Macromolecular recognition in the cytochrome P450(cam) enzyme system

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.U of I OnlyETDs are only available to UIUC Users without author permissio