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144 Chapter 6
of the haem cofactor is an iron centre which in the resting enzyme is in the þ3
oxidation state, but which is reduced to the þ2 oxidation state upon substrate
binding. This reduction is carried out by a reductase sub-unit of the enzyme
which contains a Xavin cofactor, itself reduced by NADPH.
The mechanism of hydroxylation by P 450 enzymes shown in Figure 6.30 is
explained as follows. Just as reduced Xavin is able to donate a single electron to
dioxygen in the case of the Xavin-dependent mono-oxygenases, so the reduced
iron(II) is able to donate a single electron to dioxygen, forming iron(III)-superoxide.
Transfer of a second electron from the Xavin reductase sub-unit via the iron
centre generates iron(III)-peroxide. Protonation of the peroxide generates a good
leaving group for cleavage of the O2O bond, giving formally an iron(V) oxo
species, which is believed to exist as an iron(IV) oxo/porphyrin radical cation. The
mechanism of substrate hydroxylation by this reactive intermediate has been
much debated in the literature. One possible mechanism, shown in Figure 6.30,
is the abstraction of H . from the substrate, forming a substrate radical species and
iron(IV)-hydroxide. Homolytic cleavage of the Fe2O bond and transfer of HO .
to the substrate radical gives the hydroxylated product and regenerates iron(III).
The lack of evidence for a substrate radical intermediate from radical trapping
experiments has led to the more recent proposal of a concerted mechanism
involving oxygen insertion into the C2H bond, illustrated in Figure 6.31.
C H
C H
O
OH 2
O
OH 2 N N
N N
N
Fe III 1e −
Fe II Fe III
N N
R-H N N
O 2 N N
N N
Cys-S
Cys-S
Cys-S
Fe III
N N
H + 1e −
N
Cys-S
−
CO 2
CO 2
−
C OH
N
Fe III
N N
Cys-S
N
C H
OH
O
N
Fe III
N N
Cys-S
N
H 2 O
C
N
OH
N
Fe III
N
Cys-S
N
+
C
N
H
O
N
FeIV
N
Cys-S
N
+
C
N
H
O
N
Fe V
N
Cys-S
N
Figure 6.30 Mechanism for P 450 -dependent hydroxylation.