Effector Roles of Putidaredoxin on Cytochrome P450cam Conformational States

In
this study, the effector role of Pdx (putidaredoxin) on cytochrome
P450cam conformation is refined by attaching two different spin labels,
MTSL or BSL (bifunctional spin-label) onto the F or G helices and
using DEER (double electron–electron resonance) to measure
the distance between labels. Recent EPR and crystallographic studies
have observed that oxidized Pdx induces substrate-bound P450cam to
change from the closed to the open state. However, this change was
not observed by DEER in the reduced Pdx complex with carbon-monoxide-bound
P450cam (Fe2+CO). In addition, recent NMR studies have
failed to observe a change in P450cam conformation upon binding Pdx.
Hence, resolving these issues is important for a full understanding
the effector role of Pdx. Here we show that oxidized Pdx induces camphor-bound
P450cam to shift from the closed to the open conformation when labeled
on either the F or G helices with MTSL. BSL at these sites can either
narrow the distance distribution widths dramatically or alter the
extent of the conformational change. In addition, we report DEER spectra
on a mixed oxidation state containing oxidized Pdx and ferrous CO-bound
P450cam, showing that P450cam remains closed. This indicates that
CO binding to the heme prevents P450cam from opening, overriding the
influence exerted by Pdx binding. Finally, we report the open form
P450cam crystal structure with substrate bound, which suggests that
crystal packing effects may prevent conformational conversion. Using
multiple labeling approaches, DEER provides a unique perspective to
resolve how the conformation of P450cam depends on Pdx and ligand
states.