Macromolecular recognition in the cytochrome P450(cam) enzyme system
Stayton, Patrick Sean
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Permalink
https://hdl.handle.net/2142/20509
Description
Title
Macromolecular recognition in the cytochrome P450(cam) enzyme system
Author(s)
Stayton, Patrick Sean
Issue Date
1989
Doctoral Committee Chair(s)
Sligar, Stephen G.
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
Biophysics, General
Language
eng
Abstract
The cytochrome P-450$\sb{\rm cam}$ reaction cycle is a complex set of coordinated chemical transformations requiring precise temporal and spatial control of reactivities. Cytochrome P-450$\sb{\rm cam}$ catalyzes the regio- and stereo-specific hydroxylation of camphor to form 5-exo-hydroxycamphor. The two reducing equivalents required for this reaction are supplied physiologically by putidaredoxin, a Fe$\sb2$S$\sb2$ iron-sulfur protein. The mammalian cytochromes P-450 are also known to interact with cytochrome b$\sb5$, a small redox protein for which a high resolution crystal structure is available. To characterize the molecular cytochrome P-450$\sb{\rm cam}$ binding surface, cytochrome b$\sb5$ was first genetically engineered to afford a fluorescent derivative capable of monitoring its association with cytochrome P-450$\sb{\rm cam}$. The interaction was subsequently computer modeled by looking for van der Waals complementarity and salt bridge formation between the cytochrome b$\sb5$ anionic binding surface and basic residues on the cytochrome P-450$\sb{\rm cam}$ surface. A good fit was found on the proximal surface of nearest approach to the cytochrome P-450$\sb{\rm cam}$ heme prosthetic group.
Subsequent binding competition studies demonstrated that putidaredoxin competitively inhibits the cytochrome b$\sb5$-cytochrome P-450$\sb{\rm cam}$ association, suggesting that the same P-450$\sb{\rm cam}$ surface is utilized by both partners. Site directed mutagenesis of the modeled basic residues suggested that this is indeed the site of putidaredoxin-cytochrome P-450$\sb{\rm cam}$ association. Further time resolved fluorescence studies on the putidaredoxin C-terminal tryptophan suggested that this residue is located near an anionic protein surface. This data supports a complex model featuring electrostatic complementarity between an anionic putidaredoxin surface and the cationic cytochrome P-450$\sb{\rm cam}$ binding surface, with the essential tryptophan positioned to mediate electron transfer.
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