PROBING THE INTERACTIONS OF PUTIDAREDOXIN WITH REDOX PARTNERS IN CAMPHOR P450 5-MONOOXYGENASE BY MUTAGENESIS OF SURFACE RESIDUES

The role of surface amino acid residues in the interaction of putidare doxin (Pdx) with its redox partners in the cytochrome P450(cam) (CYP10 1) system was investigated by site-directed mutagenesis. The mutated P dx genes were expressed in Escherichia coli, and the proteins were pur ified and studied in vitro. Activity of the complete reconstituted P45 0(cam) system was measured, and kinetic parameters were determined, Pa rtial assays were also conducted to determine the effect of the mutati ons on interactions with each redox partner. Some mutations altered in teractions of Pdx with one redox partner but not the other, Other muta tions affected interactions with both redox partners, suggesting some overlap in the binding sites on Pdx for putidaredoxin reductase and CY P101. Cysteine 73 of Pdx was identified as important in the interactio n of Pdx with putidaredoxin reductase, whereas aspartate 38 serves a c ritical role in the subunit binding and electron transfer to CYP101.