2026-09-20T06:52:12Z https://www.ideals.illinois.edu/oai-pmh

oai:www.ideals.illinois.edu:2142/20509 2023-07-10 col_2142_5131 col_2142_14795 com_2142_5130 com_2142_14794 com_2142_14793 com_2142_8903

Sligar, Stephen G. Stayton, Patrick Sean 2011-05-07T12:41:15Z 2011-05-07T12:41:15Z 10000-01-01 1989 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. Made available in DSpace on 2011-05-07T12:41:15Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9011038.pdf: 6223770 bytes, checksum: 4c96a7806b0245ca093b1ac5c0bb667b (MD5) Previous issue date: 1989 Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:44:22Z Item is restricted indefinitely. Restriction data tranferred 2014-07-01T11:19:32-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission ETDs are only available to UIUC Users without author permission U of I Only AAI9011038 (UMI)AAI9011038 http://hdl.handle.net/2142/20509 eng Copyright 1989 Stayton, Patrick Sean Chemistry, Biochemistry Biophysics, General Macromolecular recognition in the cytochrome P450(cam) enzyme system text Biochemistry Biochemistry University of Illinois at Urbana-Champaign Dissertation Ph.D.