Structural and Dynamic Aspects of the Cytochrome P450(cam) Protein Conformation
Fisher, Mark Thomas
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https://hdl.handle.net/2142/70567
Description
Title
Structural and Dynamic Aspects of the Cytochrome P450(cam) Protein Conformation
Author(s)
Fisher, Mark Thomas
Issue Date
1987
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
The ferric spin equilibrium of cytochrome P-450$\sb{\rm cam}$ varys from a predominantly low-spin (S = 1/2) form to a predominantly high-spin (S = 5/2) form upon binding the natural substrate camphor. Cytochrome P-450$\sb{\rm cam}$ exhibits a distribution of spin states when various camphor analogs are bound to the cytochrome. These camphor analogs provide a means of independently varying the ferric spin equilibrium of the cytochrome in order to examine the relationship between this internal protein equilibrium and important functional aspects of the cytochrome.
High pressure uv-visible difference spectroscopy has been applied to probe the extent of the conformational change associated with substrate dissociation. Results indicate that the volume change associated with the substrate dissociation event is approximately the same regardless of the initial spin equilibrium. Second derivative uv spectroscopy was used to determine the local dielectric environment of tyrosine residues as a function of the amount of high-spin form that was present. The results from the high pressure and second derivative experiments indicate that the ferric spin equilibrium of this cytochrome is a local conformational event and that the polarity of the active site increases as the low-spin ferric fraction increases.
Detailed measurement of the first electron transfer rate between the physiological electron transfer partner putidaredoxin and cytochrome P-450$\sb{\rm cam}$ bound to various camphor analogs has established that a linear free energy relationship exists between the ferric spin equilibrium and both the reduction potential of the substrate bound cytochrome and the activation energy of the forward electron transfer rate. The fastest electron transfer rate, highest reduction potential, and lowest activation energy for electron transfer occur in the high-spin ferric cytochrome.
The kinetics of the ferric spin equilibrium in the presence of the various camphor analogs was examined by temperature-jump relaxation spectroscopy. Results indicate that the most rapid recombination rates of the water-derived sixth axial ligand are observed in the predominantly low-spin form of the cytochrome. The results of the experiments outlined in this thesis suggest that the reduction potential and first electron transfer rates in cytochrome P-450$\sb{\rm cam}$ are controlled by the local polarity of the heme-containing active site.
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