M. Holden et al., PROBING THE INTERACTIONS OF PUTIDAREDOXIN WITH REDOX PARTNERS IN CAMPHOR P450 5-MONOOXYGENASE BY MUTAGENESIS OF SURFACE RESIDUES, The Journal of biological chemistry, 272(35), 1997, pp. 21720-21725
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.