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Volume 272, Number 35, Issue of August 29, 1997 pp. 21720-21725
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Probing the Interactions of Putidaredoxin with Redox Partners in Camphor P450 5-Monooxygenase by Mutagenesis of Surface Residues

(Received for publication, March 20, 1997, and in revised form, May 28, 1997)

Marcia Holden , Martin Mayhew , David Bunk ^¶ , Adrian Roitberg and Vincent Vilker

From the  Biotechnology and ^¶ Analytical Chemistry Divisions, Chemical Sciences and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001

The role of surface amino acid residues in the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450_cam (CYP101) system was investigated by site-directed mutagenesis. The mutated Pdx genes were expressed in Escherichia coli, and the proteins were purified and studied in vitro. Activity of the complete reconstituted P450_cam system was measured, and kinetic parameters were determined. Partial assays were also conducted to determine the effect of the mutations on interactions with each redox partner. Some mutations altered interactions of Pdx with one redox partner but not the other. Other mutations affected interactions with both redox partners, suggesting some overlap in the binding sites on Pdx for putidaredoxin reductase and CYP101. Cysteine 73 of Pdx was identified as important in the interaction of Pdx with putidaredoxin reductase, whereas aspartate 38 serves a critical role in the subunit binding and electron transfer to CYP101.

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