Chemistry Reference
In-Depth Information
(generally aromatic) substrates. Laccases are functionally diverse, thermostable, and environmentally friendly
catalysts: they occur naturally, use air and produce water as a by-product, and have therefore become the object
of enormous interest to biotechnologists on account of their potential applications in 'green chemistry' (
Riva,
2006
). Over 100 fungal laccases have been characterised, and to date 10 X-ray structures from nine fungal
species determined.
Figure 14.10
(
a) shows a ribbon diagram of the X-ray structure of T. versicolor laccase with the Cu atoms labeled
by type and the substrate-binding cleft highlighted in red (
Rodgers, Blanford, Giddens, Skamnioto, Armstrong, &
Gurr, 2009
)
. The detailed architecture of the four Cu sites is presented in
Figure 14.10
(
b) for three different lac-
cases. There is a variation in the redox potential of the type 1 Cu between the upper structure in
Figure 14.10
(b)
where the ligand is Phe and the lower, where it is Met of 110 mV (hence the idea of 'designer laccases').
It is suggested that dioxygen binds to the Type 3 Cu atom of the trinuclear centre. In this 'resting state' all of
the coppers will be in the oxidised
2 state (the blue colour of this intermediate confirms that the Type I copper is
certainly oxidised). Two electrons are then transferred from the substrate molecules through the Type 1 Cu to the
Type 3 coppers of the trinuclear site, where dioxygen is reduced to give a peroxo intermediate. The Cu in the type
1 site is rapidly re-oxidised by long-range intramolecular electron transfer, via a conserved His
þ
His motif to
the trinuclear Cu cluster. Two further electrons are then assimilated in a similar manner and result in splitting of
the peroxo intermediate into two hydroxyl groups. Subsequent addition of protons, provided by acidic residues in
exit channels, results in the successive release of two molecules of water. O
2
binds between the two Type 3 Cu
atoms and is reduced to water without release of reactive oxygen intermediates. In the complete catalytic cycle, the
Type I Cu must be oxidised and reduced four times. Whereas the Type 3 coppers are involved in oxygen binding
and electron transfer to the dioxygen and peroxo intermediate, the role of the Type 2 copper is postulated to be to
help to anchor the dioxygen molecule to the trinuclear cluster prior to reduction, and to temporarily bind the
hydroxyl groups arising from the reduction of the peroxo intermediate prior to their release as water molecules in
the exit channel.
Cys
e
e
(iii)
The role of Cu in Cytochrome c Oxidases
We have already discussed the terminal oxidase of the respiratory chain, cytochrome c oxidase (CcOx) in the
previous chapter. Here, we focus on the role of copper in this key metabolic enzyme. The disposition of the
different redox metal centres of bovine heart CcOx with their relative distances are represented in
Figure 14.11
.
The dimetallic Cu
A
site receives electrons directly from cyt c, and is located in a globular domain of subunit II
which protrudes into the intermembrane space (the periplasmic space in bacteria). This centre, which was orig-
inally believed to be mononuclear is in fact a di-copper site (
Figure 14.12
)
in which the coppers are bridged by two
cysteine sulfurs: each copper in addition has two other protein ligands. In the one electron reduced form, the
electron is fully delocalised between the two Cu atoms, giving rise to a [Cu
þ
1.5
.
Cu
þ
1.5
] state. The Cu
A
centre
then rapidly reduces the haem a, located some 19
˚
away (metal
metal distance) by intramolecular electron
transfer. From haem a, electrons are transferred intramolecularly to the active site haem a
3
and Cu
B
, where oxygen
binds. The Cu
B
centre (
Figure 14.13
) involves coordination of the copper atom to three His ligands and the
Fe
e
Cu
B
distance in the oxidised enzyme is 4.5
˚
, with one of the His ligands covalently linked to a nearby Tyr
residue. The mechanism of oxygen reduction has been discussed in Chapter 13.
As we saw, oxygen binds to the Fe of haem a
3
, and after cleavage of the O
e
O bond, the oxidised Cu
B
centre
binds a hydroxide ion, which is subsequently protonated, before being the first of the two water molecules to be
released from the enzyme. The coordination of the copper atom of the Cu
B
centre (
Figure 14.13
) involves three
His ligands and the Fe
e
Cu
B
distance in the oxidised enzyme is 4.5
˚
, with one of the His ligands of Cu
B
covalently linked to a nearby Tyr residue. This His
e
Tyr crosslink was first identified in the crystal structures of the
Paraccocus denitrificans and bovine heart CcO (
Figure 14.13
), and it is the source of the 4th electron for
the reduction of molecular oxygen to water by the dinuclear haem a
3
/Cu
B
. centre. The His
e
Tyr crosslink appears
to modulate the properties of the tyrosine residue, via reduction of the phenol pK
a
, facilitating proton delivery as
well as by tyrosyl radical formation.
e
