Chemistry Reference
In-Depth Information
sites occupied by displaceable water molecules on the opposite site of the facial triad available to bind exogenous
ligands, such as O
2
, substrate, and/or cofactor, thus giving the protein the flexibility with which to tune
the reactivity of its Fe(II) centre, giving rise to the extraordinary range of catalytic versatility that we have
seen above.
Figure 13.21
presents a possible mechanism for the extradiol aromatic ring-cleaving dioxygenases
(
Lipscomb, 2008
)
, illustrated by homoprotocatechuate 2,3-dioxygenase (HPCD). The active site structure with
three solvent molecules occupying the opposite side of the metal-binding triad (
Figure 13.21
(a)) is unreactive
to dioxygen. Binding of homoprotocatechuate to the resting form of the enzyme through the hydroxyl oxygens
of the catechol substrate results in the formation of a five-coordinate Fe(II) centre, which is primed for
dioxygen binding (
Figure 13.21
(b),(c)). This has two important mechanistic consequences. First, the O
2
and the
substrate are juxtaposed and presumably oriented for reaction, and second since both substrates are elec-
tronically linked through the metal, this will facilitate electron transfer from the catechol to the oxygen. This
would give both reactants radical character, allowing rapid recombination to form the alkylperoxo intermediate
(
Figure 13.21
(c),(d)) in a spin-allowed reaction. Once this intermediate is formed, fission of the O
e
O bond and
C
C bond cleavage could occur to form a 7-membered lactone. The lactone would undergo hydrolysis by the
second oxygen atom from O
2
, present bound to the metal ion, to form the open-ring product ready for release
from the enzyme (
Figure 13.21
(
e)). While the mechanism shown in
Figure 13.21
is widely accepted,
7
other
possibilities exist, including the formation of a dioxetane intermediate and the formation of a high-valent
metal-oxo species such as Fe(IV)
e
]
O, as proposed for other members of the facial triad family (
Krebs,
With an ever-increasing number of protein structures now solved, it has become clear that the 2-His-
1-carboxylate signature can be replaced by alternative metal coordination to Fe(II) or Mn(II) in a number of other
mononuclear nonhaem Fe(II) oxygenases.
There are also mononuclear nonhaem Fe(III) enzymes, including the intradiol-cleaving catechol dioxygenase
and protocatechuate 3,4-dioxygenase (PCD). PCD converts 3,4-dihydroxybenzoate (protocatechuate)
to
b
-carboxy-cis,cis-muconate, and is the best characterized of the catechol dioxygenases. The Fe(III) centre in the
isolated enzyme lies in a trigonal bipyrimidal environment coordinated by two Tyr and two His residues together
with a bound solvent molecule, probably a hydroxide. The steps in the catalytic cycle have been identified by the
crystallographic determination of the structures of PCD complexed with substrate and substrate analogs (as in the
case of HPCD (
Figure 13.22)
)
. In the enzyme
substrate complex, Tyr447 removes the second substrate proton
and is displaced as the chelated substrate complex is formed. The ternary enzyme
e
dioxygen complex
then forms. All the structures presented, except for the peroxo-model (
Figure 13.22
(e)), were generated from
X-ray crystal structures.
Lipoxygenases (LOXs), which catalyse the oxidation of unsaturated fatty acids containing the cis,cis-
1,4-pentadiene moiety to the corresponding 1-hyroperoxy-trans,cis-2,4-diene, are widely distributed in plants and
animals. The mammalian enzymes typically act on arachadonic acid to produce hydroperoxides that are
precursors of leukotrienes and lipoxins, both classes of compounds which are mediators of inflammation. The iron
active site metal is a nonhaem iron that is octahedrally coordinated by five amino acid side chains and a water or
hydroxide ligand (
Figure 13.23
)
. In plant LOXs, these residues are always three histidines, one asparagine, and the
carboxy group of the carboxy-terminal isoleucine. In mammalian LOXs, however, the iron is coordinated by four
histidines and again the carboxy-terminal isoleucine (
Andreou and Feussner, 2009
)
.
Another class of mononuclear nonhaem Fe(III) enzymes are the microbial superoxide dismutases, which have
a coordination geometry reminiscent of protocatechuate 3,4-dioxygenase, with four endogenous protein ligands,
three His and one Asp residues, and one bound water molecule (
Figure 13.24
;
Lim et al., 1997
).
e
substrate
e
7. An important advance in support of the mechanism was that HPCD remains catalytically active in the crystals, and using a poor
substrate at low O
2
concentrations, 3 different intermediates of the reaction cycle were found at high occupancy (the structures shown in
