CHAPTER

COPPER
COPING WITH

DIOXYGEN

INTRODUCTION
14
Whereas, on account of the solubility of its ferrous form, iron was widely available in the reducing
environment of the early Earth, copper which was present as highly insoluble cuprous sulphides
must have been poorly bioavailable. In contrast, once photosynthetic Cyanobacteria set off the first
major irreversible pollution of our environment with the production of dioxygen, copper became
much more bioavailable in its cupric form. Whereas the enzymes involved in anaerobic metabolism
were designed to act in the lower portion of the range of redox potentials, the presence of dioxygen
created the need for new redox systems with standard redox potentials in the range from 0 to 0.8 V,
and copper proved eminently suitable for this role. For aerobic metabolism enzymes and proteins
with higher redox potentials came to be utilized, to take advantage of the oxidizing power of dioxy-
gen. However, whereas the early evolution of life was the ‘iron age,’ it is clear that the subsequent
‘copper age,’ was in reality an ‘iron-copper age,’ where both metals were involved together. This is
well illustrated by ceruloplasmin, the principal copper binding protein in serum, which plays an
important role in iron metabolism, and by the terminal oxidase of the mitochondrial respiratory
chain, cytochrome c oxidase which requires both haem iron and copper for its activity.
Copper is present in a large number of enzymes, many involved in electron transfer, activation
of oxygen and other small molecules like oxides of nitrogen, methane and carbon monoxide, super-
oxide dismutation, and even, in some invertebrates, oxygen transport.

COPPER CHEMISTRY AND BIOCHEMISTRY

The routinely encountered oxidation states are Cu(I) and Cu(II), and as with iron, the reduced form
can catalyse Fenton chemistry with hydrogen peroxide. Cu(I) can form complexes with coordination
number 2, 3 or 4, while Cu(II) prefers coordination numbers 4, 5 or 6. Whereas four-coordinate com-
plexes of Cu(II) are square planar, the corresponding Cu(I) complexes are tetrahedral. Among the
divalent elements of the transition series, Cu(II) forms the most stable complexes. In terms of the
HSAB classification Cu(II) is ‘hard,’ while Cu(I) is ‘soft’ underlined by its preference for sulphur
ligands. Both forms have fast ligand exchange rates. It appears that throughout the living world intra-
cellular concentrations of ‘free’ copper are maintained at extremely low levels, most likely because
intracellular copper metabolism is characterized by the use of copper chaperone proteins to transport
copper towards their target proteins (cytochrome oxidase, superoxide dismutase and the multicopper
oxidases whose copper is inserted in the Golgi apparatus).
In Cu-containing proteins, three types of Cu centres (Fig. 14.1A) were classified on the basis of
their visible, UV and EPR spectra by one of the pioneers of Cu biochemistry, Bo Malmström
Biological Inorganic Chemistry. DOI: http://dx.doi.org/10.1016/B978-0-12-811741-5.00014-X
© 2019 Elsevier B.V. All rights reserved. 405
406 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

(A)
Cys L L
His L
Cu L L L L L
Cu Cu L
Cu
His L L L O
R L
R

(B)

Electron transfer Oxygen reduction to water

Blue copper CuA

(Type 1)
Trinuclear Haem-copper
oxidase
Oxygen activating

Amine Galactose Denitrification

oxidase oxidase

Nitrous Nitrite
oxide reductase
11 Å reductase (Type 2)

Coupled
Noncoupled
binuclear
binuclear
(Type 3)

FIGURE 14.1
(A) Classification of Cu sites. From left to right: Type 1, Type 2, Type 3. (B) Reactions catalysed by dinuclear
Cu enzymes.
Reprinted from Solomon, E.I., Heppner, D.E., Johnston, E.M., et al., 2014. Copper active sits in biology. Chem. Rev. 114,
3659
3853. Copyright 2014 with permission American Chemical Society.

(Malkin and Malmström, 1970). Types 1 and 2 centres have a single Cu atom which has an intense
blue colour in Type 1 centres, but the single Cu atom in Type 2 centres is almost colourless. In
contrast, Type 3 centres have a di-Cu centre which is EPR-silent:
Type 1 Cu(II): intense blue optical absorption band (λmax B 600 nm; ε . 3000 M21/cm); EPR
spectrum with an uncommonly small hyperfine splitting in gΠ region
Type 2 Cu(II): weak absorption spectrum; EPR spectrum characteristic of square planar Cu(II)
complexes
Type 3 Cu(II): coupled dinuclear copper centre; strong absorption in the near UV (λmax B 330 nm);
no EPR spectra, the two coppers are antiferromagnetically coupled.
Since this classification was proposed, it has become clear that Cu coordination in Cu proteins
is more complex than was originally thought. Copper plays a wide variety of roles in biology,
which mostly involve electron transfer (ET), O2 binding, activation, and reduction, NO22 and N2O
reduction, and substrate activation. Fig. 14.1B presents some representative active sites found in Cu
proteins, including the three classical centres (Solomon et al., 2014). The Type 1 copper ions are
normally coordinated in a distorted tetrahedral centre (Fig. 14.1B) by three strong ligands, a
 TYPE 1 BLUE COPPER PROTEINS
ELECTRON TRANSPORT 407

cysteine and two histidines, and one weaker ligand such as methionine sulphur or a nitrogen or
oxygen donor. Type 2 centres, for example in N2O reductase, have typically a square planar or
tetragonal geometry around the Cu with nitrogen or oxygen ligands. Type 3 coppers, as in the cou-
pled dinuclear Cu enzymes, are usually each coordinated by three histidines, with a bridging ligand
such as oxygen or hydroxyl anion. For an excellent recent review of copper active sites in biology
see Solomon et al. (2014).

TYPE 1 BLUE COPPER PROTEINS
ELECTRON TRANSPORT

The blue copper proteins are so-called on account of their intensely blue colour which is derived
from the strong Cys-Cu21 charge transfer band at around 620 nm in the electronic absorption spec-
trum (for reviews see Solomon and Hadt, 2011; Solomon et al., 2014). These mononuclear Type 1
Cu centres with a highly covalent Cu(II)
S(Cys) bond function exclusively as electron transporters,
as do the dinuclear CuA sites which have a Cu2(S(Cys))2 core with a Cu
Cu bond that keeps the
site delocalized (Cu(1.5)2) in its oxidized state, found in cytochrome c oxidases of the respiratory
chain. Type I Cu sites are found in mobile electron transfer proteins like azurin and plastocyanin,
as well as in more complex enzymes which contain multiple functional sites, where they serve to
deliver to, or take up electrons from, a catalytic site. An intriguing question is how Cu can function
in rapid electron transfer reactions1 when Cu(I) and Cu(II) have such drastically different prefer-
ences in coordination geometry. As we pointed out above, four-coordinate Cu(II) complexes are
square planar, while the corresponding Cu(I) complexes tend to be more tetrahedral. When the
Type I copper centre in plastocyanin was first characterized by X-ray crystallography (Fig. 14.2A
and B) it revealed a copper binding site which was virtually the same in the apoprotein as in the
copper-containing protein, whether the copper was Cu(I) or Cu(II). In other words, the protein
imposes a binding site geometry on the metal, which is in reality closer to that of Cu(I) than of
Cu(II), such that Cu(II) has no possibility to rearrange2 towards its preferred geometry. The copper-
coordination site (Fig. 14.2B) is highly distorted with two His nitrogen and one Cys sulphur donors
lying almost in a plane with the metal ion, together with a long, out of the plane axial bond,
between the sulphur of a Met residue and the Cu atom. This structure in many ways is the convinc-
ing proof of the idea that proteins can fine tune the properties of bound metal centres, imposing
what Vallee and Williams (1968) called the ‘entatic state,’ which is ‘closer to a transition state that
to a conventional, stable molecule.’ The entatic state, or strain induced by metal binding to pro-
teins, both on the metal and the protein itself, is a useful concept for explaining the generation of
metal sites in electron transfer proteins, like the blue copper proteins, which are designed for rapid
electron transfer. The trigonal pyramidal structure with three strong equatorial ligands (one Cys and

1
An important concept in enzymology is that while catalysis involving bond cleavage and formation requires conforma-
tional change, and is relatively slow (maximum B108 s21), electron transfer is much more rapid (1012 s21), which does
not allow much time for conformational change!
2
Whereas enzyme catalysis invariably involves movement and conformational change, in electron transfer, which is
orders of magnitude faster, there is no time for movement.
408 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

(B) His 87

(A)
2.90Å Met 92
2.10Å

2.13Å
2.04Å

His 37 Cys 84

(C) Ground state wave function

(x, y plane)
4% NHis

42% Cu dx 2-y 2 s 38% SCys pπ

4% NHis

FIGURE 14.2
(A) X-ray structure of poplar plastocyanin from poplar leaves, as a ribbon diagram with its ligands highlighted.
PDB code 1PLC. (B) X-ray structure of the Cu site in poplar plastocyanin. (C) Contour of plastocyanin ground
state wave function (RAMO) calculated by SCF-Xα-SW adjusted to spectroscopic data.
(B and C) Reprinted from Solomon, E.I., 2006. Spectroscopic methods in bioinorganic chemistry: blue to red to green copper sites.
Inorg. Chem. 45, 8012
8025. Copyright 2006 by permission of American Chemical Society.

two His) which the protein imposes provides a favourable geometry for both cuprous and cupric
oxidation states, and facilitates rapid electron transfer. Initially, application of molecular orbital the-
ory gave description of the ground state of the blue Cu site which is shown in Fig. 14.2C in the xy
plane defined by Cu, Cys S and the two His N. Modern DFT calculations give the same description
of the ground state of the blue Cu site, highly covalent with the covalency delocalized into the pπ
orbital of the thiolate sulphur.
A series of blue-copper-related proteins which all have the same Cys, two His, and Met ligand
set vary from blue in plastocyanin to green in some nitrite reductases (LaCroix et al., 1998). From
superposition of the crystal structures of plastocyanin, cucumber basic protein, and nitrite reductase
in Fig. 14.3A, it is observed that, in going from the blue to the green copper, the Met S
Cu bond
 TYPE 1 BLUE COPPER PROTEINS
ELECTRON TRANSPORT 409

(A) CBP SMet

NiR
Pc Axial

SMet
2.82 Å Cu
N N
2.55 Å 2.1Å 2.2Å
SCys
Cu
NHis NHis

(B)

Blue copper Green copper Red copper

π σ
σ

d–d
σ d–d

π d–d

30,000 20,000 10,000 30,000 20,000 10,000 30,000 20,000 10,000

Energy (cm–1)

FIGURE 14.3
(A) Continuum of coupled tetragonal distortions in a series of perturbed blue copper proteins show contraction of
the Cu
S(Met) bond and εu-like mode tetragonal Jahn
Teller distortion. (B) Absorption spectra of blue, green
and red Cu centres.
Reprinted from Solomon, E.I., 2006. Spectroscopic methods in bioinorganic chemistry: blue to red to green copper sites.
Inorg. Chem. 45, 8012
8025. Copyright 2006 by permission of American Chemical Society.

length decreases, the Cu
S Cys bond length increases, and there is a tetragonal rotation of the
S
Cu
S plane into the N
Cu
N plane, indicating a Jahn
Teller distortion in the green site
(Solomon, 2006). This large change in geometric and electronic structures despite having the same
ligand set clearly demonstrates that the protein can tune the structure of the metalloprotein active
site. By mutating the relatively strong Met ligand in the green site in nitrite reductase (Basumallick
et al., 2003), the site goes from having its σ charge transfer being dominant in the green site to the
π charge transfer being the dominant feature of the resultant blue site (Fig. 14.3B, left). These stud-
ies have been extended to the red copper site in nitrosocyanin (Basumallick et al., 2005). For this
class of sites, the relatively weak Met axial ligand is replaced by a strong His ligand and the strong
His equatorial ligand is replaced with a weak Glu carboxylate ligand (Lieberman et al., 2001). This
effectively rotates the equatorial plane of the copper into the Cu
S
C plane, and the copper binds
410 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

an additional equatorial water ligand. For the red copper site (Fig. 14.3B right), the thiolate is now
σ-bonding to the copper (thus, the σ-charge transfer transition is dominant) and the additional water
donor ligand raises the energy of the d manifold, which shifts the σ charge transfer up in energy
relative to this transition in the green copper site (Solomon, 2006).
A number of other Cu electron transfer proteins which contain Type 1 Cu centres (azurin,
ceruloplasmin, laccase, nitrite reductase, rusticyanin and stellacyanin) are known. They all have
three coordination positions contributed by 2 His and one Cys, similar to the copper-coordination
chemistry in plastocyanin
yet they span a range of redox potential from less than 200 mV to at
least 800 mV. One of the challenges for the future will be to determine what programmes this fine
tuning of redox properties of Type 1 copper centres.

COPPER-CONTAINING ENZYMES IN OXYGEN ACTIVATION

AND REDUCTION
There has been enormous activity in the field of Cu(I)-dioxygen chemistry in the last 30 years,
with our information coming from both biochemical/biophysical studies and to a very important
extent from coordination chemistry. This has resulted in the structural and spectroscopic characteri-
zation of a large number of Cu dioxygen complexes, some of which are represented in Fig. 14.4
(Himes and Karlin, 2009). The O2-reactive centres in Cu enzymes can be either mononuclear
(Type 2), dinuclear (Type 3) or trinuclear (Types 2 and 3). We will discuss mononuclear and dinuc-
lear Cu proteins in this section: the trinuclear sites will be included in our discussion of multicopper
oxidases.

μ-1,2-Peroxo-Cu2II μ
μ-η2:η2-Peroxo-Cu2II μ
bis-μ-Oxo-Cu 2
III

2+ 2+ O 2+
O
Cu II O CuII Cu II Cu III Cu III
O Cu II O
Electrophilic O
Nucleophilic Aromatic hydroxylation H• abstraction

End-on, η1 Side-on, η2 End-on, η2 End-on, η1

superoxo superoxo-CuII peroxo-CuIII hydroperoxo
1+
Cu II O
1+ O 1+ O 1+ Cu II O
O Cu II Cu III OH
O O
Electrophilic ?

FIGURE 14.4
Crystallographically or spectroscopically characterized Cu
O2 adduct structures found in small molecule ligand-
Cu complexes, with characteristic reactivity patterns (green).
From Himes, R.A., Karlin, K.D., 2009. Copper
dioxygen complex mediated C
H bond oxygenation: relevance for particulate
methane monooxygenase (pMMO). Curr. Opin. Chem. Biol. 13, 119
131. Copyright 2009 with permission from Elsevier.
 TYPE 2 COPPER PROTEINS 411

TYPE 2 COPPER PROTEINS

Cu enzymes involved in O2 activation and reduction, have mostly been thought to involve at least
two electrons in order to overcome spin-forbiddenness and the low potential of the one electron
reduction to superoxide (Fig. 14.2) (Koppenol et al., 2010). Since the Cu(III) redox state has not
been observed in biology, this requires either more than one Cu centre or one copper and an addi-
tional redox-active cofactor. However, there is a paradox concerning Type 2 mononuclear Cu sites
which bind dioxygen. In order to activate O2, unless they go to the unlikely Cu(III) state, they can-
not supply the 2 electrons which are required to convert the cupric-superoxo complex to the more
likely oxygen donor cupric-peroxo. There are two possible solutions to this dilemma.
The first is illustrated by galactose oxidase which converts galactose 1 O2 to the corresponding
aldehyde 1 H2O2. Originally this enzyme was thought to involve Cu(III). However, galactose oxi-
dase turns out to be a free radical metalloenzyme (Rogers and Dooley, 2003) and solves the prob-
lem via a novel metallo-radical (Fig. 14.5). There is an additional cofactor at the active site, a
Cys
Tyr covalently linked radical centre bound through the phenoxyl oxygen and antiferromagnet-
ically coupled to the copper(II) centre, which is Cu catalysed and is formed in the first turnover of
the protein. The redox potential for this cysteinated tyrosine ligand has been measured to be
0.45 V, and is thus stabilized relative to a free tyrosine radical (0.95 V) through the covalent

Trp290

Cys228
Tyr272
Cu Phe227
His581

His496

Tyr495

FIGURE 14.5
The Cu ligands of galactose oxidase (Tyr 272, Tyr 495, His 496 and His 581), the Cys228 which forms the
thioether bond to Tyr 272, the tryptophan that stacks over it (Trp 290) and Phe 227.
From Rogers, M.S., Dooley, D.M., 2003. Copper-tyrosyl enzymes. Curr. Opin. Chem. Biol. 7, 131
138.
Copyright 2003, with permission from Elsevier.
412 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

linkage with the cysteine residue in the ortho-position and through π
π stacking with a nearby
tryptophan residue (W290). As we saw in Chapter 13, Iron: Essential for Almost All Life and will
discuss later in this chapter, this strategy is also adopted by cytochrome c oxidase, which also uses
a tyrosine radical.
The second solution involves a class of dinuclear copper proteins which, unlike the Type III
dinuclear Cu proteins, have two coppers at a distance of around 11 Å with no bridging ligands such
that they are not electronically coupled. These enzymes, which in addition require ascorbate, carry
out activated C
H bond hydroxylation for the synthesis of physiologically important neurotrans-
mitters and hormones: the eukaryotic proteins dopamine β-monooxygenase (DβM) and peptidylgly-
cine α hydroxylating monooxygenase (PHM) and the recently identified insect tyramine
β-monooxygenase (TβM) (Fig. 14.6). DβM, a glycoprotein found in mammalian neurosecretory
vesicles of the adrenal gland, carries out the catalytic conversation of dopamine to noradrenaline
in the catecholamine biosynthetic pathway(540). DβM, a glycoprotein found in mammalian neuro-
secretory vesicles of the adrenal gland, carries out the catalytic conversation of dopamine to nor-
adrenaline. TβM catalyses the hydroxylation of tyramine to octopamine, an insect neurotransmitter,
responsible for a wide variety of physiological functions. Both enzymes contain two Cu atoms and
in the case of PMH (Fig. 14.7; Rosenzweig and Sazinsky, 2006), it has been established that dioxy-
gen binds to one of the two Type 2 copper atoms in an ‘end-on mode.’ A copper
dioxygen com-
plex has been trapped by freezing crystals of the enzyme which had been soaked with a slowly

DβM O2 + AscH2
H2O + AscH–
+ 2 H+
H H H OH
NH3+ NH3+

HO HO
OH OH
Dopamine Norepinephrine
O2 + AscH2
H2O + AscH–
TβM + 2 H+ H OH
H H NH3+
NH3+

HO
HO
Tyramine Octopamine
O2 + AscH2
PHM H2O + AscH–
+ 2 H+
O H H O H OH O O
RHN RHN PAL RHN H
N – N – N
COO COO H COO–
R′ H R′ H R′ H

FIGURE 14.6
Reactions catalysed by noncoupled dinuclear Cu enzymes.
Reprinted from Solomon, E.I., Heppner, D.E., Johnston, E.M., et al., 2014. Copper active sits in biology. Chem. Rev. 114,
3659
3853. Copyright 2014 with permission American Chemical Society.
 TYPE 2 COPPER PROTEINS 413

IYT

*
H2O H108
H172
CuB O2

CuA
M314 H242

H244

H107

FIGURE 14.7
Active site of PMH with coordinated O2. The CuA and CuB sites are linked by a water molecule and the substrate
analogue IYT. The position of the hydroxylated Cα is denoted by an asterisk.
From Rosenzweig, A.C., Sazinsky, M.H., 2006. Structural insights into dioxygen-activating copper enzymes. Curr. Opin. Struct. Biol.
16, 729
735. Copyright 2006, with permission from Elsevier.

reacting substrate, N-acetyl-diiodo-tyrosyl-D-threonine (IYT) in the presence of oxygen and ascor-

bate. Electron density was observed that was best modelled as O2 within coordinating distance of
the catalytic Cu in the precatalytic complex (Fig. 14.7), replacing the solvent molecule observed in
all other PHM structures. Bound substrate has been proposed to mediate electron transfer between
the two Cu centres, each of which contributes one electron for O2 reduction.
A mechanism for PHM and DβH has been proposed based on kinetic studies (Klinman, 2006)
(Fig. 14.8). Following initial reduction by ascorbate, substrate and O2 bind to the reduced enzyme
to form a ternary complex, triggering initial O2 activation involving electron transfer from the
Type 2 Cu atom, to form a Cu-superoxo intermediate coupled with abstraction of a proton from the
substrate. The following step involves one of two pathways: (1) hydroxyl transfer to form product
(O
O bond fission) by electron transfer from CuM to CuH, or (2) electron transfer which occurs
with protonation and heterolytic 0
0 cleavage followed by substrate radical coupling to a Cu
O.
species. Both pathways result in the oxidized enzyme with the product bound either to the protein
or to CuM. Dissociation of the product is the rate-limiting step in both pathways. The mechanism
of long-range electron transfer between the two Cu sites remains to be established.
414 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

O O
CuM(II)-OH CuM(I) CuM(II) CuM(II)
kred + SubH + O2 O kC-H OH
SubOH Sub•
− SubH − O2
CuH(II) 2 Asc 2 AscH CuH(I) CuH(I) CuH(I)
Pathway 1
koff
CuM(II) OH CuM(II) O• CuM(II)
O
ket OH
SubOH Pathway 2
SubOH SubOH Sub•
+
+H
CuH(II) CuH(I) CuH(I)

CuM(II) O CuM(II) O• CuM(II) O

koff ket OH
Sub
Sub• Sub•
H2O H+
SubOH H2O CuH(II) CuH(II) CuH(I)

FIGURE 14.8
Mechanistic summary of the noncoupled dinuclear Cu enzymes.
Reprinted from Solomon, E.I., Heppner, D.E., Johnston, E.M., et al., 2014. Copper active sits in biology. Chem. Rev. 114,
3659
3853. Copyright 2014 with permission American Chemical Society.

DINUCLEAR COPPER PROTEINS

If two Cu(II) atoms, each with S 5 1/2, are closer than B6 Å from one another, their electron spins
will dipole
dipole couple. This produces an S 5 1 triplet EPR signal that has characteristic spectral
features. Haemocyanin, tyrosinase and catechol oxidase all belong to this Type 3 Cu protein family,
characterized by two closely spaced antiferromagnetically coupled copper ions. However, while hae-
mocyanin is an O2 carrier protein, catechol oxidase, which converts catechols to the corresponding
o-quinones, and tyrosinase, which, in addition to converting catechols to quinones also hydroxylates
monophenols (e.g., tyrosine), are both enzymes. While both tyrosinase (Ty) and catechol oxidase
(CaOx) perform the two-electron oxidation of o-diphenols to quinones (referred to as two-electron
oxidase or catecholase activity), only Ty can catalyse the conversion of phenols to diphenols. Ty
catalyses the conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine (L-DOPA) and the subse-
quent oxidation of L-DOPA to L-DOPAquinone (3-(3,4-dioxocyclohexa-1,5-dien-1-yl)-L-alanine).
Fig. 14.9 presents the arrangements of the domains within the subunit structures of two haemocya-
nins from the haemolymph of the horse shoe crab (Limulus polyphemus) and the North Pacific giant
octopus (Octopus dofleini), a streptococcal tyrosinase and catechol oxidase from sweet potato (Decker
et al., 2007). The location of the copper centres is shown as are the amino acid residues which block
access to the catalytic site (blocking residues). The structures of the oxy forms at high resolution
confirms, as predicted from model compounds, that the dioxygen molecule is bound in a peroxo-
dicopper(II) complex, corresponding to the μ
η2:η2-peroxo, illustrated in Fig. 14.4. Each copper
atom is ligated to the protein matrix by three histidine residues (Fig. 14.9). Oxygen binding induces a
 DINUCLEAR COPPER PROTEINS 415

(A) (B)

(C) (D)

(E)
CuA CuB
F49
LimulusHC N- -C
L2830
OctopusHC
Y98
StreptTyr + MelC1
L439
IpomoeaCO

FIGURE 14.9
Arrangement of the domains within the subunit structures of different Type 3 copper proteins. (A) Limulus
polyphemus haemocyanin, (B) Octopus dofleini FU g haemocyanin, (C) Streptomyces castaneoglobisporus
tyrosinase, (D) Ipomoea batatas catechol oxidase. (E) Sequence comparison [same colour code: domain I (green),
domain II (red), domain III (cyan)]; copper centres are indicated by the hatched blocks, the blocking residues are
shown as black bars. In all cases, the domains are parts of the subunit with the exception of Streptomyces
tyrosinase, where an associated caddie protein (MelC1) provides Y98.
From Decker, H., Schweikardt, T., Nillius, D., Salzbrunn, U., Jaenicke, E., Tuczek, F., 2007. Similar enzyme activation and catalysis
in hemocyanins and tyrosinases. Gene 398, 183
191. Copyright 2007, with permission from Elsevier.

change in the valency of the copper atoms, which are in the Cu(I) state in the deoxy form, but
become Cu (II) upon oxygen bonding. This change results in the characteristic blue colour developed
by all Type 3 copper proteins upon oxygenation. The active sites of catechol oxidase, tyrosinase and
haemocyanins with oxygen bound to the copper atoms exhibit similar spectroscopic properties with
respect to UV-resonance Raman, X-ray absorption and UV/VIS spectroscopy.
416 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

This similarity in spectral properties implies that haemocyanins should also have catalytic activ-
ity. From the available body of experimental data it is clear that the distinction between the two
major functions
oxygen transport and enzymatic activity
is determined by the presence or
absence of a protein domain covering the active site. In the case of tyrosinase and catechol oxidase,
inactive proenzyme forms are activated by removal of an amino acid which blocks the entrance
channel to the active site (indicated by the black bar in Fig. 14.9). Haemocyanins behave as silent
inactive enzymes but can be activated in the same way if the blocking amino acid is removed. In
arthropods, like crabs, this is located in the N-terminal domain of a subunit whereas in molluscs,
like octopus, it is in the C-terminal domain of a functional unit.
As pointed out earlier, in addition to its natural substrate tyrosine, tyrosinases also hydroxyl-
ate other monophenols in the ortho-position to diphenols, followed by oxidation to ortho-
quinones (monophenolase activity). COs, in contrast, can only perform the latter reaction
(diphenolase activity). While the active sites of tyrosinase and of catechol oxidase are very
similar, there is a thioether bond to one of the His ligands of CuA (Cys92, His 109) in catechol
oxidase (Fig. 14.10). It had been suggested that the hydroxylation of monophenol substrates on
the o-position requires a distortion of the Cu2O2 centre and consequent reorientation of the sub-
strate (Deeth & Diedrich 2010; Rolff et al., 2011). This reorientation would not be possible in
catechol oxidase, because active site movement is restricted by this thioether bond (Goldfeder
et al., 2014). Based on the crystal structures of tyrosinase from Bacillus megaterium (TyrBm)
with and without bound mono- or diphenolic substrates it has been suggested (Goldfeder et al.,
2014) that a highly conserved Glu and an Asn are required to properly orient and activate a
conserved water molecule in order to abstract a proton from the monophenol (Fig. 14.11) The
deprotonation of a phenolic substrate in the tyrosinase cycle is illustrated in Fig. 14.12. Mutation
to introduce an Asn into a polyphenoloxidase, which exhibits only diphenolase activity, can trans-
form it into a tyrosinase (Solem et al., 2016).

MULTICOPPER OXIDASES
An important family of multicopper enzymes couple the reduction of O2, to H2O accompanied by
oxidation of a substrate. They include ascorbate oxidase, ceruloplasmin, Fet3, hephaestin and lac-
case, and contain at least four copper ions. The four Cu ions are distributed between one Type 1
blue copper site, one Type 2 site and one Type 3 copper site. The blue Type 1 site is usually
located some 12
13 Å distant from a trinuclear site which has the two Type 3 coppers, linked by a
bridging oxygen and one Type 2 copper. We illustrate this class of oxidases with laccase which
catalyses the four-electron reduction of O2 to water, coupled with the oxidation of small organic
(generally aromatic) substrates. Laccases are functionally diverse, thermostable and environmen-
tally 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.’ Over 100 fungal laccases have been characterized, and numerous
X-ray structures from different fungal species determined.
The active site structure of resting oxidized (RO) ascorbate oxidase is depicted in Fig. 14.13A,
while the global structure of laccase is in Fig. 14.13A (Rodgers et al., 2010). The redox potential of
 MULTICOPPER OXIDASES 417

(A)
Gly204

His194

CuA
CuB

His54
Glu182

(B)
Phe261

His244
His88

CuA
Cys92
CuB

His109

Glu236

FIGURE 14.10
The active site of oxy-tyrosinase (A) from the bacteria S. castaneoglobisporus and a structure of met-catechol
oxidase from I. batatas (B).
Reprinted from Solomon, E.I., Heppner, D.E., Johnston, E.M., et al., 2014. Copper active sits in biology. Chem. Rev. 114,
3659
3853. Copyright 2014 with permission American Chemical Society.

the Type 1 Cu can be varied by about 110 mV, hence the idea of ‘designer laccases’ (Rodgers
et al., 2010; Riva, 2006), which could be adapted to specific biotechnological purposes. The RO
form of the enzyme has four oxidized coppers including a hydroxide-bridged T3 centre (Jones and
Solomon, 2015; Augustine et al., 2010), but does not really figure in the generally accepted
418 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

Phenolate

HA1

HB2

2.1 Å p-tyrosol
CuA
HA2 CuB
Asn240
2.9 Å
HOH112 2.8 Å

2.7 Å HB3
HB1

HA3 Glu235

−10 0 10

FIGURE 14.11
Deprotonation and approach of p-tyrosol to the active site in the refined VvPPOg structure (PDB-ID 2P3X). The
incoming tyrosol coordinates as phenolate to CuA at a distance of 2.1 Å. The proton of the phenolic substrate is
transferred over a minimum distance of 2.9 Å to the conserved water molecule bound by Glu235 and Asn240.
The position of the p-tyrosol was adopted from the PPO structure of B. megaterium cocrystallized with p-tyrosol
(Goldfeder et al., 2014) (PDB-ID 4P6T).
From Solem, E., Tuczek, F., Decker, H., 2016. Tyrosinase versus catechol oxidase: one asparagine makes the difference. Angew.
Chem. Int. Ed. Engl. 55, 2884
2888. Copyright 2016, with permission from John Wiley and Sons.

catalytic cycle (Solomon et al., 2014) (Fig. 14.14). The starting point of the cycle is the fully
reduced enzyme. Dioxygen binds, and is reduced to the peroxy intermediate P1 by two electrons
from the T2 and T3β Cu’s. Two further electrons are then rapidly transferred to P1, one fromT3α
Cu of the trinuclear site and one from the T1 Cu, in a similar manner and result in O
O bond split-
ting and formation of N1 intermediate with all 4 Cu’s oxidized, and the O atoms reduced to the
level of water. The presence of electrons from a reducing substrate and of protons, provided by
acidic residues in the exit channels, results in the successive release of two molecules of water, and
fast reduction of N1 back to the fully reduced enzyme. Alternatively, with no excess reductant, NI
slowly decays to the thermodynamically more stable RO enzyme. Electrons derived from substrate
are transferred to the trinuclear Cu cluster from the Cu in the Type 1 site by long-range intramolec-
ular electron transfer, via a conserved His
Cys
His motif.
 THE ROLE OF CU IN CYTOCHROME C OXIDASES 419

O His
His His
I I
His CuA CuB
His
O His
His His δ+
II
O His
II
CuA CuB O
His His H H
O
His
H H2O O O
Θ NH2
H
O R Asn 241
Oδ
+
H H Glu 239
+ Phenol
O O + O2
Θ NH2
O R Asn 241
Glu 239
His His
II
O His
II
CuA CuB
His His
O His His O
His O His
II II
His
CuA CuB H O
His O His
δ–
3.5 –4.5 Å O
H 3Å H H 3Å
Oδ
+
O O
H H Θ NH2
O R Asn 241
O O
Θ NH2 Glu 239
O R Asn 241
Glu 239

FIGURE 14.12
Deprotonation of a phenolic substrate in the tyrosinase cycle.
From Solem, E., Tuczek, F., Decker, H., 2016. Tyrosinase versus catechol oxidase: one asparagine makes the difference. Angew.
Chem. Int. Ed. Engl. 55, 2884
2888. Copyright 2016, with permission from John Wiley and Sons.

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, notably the different intermediates on the pathway to reduction of
O2. Here we focus on the role of copper in this key metabolic enzyme. The disposition of the
different metal centres of bovine heart CcOx is represented in Fig. 14.15 (Brunori et al., 2005).
The four ET steps relevant to the reduction of the O2 binding site are indicated below:
Cytc - CuA - haema - haema3 - CuB
The overall structure of bovine heart CcO consists of 13 subunits (Fig. 14.16A). Of the four
metal binding sites, a3 and CuB, and the third metal centre, haem a, are in subunit I (yellow),
420 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

(A) (B)
H D94
O
O H
O
H
H O H416 L417 Type 2
NH 2 × type 3
H83 H81 N H
Cu N
H N T2
N HN
N N H418 Type 1

T3α Cu Cu T3β
F82 O
N H483 Binding pocket
N H
N
H126 N N
N N H
H485 H
H
N C484
H
S127 H128
H O
Cu
T1
H

O
E487

FIGURE 14.13
Structural insight into multicopper oxidases from X-ray crystallography. (A) Active site structure of a resting
oxidized ascorbate oxidase. (B) A ribbon model of the X-ray crystal structure from T. versicolor Lac1 (PDB 1 kya)
with the coppers (orange circles) labelled by type and the organic substrate binding cleft highlighted in red.
(A) From Augustine, A.J., Kjaergaard, C., Qayyum, M., et al., 2010. Systematic perturbation of the trinuclear copper cluster in the
multicopper oxidases: the role of active site asymmetry in its reduction of O2 to H2O. J. Am. Chem. Soc. 132, 6057
6067.
Copyright 2010 with permission from American Chemical Society. (B) From Rodgers, C.J., Blanford, C.F., Giddens, S.R., Skamnioto, P.,
Armstrong, F.A., Gurr, S.J., 2010. Designer laccases: a vogue for higpotential fungal enzymes? Trends Biotechnol. 28, 63
72.
Copyright 2007, with permission from Elsevier.

whereas the fourth metal-domain-containing CuA is positioned in subunit II (green). The four metal
sites and their ligation are shown in Fig. 14.16B. The dimetallic CuA site, located in a globular
domain of subunit II which protrudes into the intermembrane space, receives electrons directly
from cyt c. This centre, which was originally believed to be mononuclear is in fact a dicopper site
(Fig. 14.16B) in which the coppers are bridged by two cysteine sulphurs: each copper in addition
has two other protein ligands. In the one electron reduced form, the electron is fully delocalized
between the two Cu atoms, giving rise to a [Cu11.5 . . . Cu11.5] state. The CuA centre then rapidly
reduces the haem a, located some 19 Å away (metal
metal distance) by intramolecular electron
transfer. From haem a, electrons are transferred intramolecularly to the active site haem a3 and
CuB, where oxygen binds.
The mechanism of oxygen reduction has been discussed in Chapter 13, Iron: Essential for
Almost All Life, and as we saw there, oxygen binds first to the Fe of haem a3, and after cleavage
 THE ROLE OF CU IN CYTOCHROME C OXIDASES 421

Fully reduced Peroxide intermediate (PI)

D94
H2O H2O
−
O
T2 Cu1+ O2 Cu2+
O
O
1+ O
T3 Cu1+ Cu1+ Cu Cu2+
k = 2 × 106 M−1s−1

T1 Cu1+ Cu1+

H2O
FAST
k > 560 s−1
Slow 2H2O
4e−, 2H+
4e−, 4H+
D94
OH OH
OH
Cu2+ Cu2+ O
H 2O
O
Cu2+ Cu2+ Cu2+ Cu2+
O −1
k = 0.05 s
O
H H

Cu2+ Cu2+

Resting oxidized (RO) Native intermediate (NI)

FIGURE 14.14
Mechanism of O2 reduction by MCOs. Red arrows show steps in the catalytic cycle. Black arrows show reduction
of resting enzyme to enter the catalytic cycle and decay of the native intermediate which terminates catalysis.
Adapted from Augustine, A.J., Kjaergaard, C., Qayyum, M., et al., 2010. Systematic perturbation of the trinuclear copper cluster in
the multicopper oxidases: the role of active site asymmetry in its reduction of O2 to H2O. J. Am. Chem. Soc. 132, 6057
6067.
Copyright 2010 with permission from American Chemical Society.

of the O
O bond, the oxidized CuB centre binds a hydroxide ion, which is subsequently proton-
ated, before being the first of the two water molecules to be released from the enzyme. The coordi-
nation of the copper atom of the CuB centre (Fig. 14.16B) involves three His ligands and the
Fe
CuB distance in the oxidized enzyme is 4.5 Å, with one of the His ligands of CuB (His 240)
covalently linked to a nearby Tyr residue (Tyr 244). This His
Tyr crosslink was first identified in
the crystal structures of the Paraccocus denitrificans and bovine heart CcO (Fig. 14.17), and it is
the source of the fourth electron for the reduction of molecular oxygen to water by the dinuclear
haem a3/CuB centre. The His
Tyr crosslink appears to modulate the properties of the tyrosine resi-
due, via reduction of the phenol pKa, which helps in proton delivery and by tyrosyl radical
formation.
422 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

Intermembrane
CuA space

22 Å
19 Å

CuB

14 Å Heme a3

Heme a

Mitochondrial
matrix

Heme a3

H376

H378
CuB
F377

Heme a
H61

FIGURE 14.15
Schematic representation of the redox metals in bovine heart CcO with their relative distances.
From Brunori, M., Giuffrè, A., Sarti, P., 2005. Cytochrome c oxidase, ligands and electrons. J. Inorg. Biochem. 99, 324
336.
Copyright 2005 with permission from Elsevier.

SUPEROXIDE DISMUTATION IN HEALTH AND DISEASES

Superoxide is generated by a number of enzymes in the course of their reaction cycles, but by far
the greatest production of superoxide anion and the reactive oxygen species that can be derived
from it, is the respiratory chain within the mitochondria. Superoxide dismutases (SODs) lower the
levels of superoxide by catalysing the transformation of two superoxide ions into dioxygen and
hydrogen peroxide. CuZnSOD is widely distributed, located in the periplasmic space in bacterial
cells and in both the cytosol and the mitochondrial intermembrane space in eukaryotic cells. The
reaction is a two-step process in which a molecule of superoxide reduces the oxidized (Cu21) form
 SUPEROXIDE DISMUTATION IN HEALTH AND DISEASES 423

(A) (B)
CuA
H161
M207

CuA C196 C200

E198
CuB E204
Heme a H291 CuB
Heme a3
H290
H61
H240

Y244
H378 H376

Heme a Heme a3

FIGURE 14.16
Cytochrome c oxidase structure from bovine heart. (A) Overall structure where the 13 subunits are shown in
different colours [subunit I (yellow), subunit II (green), subunit III (red), subunit IV (dark blue)]. (B) Expanded
view of the redox-active metal centres. Figure generated from PDB-ID 2Y69 (Qin et al., 2009) coordinates using
VMD.
Reprinted from Solomon, E.I., Heppner, D.E., Johnston, E.M., et al., 2014. Copper active sits in biology. Chem. Rev. 114,
3659
3853. Copyright 2014 with permission American Chemical Society.

of the enzyme to give dioxygen and the reduced (Cu1) enzyme, which subsequently reduces a sec-
ond molecule of superoxide, giving hydrogen peroxide and restoring the oxidized form of the
enzyme.
2O2 1
2 1 2H
! O2 1H2 O2
Cu21 ZnSOD1O2 1
2
! Cu ZnSOD1O2
Cu1 ZNSOD1O2 1
2 1 2H
! Cu ZnSOD1H2 O2
21

Human CuZnSOD, SOD1, is a 32-kD homodimer, each subunit made up (Fig. 14.18; Strange
et al., 2003), as we saw in Chapter 3, Structural and Molecular Biology for Chemists, of an eight-
stranded β-barrel with one Cu and one Zn site, and contains an intrasubunit disulphide. The Cu site
is a typical Type 2 site with four His ligands, with His44 and His46 in trans positions of the dis-
torted square planar CuN4 coordination sphere. The tripeptide His44
Val45
His46 completely
blocks access to the Cu from one side of the CuN4 plane, while the other side is solvent accessible
via a conical channel some 4 Å wide lined by positively charged residues. The active site channel
leading to the copper atom is constructed ideally for small anionic species such as superoxide,
allowing nearly diffusion controlled rates of enzyme catalysis (rate constants B2 3 109 M21/s).
The Zn ion is also coordinated by one Asp and three His ligands, one of which, His61, bridges the
two metal ions
a structural feature that had not been seen previously in coordination geometry.
424 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

Crosslink His240

Tyr244

CuB His291

Tyr244•

His290

Heme a3

His376

FIGURE 14.17
Crystal structure of the fully oxidized dinuclear site in bovine heart CcO at 2.3 Å resolution, showing the
His
Tyr crosslink and indicating the putative Tyr•. A peroxo species is seen between Fea3 and CuB. Adapted
from PDB 2OCC using Insight.
From Rogers, M.S., Dooley, D.M., 2003. Copper-tyrosyl enzymes. Curr. Opin. Chem. Biol. 7, 131
138.
Copyright 2003, with permission from Elsevier.

Amyotrophic lateral sclerosis (ALS, also referred to as motor neurone disease or Lou
Gehrig’s disease3) is a fatal disease which targets essentially the motor neurones (which bring
messages from the brain to the muscles). As they are destroyed, denervation and muscular
atrophy causes weakness and finally paralysis. Despite the degradation of physical ability, mental
activity is not usually affected due to the selectivity of the disease for motor neurones. Most
cases of ALS, which affects worldwide 0.5
3 in 100,000 people/year, have no genetic factor
implicated (known as sporadic ALS). Of the 5%
10% remaining familial cases (fALS), around
20%
25% map to the SOD 1 gene. More than 100 mutations have been identified which can
increase aggregation of the SOD1 polypeptide for fundamentally distinct reasons (Fig. 14.19;

3
Henry Louis Gehrig a New York Yankees first baseman, inducted into the American Baseball Hall of Fame in 1939,
died 2 years later. The disease is so rare that it became known due to him and is widely known as ‘Lou Gehrig’s
disease.’
 (A)

Asp124

His120
His46

His71
W1
CU

ZN Asp83

His48 His63

His80

(B)

Zn

Cu

Zn

Cu

His78

His61
His46
Zn

Asp81
Cu

Copper His69
Zinc
His44
Nitrogen
His118
Oxygen
Carbon

FIGURE 14.18
Structure of human homodimeric SOD1 (PDB code 1PUO) and the structure of the Cu and Zn sites of human
SOD1.
From Hart, P.J. (2006) Pathogenic superoxide dismutase structure, folding, aggregation and turnover, Curr. Opin. Chem.Biol., 10,
131
138. Copyright 2006 with permission from Elsevier.
426 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

(A) ALS mutations can reduce

the net negative charge
of SOD1 without affecting
stability or metal binding

COO− → NH3+

(B) ALS mutations can

destabilize the
SOD1 native state
(C) ALS mutations can
impait Cu or Zn binding
by SOD1

FIGURE 14.19
Different ALS-associated mutations of SOD1 can increase aggregation of the SOD1 polypeptide for
fundamentally distinct reasons. The resulting amino acid substitutions can cause (A) a decrease in the net
negative charge (2q) of the protein without affecting metal binding or stability of the native state (e.g., E100K,
D101N and N139K); (B) a decrease in stability of the native fold without affecting metal binding or net charge
(e.g., A4V, L38V, L84V, G93A and I113T) or (C) disruption of Cu and Zn binding without affecting the stability
of the apoprotein (e.g., S134N). In addition, many mutations affect multiple determinants of aggregation. The
subunits of the wild-type holo-SOD1 dimer (PDB accession code 1HL5) are coloured blue and green; Cu21 and
Zn21 ions bound in the active site are shown as yellow and red spheres, respectively.
From Shaw, B.F., Valentine, J.S., 2007. How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the
protein? Trends Biochem. Sci. 32, 78
85. Copyright 2007 with permission from Elsevier.

Shaw and Valentine, 2007). It is well documented that fALS produces protein aggregates in the
motor neurons of fALS patients. Fig. 14.20 illustrates how in mutant fALS protein deficient in
copper and zinc, ultimately lead to the formation of aggregates of SOD1 protein which play a
role in the pathogenesis of ALS.

COPPER ENZYMES INVOLVED WITH OTHER LOW-MOLECULAR

SUBSTRATES
Copper enzymes are involved in reactions with a large number of other, mostly inorganic
substrates. In addition to its role in oxygen and superoxide activation described above, copper is
also involved in enzymes which activate methane, nitrite and nitrous oxide.
COPPER ENZYMES INVOLVED WITH OTHER LOW-MOLECULAR SUBSTRATES 427

SOD1 mutants lacking

metal ions

S S
S S S S

S S
S S S S S S
S S S S
S S

S S S S
S S S S

Stable amyloid-like oligomers

FIGURE 14.20
Formation of soluble oligomers occurring when apo WT SOD1 protein is kept close to physiological conditions
for an extended period of time. In the absence of metal ions, SOD1 proteins form abnormal disulphide crosslinks
though the two free cysteines (Cys 6 and Cys 111) and noncovalent associations with other SOD1 monomers or
dimers.
From Banci L, Bertini I, Boca M et al. (2008) SOD1 and amyotrophic lateral sclerosis: mutations and oligomerization. PLoS One. 3,
e1677. doi: 10.1371/journal.pone.0001677. This is an open-access article distributed under the terms of the Creative Commons
Attribution License.

There are vast reserves of methane gas in the world, which are currently underutilized as a feed-
stock for the production of liquid fuels and chemicals because of the lack of economical and sus-
tainable strategies for the selective oxidation of methane to methanol. Current processes require
high temperatures, are costly and inefficient, and produce waste, yet throughout nature methano-
trophic bacteria perform this reaction under ambient conditions using methane monooxygenases
(MMOs). We already encountered the soluble diiron MMO in Chapter 13, Iron: Essential for
Almost All Life, expressed by several strains of methanotroph under copper-limited conditions. All
methanotrophs produce membrane-bound particulate MMO (pMMO). Yet, in spite of 20 years of
research and the availability of two crystal structures, the metal composition and location of the
pMMO metal active site were still not known until very recently. In 2010, the structure of particu-
late methane monooxygenase from the methanotrophic bacteria M. capsulatus was determined at a
resolution of 2.8 Å (Fig. 14.21). It is a trimer with an α3β3γ3 polypeptide arrangement. Two metal
centres, modelled as mononuclear and dinuclear copper are located in the soluble part of each
428 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

His 72 spmoBd2

His 48

265 His 139

172
His 137
spmoBd1
His 33

FIGURE 14.21
Structure of M. capsulatus (Bath) pMMO protomer (PDB accession code 1YEW). The N-terminal cupredoxin
domain of pmoB (spmoBd1) is shown in purple, the C-terminal cupredoxin domain of pmoB (spmoBd2) is shown
in green, and the two transmembrane helices are shown in blue. In the recombinant spmoB protein, spmoBd1 and
spmoBd2 are connected by GKLGGG sequence-linking residues 172 and 265 instead of the two transmembrane
helices. Copper ions are shown as cyan spheres and ligands are shown as ball-and-stick representations. The
pmoA (transparent light green) and pmoC (transparent light blue) subunits are composed of transmembrane
helices. The location of the zinc ion (grey sphere) has been proposed to house a diiron centre. A hydrophilic
patch of residues marked with an asterisk is the site of a proposed tricopper centre.
From Balasubramanian R., Smith S.M., Rawat S., Yatsunyk L.A., Stemmler T.L., Rosenzweig A.C., 2010. Oxidation of methane by a
biological dicopper centre. Nature 465, 115
119. Copyright 2006 with permission from Nature.
COPPER ENZYMES INVOLVED WITH OTHER LOW-MOLECULAR SUBSTRATES 429

His 287(255)

Met 182(150) N

*H H*
O
S His 338(306)
Cys 167(136) Cu
His 126(95) Cu H* N*
N*
N*
S N* N
N* H
N N
H H*
H N
HN His 166(135) H
His 177(145) His 131(100)

Type-1 ∼12.5 Å Type-2

FIGURE 14.22
Schematic representation of the copper sites in nitrite reductase.
From Veselov, A., Olesen, K., Sienkiewicz, A., Shapleigh, J.P., Scholes, C.P., 1998. Electronic structural information from Q-band
ENDOR on the type 1 and type 2 copper liganding environment in wild-type and mutant forms of copper-containing nitrite reductase.
Biochemistry, 37, 6095
6105.

β-subunit, which resembles cytochrome c oxidase subunit II. A third metal centre, occupied by Zn
in the crystal, is located within the membrane. From a series of site-directed mutants, it has been
shown that methane oxidation occurs at the dinuclear copper site in the soluble domain of the
pmoB subunit (Balasubramanian et al., 2010).
Nitrite reductases catalyse the reduction of nitrite to nitric oxide:
NO2 1 2
2 1 2H 1 e
!NO1H2 O

Nitrite reductases in many bacteria are haem proteins, However, some are copper-containing
homotrimers which bind three Type I and three Type II copper centres The Type 1 copper centre
serves to transfer electrons from donor proteins to the Type 2 centre which has been proposed to be
the site of substrate binding. Fig. 14.22 shows a schematic representation of the copper sites in
nitrite reductase (Veselov et al., 1998).
Nitrous oxide reductases catalyse the final step in the denitrification4 process, reducing N2O to
N2. Organisms which carry out denitrification use oxidized forms of nitrogen instead of oxygen as
the terminal electron acceptors for anaerobic respiration, which is coupled, via proton-pumping, to
ATP synthesis. N2O reductase is also of environmental interest, since not only is N2O the third
most important greenhouse gas (after CO2 and CH4) it is a potentially attractive oxo-transfer
reagent to oxidize organic substrates in a green reaction where the only by-product is N2. Nitrous

4
In denitrification, part of the biological nitrogen cycle, nitrate in the soil is converted via four enzymatic reactions
stepwise to nitrite, nitric oxide and nitrous oxide to finally yield gaseous nitrogen.
430 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

z⬘ y⬘
C-terminal domain x⬘ x⬘
CuA
N-terminal domain

CuI CuI
O/OH/H2O y
x
Cuz S
CuIV CuIII CuII/IV x
y

CuII CuII

Cuz Front view Side view

FIGURE 14.23
Crystal structure of the CuZ site from Pn. The two subunits in the homodimeric protein are indicated in red and
blue. The Cu4S cluster has approximate Cs symmetry with CuI
S
CuII defining the mirror plane. The water-
derived ligand L (O22, OH2, H2O) is weakly bound according to the higher resolution structure of Pd (1.6 Å
resolution), however its nature has not been assigned. Molecular (x0 ,y0 ,z0 ) and local (x,y,z) coordinate systems are
indicated, which are used for labelling orbitals.
From Chen, P., Gorelsky, S.I., Ghosh, S., Solomon, E.1., 2004. N2O reduction by the μ4-sulfide-bridged tetranuclear CuZ cluster
active site. Angew. Chem. Int. Ed. 43, 4132
4140. Copyright 2004 with permission from John Wiley and Sons.

oxide reductase contains two copper sites designated CuA and CuZ. The CuA site is the well-
characterized mixed-valence dinuclear electron transfer site with two coppers bridged by two Cys
ligands which we already encountered in cytochrome oxidase (and which is also found in NO
reductase). The reduction step is catalysed by the Cuz cluster of N2O reductases (N2OR). The
structure of the CuZ proved to be quite unusual, namely a μ4-sulphide bridged tetranuclear copper
cluster. The CuZ centre is located in the N-terminal domain of the dimeric enzyme whereas the
CuA centre is located in the C-terminal domain of each subunit (Fig. 14.23; Chen et al., 2004).
Thus in the dimeric protein structure the neighbouring CuA and CuZ sites are contributed by differ-
ent subunits. While the [Cu4S] cluster has approximate two-fold symmetry, with very similar
Cu
S bond lengths, the Cu
Cu distances are very different with three copper centres, designated
as CuII, CuIII and CuIV, closer to one another, with CuI further away. The entire [Cu4S] cluster is
coordinated by seven His ligands to the protein, with an additional as yet unidentified oxygen
ligand at the CuI/CuIV edge. This CuI/CuIV edge is thought to be the substrate binding site. The cat-
alytically relevant form of the CuZ is the fully reduced state with Cu1 at each of the four coppers.
In the proposed mechanism (Fig. 14.24), the reduction of N2O to N2 is assumed to involve binding
of the N2O substrate at the CuI/CuIV edge where it could interact with CuI and CuIV in a bridged
binding mode. Simultaneous donation of electrons from CuI and CuIV would allow the two-electron
reduction of N2O. Good electron transfer pathways exist from the neighbouring CuA centre in the
second subunit of the dimeric protein to CuII and CuIV to allow rapid rereduction of the CuZ centre.
 REFERENCES 431

CuI
e–

N2 O
CuIII
e–
CuIV

CuA CuII

S
Cu Cu
S His

FIGURE 14.24
Reduction of N2O at the CuZ site.
From Chen, P., Gorelsky, S.I., Ghosh, S., Solomon, E.1., 2004. N2O reduction by the μ4-sulfide-bridged tetranuclear CuZ cluster
active site. Angew. Chem. Int. Ed. 43, 4132
4140. Copyright 2004 with permission from John Wiley and Sons.

MARS AND VENUS
THE ROLE OF COPPER IN IRON METABOLISM

Very early studies established that copper deficiency is associated with anaemia in a number of ani-
mals. However the key to understanding the interaction between copper and iron came from the
observations that in yeast mutations affecting copper metabolism blocked the high affinity iron uptake
system. Whether the mutations were in the plasma membrane copper transporters or in the copper
chaperone P-type ATPase Atx1, which inserts iron into the Fet3 oxidase, the outcome was the same,
and for the same reason
a multicopper oxidase is required for high affinity iron uptake into yeast.
It then came as no surprise to find that in the rare human neurological disease aceruloplasminaemia,
iron accumulated in brain and liver, indicative that a key role of ceruloplasmin was in tissue iron
mobilization. This was convincingly shown by studies in which the yeast Fet3 oxidase was shown to
restore iron homeostasis in aceruloplasminaemic mice. The likely mechanism, involved in the export
of iron via the Fe21 transporter ferroportin is thought to require the ferroxidase activity of ceruloplas-
min to ensure its incorporation into apotransferrin.

REFERENCES
Augustine, A.J., Kjaergaard, C., Qayyum, M., et al., 2010. Systematic perturbation of the trinuclear copper
cluster in the multicopper oxidases: the role of active site asymmetry in its reduction of O2 to H2O. J. Am.
Chem. Soc. 132, 6057
6067.
Balasubramanian, R., Smith, S.M., Rawat, S., Yatsunyk, L.A., Stemmler, T.L., Rosenzweig, A.C., 2010.
Oxidation of methane by a biological dicopper centre. Nature 465, 115
119.
432 CHAPTER 14 COPPER
COPING WITH DIOXYGEN

Basumallick, L., Szilagyi, R.K., Zhao, Y., et al., 2003. Spectroscopic studies of the Met182Thr mutant of
nitrite reductase: role of the axial ligand in the geometric and electronic structure of blue and green copper
sites. J. Am. Chem. Soc. 125, 14784
14792.
Basumallick, L., Sarangi, R., George, S.D., et al., 2005. Spectroscopic and density functional studies of the red
copper site in nitrosocyanin: role of the protein in determining active site geometric and electronic struc-
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