A Self-sufficient Cytochrome P450 with a Primary Structural Organization That Includes a Flavin Domain and a [2Fe-2S] Redox Center
Date
27/09/2003Author
Roberts, Gareth A
Celik, Ayhan
Hunter, Dominic JB
Ost, Tobias WB
White, John H
Chapman, Stephen K
Turner, Nicholas J
Flitsch, Sabine L
Metadata
Abstract
P450 RhF from Rhodococcus sp. NCIMB 9784 is the
first example of a new class of cytochrome P450 in which
electrons are supplied by a novel, FMN- and Fe/S-containing,
reductase partner in a fused arrangement. We
have previously cloned the gene encoding the enzyme
and shown it to comprise an N-terminal P450 domain
fused to a reductase domain that displays similarity to
the phthalate family of oxygenase reductase proteins. A
reductase of this type had never previously been reported
to interact with a cytochrome P450. In this report
we describe the purification and partial characterization
of P450 RhF. We show that the enzyme is selfsufficient
in catalyzing the O-dealkylation of 7-ethoxycoumarin.
The P450 RhF catalyzed O-dealkylation of
7-ethoxycoumarin is inhibited by several compounds
that are known inhibitors of cytochrome P450. Presteady
state kinetic analysis indicates that P450 RhF
shows a 500-fold preference for NAPDH over NADH in
terms of Kd value (6.6 -mu M versus 3.7 mM, respectively).
Potentiometric studies show reduction potentials of
-243 mV for the two-electron reduction of the FMN and
-423 mV for the heme (in the absence of substrate).