B$) substates of the protein-ligand system. The spectral mapping between the $A$ and the $B$ substates of the MbCO is derived by simultaneously measuring the low temperature CO rebinding kinetics in both sets of bands. The peak enthalpy of the $g(H)$ for rebinding to each $A$ substate, $H\sb{p\sb{A\sb i}}$, is found to pH independent. In addition new substates are found hidden beneath the observable $A$ and $B$ bands. One of these new bands, $A\sb{X}$, may present a more open state of the MbCO structure. The low temperature CO rebinding kinetics to $\alpha\sp{SH}$HbCO and $\beta\sp{SH}$HbCO are also studied. $\alpha\sp{SH}$CO displays a single $A$ band and two $B$ bands. The single $A$ band is shown to actually represent two distinct $CS\sp0$ substates. The photolyzed IR spectrum of the model compound microperoxidase(11)-CO ($\mu$p-CO) is measured for comparison to the $B$ bands of MbCO and the Hb subunits. Finally, the His E7 residue, which is opposite the binding site of the CO, is shown to be nonessential in governing the pH dependence the $A$ substates of a site-directed mutant of MbCO, (Met$\sp{E7}$) MbCO, which has a methionine (Met), in place of the His E7.
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