comprehensive inorganic chemistry ii || copper enzymes
TRANSCRIPT
Co
3.07 Copper EnzymesRL Peterson, S Kim, and KD Karlin, Johns Hopkins University, Baltimore, MD, USA
ã 2013 Elsevier Ltd. All rights reserved.
3.07.1 Introduction 1493.07.1.1 Scope of the Chapter 1493.07.1.2 Copper Chemistry Basics 1503.07.2 O2 Utilization 1523.07.2.1 Fundamental Properties of Dioxygen 1523.07.2.2 Hemocyanin 1523.07.2.3 Tyrosinase 1533.07.2.4 Other Monooxygenases 1563.07.2.4.1 Noncoupled dinuclear monooxygenases 1563.07.2.4.2 Particulate methane monooxygenases 1583.07.2.4.3 Cellulose monooxygenase 1593.07.2.5 Dioxygenases (Quercetin 2,3-Dioxygenase) 1603.07.2.6 Copper Oxidases 1613.07.2.6.1 Mononuclear copper oxidases 1613.07.2.6.2 Galactose oxidase 1623.07.2.6.3 Multicopper oxidases 1653.07.2.6.4 Heme–copper oxidases 1663.07.2.7 Superoxide Dismutase 1703.07.3 NOX Processing 1713.07.3.1 Nitrite Reductase 1713.07.3.2 Nitrous Oxide Reductase 1733.07.4 Conclusion 174Acknowledgment 175References 175
3.07.1 Introduction
3.07.1.1 Scope of the Chapter
Copper is the third most abundant transition metal found
in biology. This essential micronutrient is vital constituent
of metalloprotein active sites, which are necessary to support
aerobic eukaryotic life. Some of the many biological roles
of copper proteins include electron shuttling/trafficking (i.e.,
cupredoxins), small-molecule processing (i.e., dioxygen (O2)
and nitrogen oxides (NOX)), oxidative transformation of biolog-
ical/organic substrates (i.e., pigment production, activation/
production of neurotransmitters and hormones, etc.), as well as
production and scavenging of reactive oxygen species (ROS)
(superoxide dismutase (SOD), ceruloplasmin). (See Table 11–5
for a listing of copper proteins and their properties.) Much of the
biochemistry of copper surrounds the rich one-electron (e�)redox chemistryof copper shuttlingwithin theCuI/CuII oxidation
states, which can be easily accessed under physiological con-
ditions using biologically available oxidants (i.e., O2) and reduc-
tants such as ascorbate and glutathione. Through evolution,
Nature tunes the driving force of these electron-transfer events
throughmodulation of the protein active site by the use of ligand
coordination (number and type) and geometric constraints.
The major focus of this chapter surrounds the presentation
of the bioinorganic chemistry of dioxygen (O2) and nitrogen
oxide (NOX) processing by copper proteins along with our
presentation of bioinspired studies using synthetic copper
mprehensive Inorganic Chemistry II http://dx.doi.org/10.1016/B978-0-08-09777
‘model’ complexes. Highlights into the bioinorganic chemistry
of copper including coordination and spectroscopic properties
of major classes of copper proteins are briefly discussed. Other
classes of copper proteins and copper-related biochemistry not
included in this chapter are chaperone and transport proteins
involved in copper ion homeostasis, copper-related diseases
such as Menkes and Wilsons disease that result in imbalances
in this metal ion’s local or global concentration and copper
chemistry associated with aging and oxidative stress including
‘Lou Gehrig’s’, Alzheimers’, and Prion diseases.6
The reaction of copper(I) with molecular oxygen is a
fundamental process in harnessing the oxidative power of
molecular oxygen for chemical and biological energy transfer.
In addition, O2 is a source of oxygen atoms for insertion into
organic substrates. There are a number of copper proteins
that reductively bind, activate and transform dioxygen to
different physical states and entities (Table 1 and Figure 17).
Hemocyanins (Hcs) are found in mollusks and arthropods and
reversibly bind/transport molecular oxygen at active sites pos-
sessing two proximal copper ions (i.e., dinuclear sites). Copper
oxygenases or oxidases play crucial roles in the activation of
molecular oxygen and oxidative transformation of organic sub-
strates. Copper mono- or dioxygenases functionalize organic
substrates through the incorporation of one (mono-) or two
(di-) oxygen atoms derived frommolecular oxygen. The copper
monooxygenases dopamine-b-monooxygenase (DbM),
peptidylglycine-a-hydroxylating monooxygenase (PHM), and
4-4.00309-0 149
Table 1 Biologically important proteins with copper ion active sites
Protein PDB # a Biological function
Electron-transfercenters1,2
Plastocyanin, azurin 1PLC, 2AZA 1 Electron transferCuA; in CcO and N2OR 2OCC 2 Electron transfer
Oxygen carriers Hemocyanin (Hc) 1LLA, 1OXY 2 O2 transportMonooxygenases Tyrosinase (Tyr) 1WX2 2 Phenol o-hydroxylation
Dopamine b-monooxygenase (DbM) – 2 Dopamine!norepinephrinePeptidylglycine-a-hydroxylating monooxygenase(PHM)
1PHM,1SDW
2 Glycine-extended prohormone peptidehydroxylation
Particulate methane monooxygenase (pMMO) 1YEW 2,1 Methane!methanolAmmonia monooxygenase (AMO) – ? Ammonia!hydroxylamine
Dioxygenases Quercetinase (2,3QD) 1JUH 1 Quercetin oxidative cleavageOxidases O2!H2O 4e�/4Hþ processBlue MCOsb Catechol oxidase (CO) 1BT3 2 o-Catechol!o-quinone
Laccase (Lc) 2Q9O 3,1 Oxidation of phenolAscorbate oxidase (AO) 1AOZ 3,1 Oxidation of L-ascorbateCeruloplasmin (Cp) 1KCW 3,3 Fe(II)!Fe(III)
O2!H2O2 2e�/2Hþ process
Non-blue Copper amine oxidase (CAO) 1KSI 1 1� amine!aldehydeGalactose oxidase (GAO) 1GOG 1 1� alcohols!aldehydeGlyoxal oxidase (GLO) – 1 Aldehyde!carboxylic acid
Heme–Cu oxidases3–5 Cytochrome c oxidase (heme–Cu site) (CcO) 2OCC 1 O2!2H2O, proton translocationOther Cu,Zn-superoxide dismutase (Cu,Zn-SOD) 2SOD 1 O2•� dismutation (detoxification)
Nitrite reductase (NiR) 2AFN 1,1 NO2�!NO
Nitrous oxide reductase (N2OR) 1QNI 2,4 N2O!N2
a# of active-site Cu ions with multiple entries indicating more than one type of site.bFET3, phenoxazinone synthase, bilirubin oxidase, dihydrogeodin oxidase, and sulochrin oxidase also belong to this class.
Oxidases:
Monooxygenases:
O2+ 4H++ 4e-
O2+ 2H++ 2e-
R–H + O2+ 2H++ 2e-
R–H + O2 R(O)OH
R–OH + H2O
2H2O
Laccase, ascorbate oxidase
Amine oxidase, GO
Superoxidedismutase
+1.20
+0.64
+0.38 +2.31
+1.35
+0.82 (+0.86)+0.31 (+0.37)
+0.28 (+0.36)O2
O2·- H2O2 H2O + ·OH 2H2O-0.33 (-0.16) +0.89
H2O2
Dioxygenases:
Figure 1 Standard aqueous reduction potentials of dioxygen-derived species (volts, pH 7) dioxygen at 1 atm (or unit activity), adapted from Feig, A. L.;Lippard, S. J. Chem. Rev. 1994, 94, 759–805. Copper-containing metalloenzymes facilitating specific reactions are also shown.
150 Copper Enzymes
tyrosinase play important roles in the regiospecific transforma-
tion of biological signaling molecules. Other important mono-
oxygenases include particulate methane monooxygenase
(pMMO) and ammonia monooxygenase (AMO), which are
key enzymes in the global carbon and nitrogen cycles; these
functionalize alkanes or ammonia for further biological pro-
cessing. Copper oxidases couple the dehydrogenation of or-
ganic substrates with the formation of hydrogen peroxide
(H2O2) or water (H2O). Many different forms of copper oxi-
dases exist in Nature, including copper only (laccase and ascor-
bate oxidase), copper–organic cofactor (amine oxidase and
galactose oxidase), and heterobimetallic (Fe-heme/Cu site in
cytochrome c oxidase (CcO)) sites. Cu–Zn SODs
are responsible for scavenging the ROS superoxide (O2•�) by
facilitating its disproportionation to the less reactive products,
hydrogen peroxide (H2O2) and molecular oxygen.
3.07.1.2 Copper Chemistry Basics
In copper metalloproteins, copper ions are most commonly
found in the cuprous Cu(I) d10 and the cupric Cu(II) 3d9
oxidation states; a high-valent Cu(III) 3d8may also be relevant.
Copper Enzymes 151
In an aqueous coordinating environment, Cu(I) is ‘unstable’
and readily disproportionate to Cu0S and Cu2þaq (Figure 2).
Copper(I) and copper(II) have very different ligand donor
atom preferences and normally exhibit differing coordination
geometries. The 3d10 cuprous ion can tolerate a wide variety of
coordination environments but is most commonly found in
linear two-coordinate, T-shaped three-coordinate, or tetrahe-
dral four-coordinate complexes. Copper(I) is a ‘soft’ acid and
prefers softer bases such as thioethers, phosphanes, and unsat-
urated amines such as nitriles, pyridine, and imidazole. On the
other hand, for electronic structure stabilization, the cupric 3d9
ion prefers tetra-coordination (planar or sometimes pseudote-
trahedral) or pentacoordinate in square-pyramidal or trigonal-
bipyramidal environments. Relatively speaking, copper(II) is
considered a borderline or ‘hard’ acid because of the smaller
ionic radius and higher charge, thus preferring bases such as in
carboxylates, saturated amines, alkoxides as well as deproto-
nated amides; however, N atoms from pyridines and imidaz-
oles also make good ligands for copper(II). The most common
active-site-coordinating biological amino acids are shown in
Figure 2. For the amino acid histidine (His), there are two
modes of nitrogen coordination from the heterocyclic imidaz-
ole donor, via the d-NHis or e-NHis atoms; the observed prefer-
ence varies depending on the protein. Glutamate (Glu) and
aspartate (Asp) are common ligands in iron biochemistry but
are rare in copper biochemistry and are thus far only found in
the so-called red electron-transfer protein nitrosocyanin and
the copper dioxygenase, quercetin 2,3 dioxygenase.
In copper biochemistry, proteins have been classified by
their distinctive spectroscopic features, which are dictated in
part by the type of coordinating amino acids and geometric
constraints at the protein active site. Historically, since Mal-
mstom’s classic studies,8 copper proteins are divided into
three classes of copper proteins, named type 1 (i.e., T1), 2, or
3, which contain one to two copper ions (see Figure 3 for
depiction of each site type). More recently, ‘new’ classes of
copper sites have been defined; these are called type 0, CuA,
and CuZ. The latter are described below, in more detail.
T1 copper sites (also called blue copper) are electron-transfer
proteins containing a mononuclear copper site, which is
CuIaq + e−
CuIIaq + e−
Cus
CuIaq
2CuIaq
E� = 0.52 V
E� = 0.153 V
Cus + CuIIaq E� = 0.37 V Keq ~ 106
CHN
O
R
R =NHδN
SH
O O
OHOH OH
HN NεS
εHisδHis Met Cys
Tyr Asp Glu
Figure 2 Top: Redox chemistry of copper(I) in aqueous systems.Below: Protein amino acid residues commonly observed as ligand donorsto copper ion. As appropriate, the actual donor may be in itsdeprotonated and not neutral state.
coordinated by a His2,�S-Cys, X-binding motif that is orientated
in a distorted tetrahedral ‘entatic’ state.1,2 Blue copper proteins
are commonly found in photosynthetic organisms and are evo-
lutionarily tuned over awide range of redox potentials (180–800
mV (vs. normal hydrogen electrode (NHE))), which result
from variations in the nature of the X moiety and secondary
coordination sphere. T1 copper centers have two distinct spec-
troscopic properties that can be observed in their electronic
absorption (UV–Vis) and electron paramagnetic resonance
(EPR) spectra. They have�100–1000 times highermore intense
electronic transitions thanwhat is found for typical copper(II) T2
ligand field (d–d) transitions (Figure 3). These occur at a lmax
�600 nm (and/or �450 nm) with emax�2000–5000 and are
attributed to a Cys-S!Cu(II) ligand-to-metal charge-transfer
(LMCT) bands. In addition, the EPR spectra of blue copper pro-
teins possess a four-line copper hyperfine pattern with unusually
small splittings (A║<80�10�4 cm�1), that is, less than half
of what is found in normal copper(II) T2 complexes. Both of
these unique spectroscopic properties are associated with a
highly covalent Cu–S bond held in a distorted tetrahedral ligand
field.1,2,9
T2 (‘normal’) copper centers possess lower (i.e., more neg-
ative) redox potentials when compared to T1 sites. These typ-
ically have three to five coordinating moieties (usually three
coordinating amino acid side-chain donors with an additional
one or two labile water ligands) and can easily adopt different
active-site geometries including distorted tetrahedral, square
pyramidal, or trigonal bipyramidal. T2 copper centers possess
weak electron absorption bands at �700 nm and show a four-
line EPR signal with larger hyperfine splitting patterns of
A║�(130–180)�10�4 cm�1. T2 copper centers are involved
in the reductive activation of dioxygen including copper diox-
ygenases, monooxygenases, and oxidases as well as copper
enzymes that activate other small molecules including nitrite.
Type 3 copper centers are dinuclear that show no EPR spec-
troscopic signal due to strong antiferromagnetic coupling be-
tween the two cupric S¼1/2 sites leading to an S¼0 ground
state. Each copper ion is usually three to five coordinate, depend-
ing on its ligands andoxidation state.9–11 As a dicopper center, T3
sites are capable of donating 2e� (one from each copper center)
and efficiently reducing dioxygen to the peroxo* (O22�) oxida-
tion state level without the leaking of partially reduced oxygen
species, such as superoxide radical anion (O2•�). In multicopper
oxidases (MCOs), a T3 copper center is in close proximity to an
additional single T2 copper site that assists with electron transfer
anddioxygen reduction. The T2andT3 copper ions constitute the
Type 1R = Met, Gln, Glu
Type 2L = N, O ligandsHis, Glu, Met, Tyr
Type 3L = His
X = other ligands or O22−
R
CuCysS
NHisNHis
L
L
L
CuL
L
X
L
CuOH
LL
XL
CuL
L
Figure 3 Traditional classification of biological copper sites.
*In this chapter peroxido and peroxide are often shortened to peroxo;
hydroxo, oxo and superoxo are used likewise.
152 Copper Enzymes
trinuclear cluster (TNC), which altogether bind O2 and effect its
reduction in MCOs. T3 centers are commonly associated with
description of the proteins, Hc and tyrosinase; however, the
term T3 originally derived from classification of the spectro-
scopically distinct dinuclear centers in MCOs.
Type zero copper sites. As described, T1 copper centers occur
in ‘blue’ electron-transfer proteins, and typically possess His2,�S-Cys, and X ligation, with the His2, Cys essentially in a trigo-
nal plane and possessing a long-axial ligation to a Met thioether
S-ligand or another residue X. These show a tetrahedral geome-
try type of EPR spectrum, also with a very narrow EPR hyperfine
splitting (A||), from unpaired spin density interaction with the
copper nucleus (I¼3/2), and high (positive) redox potentials
indicating a site very favorable for reduced copper(I). Type 2
copper, so-called ‘normal’ copper(II), has only N or O strong
ligands, and such complexes possess a tetragonal EPR spectrum
with much larger and ‘normal’ A|| values.
A new type of mononuclear Cu center has been recently
described by Gray and coworkers,12,13 referred to as type
0 copper.14 These do not occur naturally, but were conceived
with the long-term goal of using MCOs, which after designed
mutation possess a more robust T1 center (i.e., without an
S-ligand, which may be susceptible to oxidative degradation)
potentially useful for O2-reduction catalysis, i.e., in fuel cell
applications. Type 0 copper ions arise from double mutagene-
sis of the type 1 copper electron-transfer site in azurins, leading
to the following changes: (His2, Met(121), Cys-S-(112)) to a
(His2, (Phe, Ile, Leu)(121), Asp(112)). The resulting constructs
have unique properties including relatively high redox poten-
tials (E1/2�300 mV vs. NHE), similar to copper T1 sites, while
their electronic absorption features are similar to T2 copper
sites, which are weakly absorbing (lmax�800, e�100). Their
axial EPR signatures exhibit A|| values that are narrower than
is typically observed for T2 copper sites (Figure 413). This new
class of copper sites also contains an unusually short contact
(�2.35 A) to the carbonyl oxygen of Gly45 compared to
2.84 A in wild-type azurin leading to significant copper distor-
tion out of the N–N–O(His46–His117–Asp112) trigonal
plane. This increased bonding interaction is most likely due
to the substitution of a hydrophobic noncoordinating residue
at the Met121 position, forcing the carbonyl group lone pair of
electron on Gly45 to act as the fourth ligand to the copper site.
Type 1
His2, Cys,Met, Gly
Type 0
Type 2
His2, Asp,Met, Gly
His2, Asp,Gly
Figure 4 Type 0 copper and EPR spectroscopic comparison summary(PDB code: 3FQ1). Adapted from Lancaster, K. M.; Farver, O.;Wherland, S.; Crane, E. J.; Richards, J. H.; Pecht, I.; Gray, H. B. J. Am.Chem. Soc. 2011, 133, 4865–4873.
3.07.2 O2 Utilization
3.07.2.1 Fundamental Properties of Dioxygen
Dioxygen is a major component of the earth’s atmosphere
comprising �20% by volume. Molecular oxygen is a ground-
state triplet, consisting of two unpaired electrons one in each of
the doubly degenerate p* HOMOs. Formally, molecular oxy-
gen has a double bond and is thermodynamically a powerful
oxidant. However due to spin conservation, the reaction of O2
with ground-state singlet molecules is kinetically unfavorable
and requires the reaction with other ground-state radicals, such
as flavins, pterins, or metal ions. As shown in Figure 1, the one-
electron reduction of dioxygen to superoxide is thermodynam-
ically unfavorable. However, the two-electron reduction of
dioxygen and the one-electron reduction of superoxide to per-
oxide are thermodynamically favorable and lead to O–O bond
elongation as electrons are added to the antibonding p* or-
bitals (see Figure 515 for the physical properties of dioxygen-
containing moieties).
The coordination of O2 to copper in both enzymatic and
synthetic systems involves a large degree of electron transfer
from the reduced copper center to molecular oxygen in what is
most often believed to be an inner-sphere mechanism, i.e.,
complex formation includes bond formation accompanied
by electron transfer. The nature of the copper–dioxygen adduct
formed is highly variable and depends on many factors includ-
ing the ligand/protein type (e.g., S vs. N), resulting coordina-
tion number and geometry, the number of copper ions in close
proximity, etc. (see Figure 6).
3.07.2.2 Hemocyanin
Hcs are extracellular dioxygen transport proteins found in the
blood of many arthropods and mollusks.9 These large, multi-
subunit, highly cooperative proteins bind dioxygen at a T3
dicopper site where each copper ion is ligated by three e-Nhistidines. There are two distinct classes of Hcs found in bio-
logical systems: one found in mollusks (e.g., snail, bivalves,
and octopi) and the other found in arthropods (e.g., lobsters,
Bond order
2
2
1.5
1
1
nO–O (cm−1)
1555
1484
1145
794
880
O–O distance(pm)
121
122
134
149
146
Dioxygen species
LUMO:
HOMOs: • • • •
π*2p π*2p
• •σ*2p
(a) (b) (c)
MOoxygen
fragment
Triplet O2 (3Σg−) (a)
Singlet O2 (1Δg)
Superoxide in [K+][O2•−] (b)
Peroxide in [Na+]2[O22−] (c)
Peroxide in H2O2
Figure 5 Physical properties of molecular oxygen and reduceddioxygen moieties. Adapted from Conry, R. R. Encyclopedia of InorganicChemistry, John Wiley and Sons: Hoboken, 2006.
O
Ar
OAr
OOH
OOH
1+ 2+ 2+
O
OO
O
O
O
2+ 2+ 2+
O
O
OO
O
1+ O
O
0 1+1+
OAr
0
Dinuclearcopper
Mononuclearcopper
Peroxo Hydroperoxo Superoxo
CuII CuII CuII
CuII
CuIICuII
CuIICuII
CuIICuIICuIICuIICuIICuII
CuIII–hydroxide
CuIII–OH
m-1,2-peroxo m-h2 : h2-peroxo Bis-m-oxo
CuIII
CuIII
CuIII
End-on, h1
superoxoSide-on, h2
superoxoSide-on, h2
peroxo-CuIIISide-on, h2
peroxo-CuII
End-on, h1
hydroperoxo
(O2·-)
O·-
(O22-)
Figure 6 Synthetically derived mono- and dinuclear copper complexes ligated to O2-derived species. Underlined species have been observed inbiological systems.
DeoxyCu–Cu 4.6 ÅColorless
+O2
-O2
NHis
NHisNHis
NHis
NHis
NHis
NHis
NHis
HisN HisN
HisN
HisNCuI CuI CuII CuII
OxyCu–Cu 3.6 Å
Blue lmax= 350, 550 nmn(O–O) ~ 750 cm-1
O
O
Figure 7 Top: Reaction overview in Hc, involving deoxy and oxy-Hc.Bottom: Diagram from the x-ray structure of oxy-Hc from Octopusdofleini showing (i) the side-on binding mode of dioxygen (as peroxide)to the dicopper site and (ii) the C2His/S-Cys crosslink (PDB code: 1JS8).
Copper Enzymes 153
crabs, and insects). Some molluskian Hcs contain a unique
C2His/S-Cys crosslink in the so-called CuA site, which is not
commonly found in biological systems. Despite this unique
feature, the spatial arrangements of the copper ions in relation
to the binding histidines’ N of both classes of Hcs are nearly
identical.16 The fully reduced colorless dicuprous state, deoxy-
Hc, is EPR silent with the two copper ions separated by�4.6 A.
Oxy-Hc is formed upon dioxygen exposure, which facilitates a
redox event in which dioxygen is formally reduced to the
peroxo state and where each copper ion is oxidized by one
electron forming an intense purple m-Z2:Z2-peroxo-dicopper
(II) adduct (Figure 7).
There are many unique spectroscopic features of oxy-Hc,
which come about as a result of the unique geometry and
electronic properties of the purple side-on m-Z2:Z2-peroxo-
dicopper(II) core. The Cu–Cu distance in oxy-Hc is �3.6 A16–19 and has an O–O bond length of �1.4 A. The latter is
consistent with the dioxygen-derived fragment being in the
peroxide state. Resonance Raman (rR) spectroscopic experiments
show an oxy-Hc O–O stretching frequency at �750 cm�1
(D��40 cm�1 upon 18O2 isotope labeling).20–22 This value is
unusually low when compared to other end-on bound metal
peroxo species and is �100 cm�1 lower than what is observed
for the nonheme iron dioxygen transport protein hemerythrin,
which is thought to bind dioxygen as an end-on bound peroxide
at single iron center.23 The unusually low O–O stretch in oxy-Hc
is attributed to significant backbonding from a copper d-orbital
to the peroxide s* orbital.24 The electronic absorption spectrum
of oxy-Hc reveals two charge-transfer bands that are assigned to a
peroxo-s and -p charge transfer to the cupric ion and these are
observed at�350 (e�20 000M�1 cm�1) and 550 nm (e�1000
M�1 cm�1), respectively. The latter are responsible for the
purple color of oxy-Hc. The side-on peroxide moiety bridging
the two copper(II) centers leads to a metal ion separation of
�3.6 A and efficient Cu(II)���Cu(II) antiferromagnetic coupling,
J>600 cm�1; the active site is essentially a diamagnetic species.25
3.07.2.3 Tyrosinase
Tyrosinase (Tyr) and catechol oxidase (CO) make up a unique
class of dinuclear T3 copper monooxygenases and oxidases
Figure 8 Crystal structure of the oxy-Tyr active site, which wasco-crystallized with the ‘caddie’ protein ORF378. A Tyr from ORF378,modeling an approach of a tyrosine substrate to the dicopper active siteis depicted in pink (PDB code: 1WX2).
154 Copper Enzymes
that have active sites structurally related to Hc. The His6 dicop-
per active sites also form a reactive side-on peroxo-dicopper(II)
intermediate (oxy-Tyr or oxy-CO) (Figure 8) possessing struc-
tural and spectroscopic properties similar to those found
in oxy-Hc, lmax �345 nm (e�18000 M�1 cm�1), Cu–Cu dis-
tance �3.6 A, and with an O–O bond distance of �1.4 A.9 In
addition to the oxy form, two other enzyme states have been
structurally characterized. The deoxy-dicuprous centers of both
Tyr and CO are structurally similar to deoxy-Hc where each
cuprous ion is three-coordinate, diamagnetic, and contains a
large Cu–Cu distance that is >4.0 A.26,27 In the met (resting)
form of CO, the two cupric ions form a diamagnetic core that is
bridged via a hydroxide or aqua ligand.27
Unlike Hc that just reversibly binds molecular oxygen,
Tyr is capable of exogenous substrate oxidation including
the o-hydroxylation and sequential oxidation of phenols to
o-quinones. Tyr’s are found in many organisms including
plants, animals, insects, and fungi and are associated with
insect resistance to pathogens26 and more commonly, the
pigmentation of skin and hair in animals. Biotechnological
application of tyrosinases in phenol dehalogenation may also
be possible.28 They are responsible for catalyzing the first steps
in the melanin biosynthetic pathway, which involves the four-
electron oxidation of tyrosine to L-dopaquinone.9 This net
four-electron oxygenation–oxidation is also known as the enzy-
me’s cresolase activity and the proposed Tyr mechanism is
shown in Figure 9. Kinetic and mechanistic studies using mush-
room Tyr have shown that the o-hydroxylation of the phenol is
rate limiting and in studies using a variety of substituted phe-
nols, a Hammett value of r¼�2.4 supports a rate-limiting
electrophilic aromatic substitution mechanism.29,30
COs are commonly found in plant tissues; however, their
biological function is not fully understood but they are
thought to play roles in plant disease resistance, flower color-
ation, and photosynthetic pathways.31 CO has a limited sub-
strate pool and is only capable of the two-electron oxidation of
catechols to quinones, as is shown in Figure 9, and which is
known as the catecholase activity of COs or Tyr’s.
The ability of Hc, CO, and Tyr to form the same side-on
peroxo intermediate while facilitating dramatically different
chemistry has led to great efforts to better understand those
factors dictating substrate oxidation and specificity. Based on
several crystal structures and mutagenesis experiments on Try
and CO, it has been proposed that solvent (and therefore sub-
strate) accessibility to the dinuclear copper core plays a crucial
role in dictating substrate reactivity.32 Both the catecholase and
cresolase activities have been observed in Hc when the dinuclear
copper site has been made more accessible to the solution by
either proteolytic cleavage or the addition of chemical denatur-
ants including SDS or urea.33–35 Itoh and coworkers33 have
shown that octopus Hc is capable of monooxygenase activity
via an electrophilic aromatic substitution mechanism with an
observed Hammett constant of r¼�2.0, similar to what is
observed in mushroom Tyr (vide supra), suggesting a common
reaction pathway. Further proposals by Decker and coworkers
suggest that substrate accessibility and the positioning of key
amino acid residues nearby properly orient the substrate
relative to the CuA active-site position that dictates cresolase
(monooxygenase) activity in Tyr; when access to CuA is more
sterically hindered, a decrease in phenolase activity and an
increase in catecholase activity is observed.32
The ability of synthetic copper/dioxygen adducts to facili-
tate the hydroxylation of phenolates was first reported in the
1950s by Brackman and Havinga.36–41 In the 1980s, Karlin and
coworkers reported an intramolecular arene hydroxylation
reaction facilitated by a dicopper–dioxygen complex with
a m-xylyl-linked binucleating XYL ligand. Later, kinetic and
mechanistic studies suggested an electrophilic aromatic substi-
tution mechanism facilitated by a m-Z2:Z2-peroxo-dicopper(II)
intermediate (Figure 10). The conclusion was supported
by Hammett analysis involving para-substituted arenes giving
rise to r¼�2.1.42
Several examples of mechanistic studies by the Itoh and
Stack laboratories have involved the intermolecular oxidation
and hydroxylation of phenols and/or phenolates by biomi-
metic copper(I)-dioxygen-derived complexes. The Itoh labora-
tory has shown in small-molecule complexes that the charge
and identity of the substrate have profound effects in dictating
the preferred oxidative mechanism and nature of products
when comparing anionic phenolate versus neutral phenol sub-
strates (Figure 11). The former react with a m-Z2:Z2-peroxo-
dicopper(II) complex where the major oxidative product is
the o-catechol, which proceeds via an electrophilic aromatic
substitution mechanism.43,44 However, when neutral phenols
were employed, the primary oxidative product is a coupled
dimer phenol consistent with radical formation.44 Support
for this conclusion came from Marcus analysis indicating
oxidation of the neutral phenol occurs via hydrogen-atom ab-
straction and a proton-coupled electron transfer (PCET) process.
Work by the Tolman47 and Stack46 laboratories demon-
strated that a rapid equilibrium can exist between the
m-Z2:Z2-peroxo-dicopper(II) and bis-(m-oxo)-dicopper(III) core(Figure 1245,46). Many factors dictate the position of this equi-
librium including the ligand donor ability (electronic effects),
solvent, sterics, and counteranion influences. Low-temperature
studies carried out by Stack and coworkers and illustrated in
Figure 13 provide evidence that coordination of the phenolate
to a copper ion in the m-Z2:Z2-peroxo-dicopper(II) core facili-
tates O–O bond cleavage forming a phenolate-bound bis-
(m-oxo)-dicopper(III) species.48 This can undergo internal
hydroxylation of the phenolate to give the corresponding
o-catechol. In addition to this product, the over-oxidized
CuII
CuII CuIICuII CuII
CuII
O
OCuI CuI
+O2
R
OHOH
H+
R
OH
H+
O
O
R
OHisN HisN
HisNHisNOH
O
R
O
R
O
H+
O
+ H2O
O
O
R
OOH
2H+
R
OO
H2O +
CuII CuII
CuII CuII
HisN
HisNHisN
HisN
HisN
HisN
HisN
HisN
NHis
NHis
NHis
NHis
NHis
NHis
NHisNHis
NHis NHis
NHis
NHis
NHis
NHis
NHisNHis
NHisNHis
NHis
NHisNHis
NHis
NHis
NHis
OH
R
OO
H2O + H+
R
OHHO
Oxidase/catecholaseactivity
Catechol oxidase(CO)
Monooxygenase/cresolaseactivity
Tyrosinase(Tyr)
deoxy oxy
met
Figure 9 Top: The proposed mechanism of oxidation of o-catechols to o-quinones by catechol oxidase (CO). Bottom: Proposed mechanism forthe oxidation of phenols to o-quinones by tyrosinases.
N
PY
PY
CuIIN
PY
PY
CuIIO
O
N
PY
PY
CuI
N
PY
PY
CuI
2+ 2+
N
PY
PY
CuII
O N
PY
PY
CuII
OH
2+
O2
N= Py
Figure 10 XYL model system demonstrating the intramolecular hydroxylation of arenes by a m-Z2:Z2-peroxo-dicopper(II) complex intermediate.See text.
Copper Enzymes 155
ND
D
N
N
= L LCuII O
OCuIIL
O
OHOH
OH
OHOH
Catecholmonooxygease activity
Phenol dimeroxidation via HAT
Anionic
Neutral
Figure 11 Monooxygenase versus HAT from phenolic substrates as described by Itoh and coworkers.
lmax ~ 360 nm
m-h2:h2-peroxo Bis-m-oxo
2+ 2+
~ 520 nm
nO–O ~ 750 cm-1
nCu–O ~ 280 cm-1
Cu–Cu ~ 3.6 Å
O–O ~ 1.4 Å
lmax ~ 300 nm
~ 400nmnO–O ~ NA
nCu–O ~ 600 cm-1
Cu–Cu ~ 2.8 Å
O–O ~ 2.3 Å
OO
CuII CuIII CuIIICuIIO
O
Figure 12 Equilibrium and physical properties comparing m-Z2:Z2-peroxo-dicopper(II) and bis-(m-oxo)-dicopper(III) complexes.Reproduced from Henson, M. J.; Vance, M. A.; Zhang, C. X.; Liang, H.-C.;Karlin, K. D.; Solomon, E. I. J. Am. Chem. Soc. 2003, 125, 5186–5192;Mirica, L. M.; Ottenwaelder, X.; Stack, T. D. P. Chem. Rev. 2004, 104,1013–1045.
156 Copper Enzymes
o-quinone is also detected in modest yield. These results pro-
vided the first experimental evidence evoking a bis-(m-oxo)-dicopper(III) complex as the active species for monooxygenase
activity in copper systems, possibly even in Tyr’s. It should
be noted also that Casella and coworkers have provided
an example of a copper(III)-oxo species that is capable of
o-hydroxylation of phenolates.49
3.07.2.4 Other Monooxygenases
3.07.2.4.1 Noncoupled dinuclear monooxygenasesAn important class of upper eukaryotic copper-dependent
monooxygenases that utilize molecular oxygen for the oxida-
tive transformation of neurological chemical signaling agents
yielding water and hydroxylated products includes DbMand PHM. DbM is a tetrameric glycoprotein (75 kDa per
monomer) and is involved in the catecholamine biosynthetic
pathway catalyzing the benzylic hydroxylation of dopamine to
norepinephrine (Figure 14). PHM is part of a larger enzymatic
complex called peptidylglycine-a-amidating monooxygenase
(PAM) in which a two-step process is responsible for the hy-
droxylation and N–C cleavage of the a-carbon of glycine-
extended pro-hormones; the product is a biologically active
amidated hormone; PAM consists of two domains – PHM is
responsible for the hydroxylation of the peptide backbone and
peptidylglycine-a-hydroxyglycine a-amidating lyase facilitates
the subsequent C–N bond-cleavage reaction.50,51 These
enzymes use ascorbate as exogenous electron donor to regen-
erate the reduced, so-called dinuclear copper active site,
which contains two chemically distinct and spatially separated
type 2 copper ions spanning an �11 A solvent-exposed cleft
(Figure 15).10,50,51 Although there are considerable differences
in size and substrate specificity, DbM and PHM contain �30%
sequence homology and the mechanism leading to substrate
hydroxylation is thought to be interchangeable.
The two chemically distinct copper centers in PHM/DbMare thought to play exclusive roles during enzymatic turnover.
CuH (¼CuA) is thought to serve as the electron-transfer site,
accepting reducing equivalents from ascorbate, and is coordi-
nated by two or three (depending on oxidation state) d-NHis
residues. Solution-state studies indicate that the CuH site loses
His172 as a ligand upon CuH reduction. However, exogenous
ligand binding/exchange at the reduced CuH site does not
occur even in the presence of strong copper chelators (i.e.,
carbon monoxide, azide, isocyanide, and nitrite) assuring a
narrow CuI/CuII redox couple for efficient electron transfer
during catalysis to the CuM site.52,53 At CuM (¼CuB), dioxygen
binding and substrate hydroxylation occur. The CuM site has a
unique coordination environment and in the oxidized form
of the enzyme is coordinated by two e-NHis, SMet, and a water
molecule. The Cu–SMet bond at >2.4 A shortens to �2.24 A
upon reduction, which is also accompanied by a loss of the
coordinated water molecule.54 The role of the methionine
coordination is not fully understood but must be important
during catalysis. One proposal is that it stabilizes the cuprous
oxidation state and inhibits the loss of copper upon reduction.
Significant insight into the mechanism of PHM occurred from
a 2004 crystal structure from the Amzel laboratory,55 which
shows a dioxygen-derived species, most likely superoxide,
bound in an end-on fashion at the CuM site, with ∠Cu–O–
O�110� and an O–O distance of 1.23 A (Figure 15).
Several mechanisms have been proposed over the past three
decades based on experimental (both on enzymatic and model
systems), structural, and theoretical studies. The generally ac-
cepted reaction mechanism proposed by Klinman51 for the
hydroxylation of C–H substrates by PHM/DbM is shown in
Figure 16 invoking substrate hydrogen-atom abstraction/
transfer (HAT) by a cupric-superoxo complex, i.e., the initially
formed copper(I)/O2 adduct. The Klinman and Blackburn54
laboratories demonstrated a reversible reaction with dioxygen
occurs prior to substrate activation.56,57 Computational studies
by Chen and Solomon suggest that C–H activation occurs via a
hydrogen-atom abstraction mechanism by most likely a side-
on Z2-bound cupric-superoxo species.58 Other CuI/O2-derived
species following reduction–protonation of the initial
LCuIIO
OCuIIL= L
NH
NHLCuIII
O
OCuIIIL
OBut
tBu
OHBut
tBu
OHO
But
tBu
O+
PhenolateO–O bondcleavage
30% 30%
Figure 13 Dioxygen bond cleavage induced via phenolate binding to a m-Z2:Z2-peroxo-dicopper(II) complex as described by Stack and coworkers.
O2 H2OAscorbate
N
O
H O
NH
CO2HR
HHN
O
H O
NH
CO2HR
OHHPHM
Glycine extendedpro-hormone
Active hormone
Dopamine Norepinephrine
NH2HO
HO
NH2HO
HO
OH
DβM
O2 H2O
N
O
H O
NH2R
PAL
Glyoxylic acid
PAM
Ascorbate
Hydroxylated product
Figure 14 Chemical transformations catalyzed by the noncoupled dinuclear monooxygenases. Top: DbM-catalyzed benzylic hydroxylation ofdopamine. Bottom: Oxidation of glycine-extended peptides yielding active amidated peptides mediated by the enzyme complex PAM, consisting of amonooxygenase domain PHM and lyase domain PAL.
CuM site CuH site
~11 Å
His172
Figure 15 The active site of PHM showing the spatial separationbetween the two copper ions and that an inhibitory substrate analog ispositioned in close proximity to the CuM site. Bottom left: The CuM siteexhibiting dioxygen binding in an end-on fashion. Bottom right: Expandedview of the electron-transfer CuH site (PDB code: 1SDW).
H-R O2
H2O OH+
Alkoxide Cuproduct
H-R •R
•R •R
H
H
HAT
Electrontransfer
CuMI CuMII
CuMII CuMII CuMII
CuMIICuHI CuHI
CuHII CuHII CuHI
CuHI
H+,+2e-,R–H
-R-OH
OR O• O
O OO•- O
Figure 16 Mechanism for the PHM mediated oxidation of substrateas proposed by Klinman.51
Copper Enzymes 157
superoxo–copper(II) complex, including a Cu(II)–OOH or Cu
(II)–O•$Cu(III)¼O species, have also been proposed to be
the active oxidant responsible for initiating hydrogen-atom
abstraction from the substrate.59,60 As a consequence, the Cu/
O2 species responsible for C–O bond formation is still under
some debate. A Cu(II)–O• or Cu(II)–OOH entity may facili-
tate C–O bond formation by attack/rebound to the substrate
radical. The different proposals are due to variations in the
mechanistic details concerning the ‘timing’ of electron transfer
from the CuH and the O–O cleavage event. A recent report
provides evidence in PHM for the formation of an inner-sphere
Cu–alkoxy complex formed during catalytic turnover.61,62
Significant efforts have been made to synthesize and
characterize mononuclear copper–dioxygen adducts such as
Cu(II)–O2•�, Cu(II)–OOH, and high-valent copper(III)-oxo
complexes as they may provide chemical insights into the
relevant Cu/O2 intermediates formed during PHM/DbM catal-
ysis. There are several examples of cupric-superoxo complexes
(with Z1 and Z2 binding modes) that have been described.
However, only a few have been shown to oxidize C–H sub-
strates (Figure 17(a)). The Itoh laboratory demonstrated
ligand hydroxylation facilitated by a cupric-superoxo species,
N
N
N
N
N
CuII CuII
CuII CuII
CuII –O2•- CuIII –O2
2-
O
O O
O O
OO
O
O•
NN
N
N
NN
N
N
N
NN
N N
NN
NN N
N
NN N
N
N
CuIIIOH
OH
NN
H
RR
HB
+
+
Bulky aryl substituentsnot shown
(a) (b)
(c) (d)
+
Figure 17 Synthetically derived copper(II) superoxo, peroxo, or hydroperoxo complexes, discussed in relationship to the PHM reactionmechanism. See text.
158 Copper Enzymes
which occurs at the benzylic position of the phenethyl group
attached to the tridentate ligand (Figure 17(a)).62 An example
of exogenous C-H activation by an end-on cupric superoxo
complex was reported by the Karlin laboratory suggesting rate
limiting hydrogen atom abstraction from the C-H substrate
was supported. An observed primary kinetic isotope effect
(KIE) of �12 was found upon selective isotopic labeling of the
substrate, this value is close to what is found for PHM/DbM for
their native substrates.63,64 Kitajima, Fujisawa and coworkers
characterized a cupric superoxo complex with side-on bound
dioxygen coordination mode exploiting the use of anionic tris
(pyrazolyl)borate ligands (Figure 17(b)).65,66 Tolman and
coworkers67 have also shown that the dioxygen fragment can
be formally reduced by 2e� to the peroxo state by a cuprous
diketiminate precursor, yielding a stable Cu(III)-peroxo com-
plex (Figure 17(b)). These latter examples have not yet been
demonstrated to exhibit substrate reactivity, probably because of
the ligand steric bulk employed in stabilizing the complexes.
Several examples of copper(II)-hydroperoxo complexes
have been reported in the literature with significant contribu-
tions coming from the Masuda, Itoh, and Karlin research
groups. Such complexes are typically formed by the deproto-
nation of hydrogen peroxide via addition of an organic base
such as triethylamine in the presence of a ligand–copper(II)
complex. However, most of them have little reactivity toward
organic substrates. A copper(II)-hydroperoxo complex that
has been structurally characterized employs an extensive
hydrogen-bonding network to stabilize the hydroperoxide
anion at the cupric metal center (Figure 17(c)). More extensive
investigations have demonstrated that hydrogen bonding to
the Cu–OOH proximal O-atom leads to stabilizing interac-
tions, whereas H-bonding interactions to the distal O-atom
results in destabilization.68 Reactivity studies performed by
Karlin and coworkers demonstrated intramolecular C–H
monooxygenase activity in hydroperoxo complexes of the 60-NR2–TMPA (TMPA¼ tris(2-pyridylmethyl)amine; R¼Me, Bz)
ligand, resulting in oxidative N-dealkylation and release of
formaldehyde (R¼Me) or benzaldehyde (R¼Bz).69,70 The
Karlin laboratory has also shown that a putative copper
(II)–OOH intermediate formed by the reduction/protonation
of the cupric-superoxo complex of TMG3tren leads to rapid
ligand oxidation and the formation of a structurally character-
ized copper–alkoxide complex (Figure 17(d)).71
3.07.2.4.2 Particulate methane monooxygenasesMethanotrophic bacteria utilize methane for growth and en-
ergy, with CH4 as their sole carbon source. The initial step in
the metabolism of methane by these organisms involves the
selective oxidation to methanol by methane monooxygenase
enzymes (MMOs) at ambient temperature and pressure.72–74
CH4 þNADHþO2 þHþ ���!MMOCH3OHþH2OþNADþ
Two broad classes of MMOs have evolved to perform this
function: a membrane-bound pMMO is present in most
methanotrophs and a soluble, cytoplasmic MMO (sMMO) is
expressed in several methanotrophs under conditions of cop-
per starvation.72–74 Although both MMOs utilize a metal
center to activate atmospheric dioxygen for attack on the strong
C–H bond in methane (104 kcal mol�1), their overall struc-
tures are completely different. The well-understood sMMO
employs an active-site diiron cluster to bind and activate dioxy-
gen for methane hydroxylation. Extensive bioinorganic re-
search over several decades has involved investigation of the
sMMO mechanism.75 In contrast, the metal content, organiza-
tion, and location of the active site in pMMO have been
surrounded by controversy in part due to the difficulties in
the isolation and purification of the enzyme.72,76–78
The first crystallographic study of pMMO, on a protein
isolated from Methylococcus capsulatus Bath (2.8 A resolution),
shows a structure comprised of three subunits, pmoA (b),pmoB (a), and pmoC (g), with molecular masses �24, �47,
and �22 kDa, respectively (Figure 18).79 Two copper centers
(a mononuclear copper and a dinuclear copper center) are lo-
cated in the pmoB subunit. The dicopper center exhibits a short
Cu–Cu distance of 2.5–2.7 A, where the ligands are provided by
His33, His137, and His139. The same structure was also
observed in a later study on the enzyme from Methylosinus
His 139
His 33 His 137
Glu 35
His 160
Asp 156
His 173
Glu 195
His 48
Gln 404
His 72
(a)
(c)
(b)
Tyr 373
Figure 18 The structure of the M. capsulatus (Bath) pMMO protomer. (a) Monocopper, (b) dicopper, and (c) zinc sites (PDB code: 1YEW).
His137
His139
Glu35Tyr373
Cu–Cu = 2.6 Å(2.57 Å, EXAFS)
Cu–Cu = 2.775 Å(DFT)
CuI
CuII CuIII
CuIHis33
CH4
CH3OH
O2, e-
O
O
Figure 19 Left: The dicopper active site as determined from thelow-resolution pMMO x-ray structure (PDB code: 1YEW). Right:A computationally derived suggested mixed-valent CuII–CuIII bis-m-oxoreactant complex for methane hydroxylation. See text.
Copper Enzymes 159
trichosporiumOB3b.80 Themonocopper site shows ligation from
His48 and His72 but this copper ion is absent in pMMO
from M. trichosporium OB3b.80 A third metal center occu-
pied by zinc is located �19 A from the dinuclear copper
site and ligated by Asp156, His160, and His173 from
pmoC and possibly Glu195 from pmoA. This zinc site is
replaced by copper ion in the structure of M. trichosporium
OB3b pMMO.80,81 More recently, a third crystal structure of
pMMO from Methylocystis species strain M has been deter-
mined to 2.68 A resolution and is the highest-quality
pMMO structure obtained to date providing a revised
model for the pmoA and pmoC subunits and leading to
an improved model of the M. capsulatus (Bath) pMMO
structure.81 It binds around two copper ions per 100 kDa
protomer. As in the M. trichosporium OB3b and
M. capsulatus (Bath) pMMOs, the pMMO from Methylocystis
sp. strain M contains a mixture of CuI and CuII and
exhibits a T2 CuII EPR signal. The recent investigation
included detailed activity studies, where copper ion loading
was varied, with maximal activity at 2 equiv. Cu, per sub-
unit, leading to the strong conclusion that the active site is
indeed a dicopper center. Still, there is controversy and a
lack of detailed understanding concerning the mechanism
of C–H activation in this enzyme.
Based on calculations, Yoshizawa and coworkers82,83 sug-
gested that following the dicopper(I)/O2 reaction, a one-
electron reduced CuIICuIII(�(O)2) species (Figure 19) formed
by electron injection into a bis-m-oxo-dicopper(III) intermedi-
ate would most likely facilitate methane oxygenation. Based on
their biochemical, spectroscopic, and computational studies,
Chan and coworkers76 also have suggested the presence of
a mixed-valent dicopper site in pMMO, although this as a
portion of a tricopper center.84 Solomon and coworkers85,86
deduced that a relatively simple CuII–O–CuII moiety on a
copper-loaded zeolite (CuZSM-5) is an active oxygenating
agent for the conversion of methane to methanol; perhaps
such a CuII–O–CuII moiety is relevant to pMMO (bio)
chemistry.77
3.07.2.4.3 Cellulose monooxygenaseThe processing of cellulose-containing material from naturally
occurring lignocellulosic biomass is considered a major chal-
lenge for the industrial production of sustainable liquid
biofuels.87 Nature uses a synergetic complex cocktail of
OHO
H
O
OHHO
ROO
OHHO
OR
OHH
OHO
O
OHHO
ROO
OHHO
OR
OHH
OHO
O−
O
HOHO
ROO
OHHO
OR
OHH
PMO−CuII
PMO−CuI
O2
PMO−CuII OO
H+, e−
H2O
PMO−CuII OOH
PMO−CuII O
HAT
e−
Figure 20 A cellulose oxidative breakdown mechanism proposed for GH61. See text.
2.9
2.9
Figure 21 Active site of the copper form of the GH61 cellulosedegrading enzyme (PDB Code: 3ZUD). See text.
O
OHOH
OHOOH
HO+ O2 + CO
O
OHOH
HO
OH
O2,3-QD
1
23
4
160 Copper Enzymes
cellulolytic enzymes to break down cellulose material, ulti-
mately leading to cellulose hydrolysis and the release of simple
sugars. A new family of metal-dependent glycoside hydrolases
was recently discovered, termed GH61; these enzymes are ca-
pable of degradation of crystalline cellulose.88 Zn,88,89 Ni,89 or
Cu90 bind at the N-terminus of the enzyme and x-ray crystal
structures have been reported. It was only shown recently that
the enzyme has the highest affinity for copper, with a
kD<1 nM, and that a sacrificial reductant is required to break
down cellulose.90,91 Analysis of the cellulose degradation
products indicates that it contains multiple oxidation products.
Thus, a general oxidative mechanism for the breakdown of
cellulose by GH61, as proposed by Marletta and coworkers,91
is shown in Figure 20. The crystallographic study revealed that
a single copper ion at the active site occurs in a near-tetragonal
D4h symmetric environment, also with a chemically modified
N-terminal histidine (Ne-methylated) binding in a bidentate
fashion through the d-nitrogen and amino nitrogen terminus.
A third histidine (His86) binds via its N-e atom and a polyeth-
ylene glycol molecule fills out the ligand equatorial plane.
A water molecule and Tyr residue show weak axial binding
interactions with Cu–O distances of �2.9 A (Figure 21). This
copper-binding motif in GH61 has structural characteristics
which in part resemble those of the so-called dicopper active
site of pMMO (vide supra), which facilitates the oxidative trans-
formation of hydrocarbons.
OHOQuercetin Depside
Figure 22 Quercetin 2,3-dioxygenase substrate and products.
3.07.2.5 Dioxygenases (Quercetin 2,3-Dioxygenase)Flavonol 2,3-dioxygenase, also called quercetin 2,3 dioxygen-
ase (2,3-QD), is the only copper dioxygenase enzyme found
in biology. 2,3-QDs play important roles in the degradation
of heterocyclic aromatic flavonols by cleaving their C2–C3
double bond, producing carbon monoxide and a more
easily degradable depside (phenolic carboxylic acid ester)
(Figure 22). In addition to copper, multiple other redox-active
metal ions including Fe, Mn, and Co have been shown to have
catalytic activity in 2,3-QDs.92,93
A few x-ray crystal structures of copper 2,3-QDs
are available.94,95 One of these supports an active site with
two different preferred conformations in an �70:30 ratio. The
dominant conformation has the copper active site in a pseudo-
tetrahedral coordination environment with three ligating histi-
dine residues and a fourth aqua ligand and the less favorable
conformation evokes the additional binding of a proximal
Copper Enzymes 161
glutamate to form a trigonal bipyramidal-coordinated copper
active site.94 Glutamate coordination to copper is rare in copper
biochemistry with the only other example found in the red
copper protein, nitrocyanin.96 The reactive species in the copper
2,3-QD enzyme–substrate complex forms upon displacement of
the water ligand by the C3-hydroxy group of quercetin.95,97
Substrate activation occurs when copper(II) binds as a mono-
anionic chelate with the ring 3- and 4-oxygen atoms, resulting in
the development of radical character on the 2-carbon as copper
ion is (formally) reduced. This facilitates dioxygen binding to
the cuprous ion giving a copper(II)-superoxide anion complex
where the distalO-atomattacks theC-2 radical, to formaCu–O–
O–C peroxide moiety. A series of subsequent steps lead to the
products, including carbonmonoxide (Figure 22). A large num-
ber of elegant model compound studies have been carried out
by Speier and coworkers.93
3.07.2.6 Copper Oxidases
3.07.2.6.1 Mononuclear copper oxidases3.07.2.6.1.1 Copper amine oxidase
Copper amine oxidases (CAOs) are ubiquitousmetalloenzymes,
found in bacteria, yeast, fungi, plants, and mammals,50,98,99
which catalyze the oxidative deamination of primary amines to
aldehydes via the reduction of O2 to H2O2.
RCH2NH3þ þO2 þH2O���!CAO
RCHOþH2O2 þNH4þ
CAO is a homodimer of 140–180 kDa subunits, and each
subunit contains a mononuclear (T2, ‘normal’) copper(II) site
and a quinone cofactor, 2,4,5-trihyroxyphenylalanine quinone
(TPQ) that is derived from an active-site tyrosine residue. The
copper ion is ligated by three histidines, a well-ordered axial
water ligand, and a more labile equatorial water ligand held in
a distorted square-pyramidal geometry; the TPQ cofactor lies
within 6 A of the Cu ion and is connected by a hydrogen bond
to the Cu ion via two water molecules (Figure 23). The CAO
TPQ
H2O
H2O
Figure 23 The crystal structures of the active site of CAO from Escherichiastructure of CAO using flash-freezing after exposure to substrate (PDB code:
catalytic reaction follows a classical ping-pong mechanism
involving two distinct half-reactions (Figure 24). In the reduc-
tive half-reaction, the enzyme oxidizes a substrate amine to an
aldehyde, generating the two-electron reduced aminoquinol
form of the TPQ. In the oxidative half-reaction, O2 oxidizes
the reduced enzyme back to its resting state, releasing NH4þ
and H2O2 (Figure 24).
The mechanism of oxidative deamination by CAOs is
well understood, but there is still some controversy surrounding
the role of the copper ion in cofactor reoxidation. The two
plausible pathways for the oxidative half-reaction mechanism
are illustrated in Figure 24. Does the initial electron-transfer step
occur via an inner- or outer-sphere pathway during the step
where reduction of O2 to H2O2 occurs?99,100 In the inner-sphere
pathway, with the release of aldehyde, the reduced enzyme
exists in an equilibrium between the two-electron-reduced
aminoquinol state (TPQAMQ) and the one-electron-reduced
semiquinone state (TPQSHQ). If TPOSHQ is involved, then one-
electron reduction of copper(II) would have occurred and a Cu
(I)-semiquinone intermediate (TPQSHQ) reduces O2 generating
a Cu(II)-superoxide intermediate (TPQSQ) and then via electron
transfer accompanied by protonation, a Cu(II)-hydroperoxide
iminoquinone species (TPQIMQ) is formed. The latter may un-
dergo hydrolysis, liberating NH4þ leaving the resting cofactor,
TPQOX.
On the other hand, the outer-sphere pathway does not
involve Cu(I) reacting with O2 in the initial electron-transfer
reaction. Rather, TPQAMQ directly reduces O2 to O2•� bound
in a hydrophobic pocket near the quinone cofactor.101,102 In
spite of those mechanistic differences, both pathways converge
at the CuII–OOH iminoquinone intermediate (TPOIMQ).
Notably, one of the x-ray crystal structures of CAOs was
found from Escherichia coli by flash-freezing after exposure to
substrate. It revealed a dioxygen-derived moiety bound close
to the active site; the oxygen species might be a hydroperoxo
moiety indicating that the TPQ group is in the TPQIMQ state
(Figure 23).103 There are still significant questions considering
TPQIMQ
O2
coli. Left: Reduced AO (PDB code: 1D6U). Right: The x-ray crystal1D6Z).
OH
NH2 NH2
OHCuII
CuII
CuII CuII
CuII
CuII
O2 O2TPQAMQ
CuI
TPQSHQ
O
O
O2-·
O2-·
TPQSQ
O
NH2
· +
O
2H+
2H+RCH2NH3+
RCHO
OH
NH2 NH2
OH
O2 TPQAMQ TPQSQ
O
O
2H+
O
NH2
OHCuII
OHO
RCH2NH3+
RCHOOH
H+
OH2
H2O
TPQIMQ
O
O
O
Inner-sphere pathway
Outer-sphere pathway
O
O
O
H2O
H2O2+ NH4+
TPQOX
TPQOX
Reductive half-reaction:E_TPQox + RCH2NH3+ E_TPQred + RCHO + H2O
E_TPQox + NH4+ + H2O2Oxidative half-reaction:E_TPQred + O2 + H2O
Figure 24 Proposed CAO catalytic mechanism illustrating the two proposed pathways for cofactor reoxidation.
162 Copper Enzymes
which species initially reduces O2 and whether the reoxidation
mechanism differs for CAOs purified from various sources.
Recently, Dooley and coworkers provided kinetic and
spectroscopic evidence supporting an inner-sphere reaction
pathway where the copper(I)-semiquinone intermediate re-
duces dioxygen.100,104
There are a few relevant model systems for CAO chemistry.
In order to provide mechanistic insight into the biosynthesis of
the TPQ cofactor in CAO, Itoh and coworkers showed that they
could generate a (methoxy-substituted) para-quinone ligand
derivative by reacting a copper(II)–phenol complex (meta-
hydroxyphenyl) with O2 in the presence of triethylamine in
CH3OH solvent at 60 �C (Figure 25).105 The ligand hydroxyl
group regiochemistry was found to be important for the suc-
cess in generating a para-quinone, that is, a TPQ cofactor
model. Further, this para-quinone copper complex derivative
can act as a catalyst for the aerobic oxidation of benzylamine to
N-benzylidene benzylamine as suggested for biological amine
oxidation involving copper and TPQ cofactors (Figure 26).105
3.07.2.6.2 Galactose oxidaseGalactose oxidase (GAO) is a well-studied radical monocopper
oxidase, which performs the efficient oxidation of alcohols to
aldehydes facilitated via an unusual free radical-coupled cop-
per motif.99,106–109 The active site of GAO consists of two
distinct one-electron acceptors, a Cu(I) metal center and a
relatively stable protein free radical forming a metalloradical
complex that acts as a two-electron redox site during catalytic
turnover. The overall catalytic reaction is comprised of two
separable half-reactions, oxidation of alcohol and reduction
of O2, via a ping-pong mechanism (Figure 27).107
In the first half-reaction, the alcohol is oxidized to the
aldehyde via the reduction of the Cu(II)-radical active site.
In the second half-reaction, O2 is reduced to H2O2, by the
reduced Cu(I)–tyrosine site reforming the oxidized Cu(II)–
tyrosyl radical state. In the x-ray structure of an ‘inactive’
form, GAO was shown to possess a unique active site contain-
ing a mononuclear copper ion coordinated by two histidines,
two tyrosines, and an exogenous H2O ligand, all held in a
square-pyramidal geometry (Figure 28).110 The most interest-
ing feature in the active site is the presence of a novel thioether
bond, forming a Tyr–Cys crosslink. This cofactor arises during
the protein biosynthesis through posttranslational covalent
modification of a tyrosine residue that becomes the redox
cofactor; a mechanism for Cu/O2 chemistry leading to the
Tyr–Cys moiety has been proposed.99,108 The proposed cata-
lytic mechanism for GAO has been deduced based on enzyme
research investigations, synthetic modeling,106,111 and theoret-
ical calculations112 (Figure 29).99,106–109 In the active form of
GAO, Try272 is oxidized to generate a Cu(II)-coordinated
phenoxyl radical species possessing a diamagnetic ground
state (i.e., EPR inactive) due to strong antiferromagnetic cou-
pling between the copper(II) ion and the tyrosyl radical.113
This Cu(II)–tyrosyl radical is responsible for alcohol oxidation
in the oxidative half-reaction of the catalytic cycle. In the
reductive half-reaction, electron transfer from Cu(I) to coordi-
nated dioxygen yields a CuII–OO•� species and this abstracts a
hydrogen atom from the tyrosyl hydroxyl group of the Tyr–Cys
cofactor to produce a CuII–OOH plus tyrosyl radical. The Cu
(II)–tyrosyl radical moiety is regenerated via release of H2O2
following proton transfer from the axial Tyr–OH to the CuII–
OOH complex (Figure 29).
N
N
N
OO
O
O
N
N
N
HO
CuII
CuII CuIICuII CuII
CuII
N
N
N
O O
N
N
N
-O
O
N
N
N
H
O
O
N
N
N
·O
CuI
O
CH3
CH3
2 equiv. NEt3
In MeOH
ET
N
C
CH3
CH3 CH3 CH3
CH3
O2
OOO
O
CH3OH
O2-H2O
Figure 25 The formation of TPQ model.
NN
N
O O
O
L
LCuII
LCuIIred
H2O2
O2
NH22
N
Figure 26 The catalytic reaction for the aerobic oxidation of benzylamine to N-benzylidene benzylamine.
HO HO
OH OH
OH OHOH
OH
OH
O
O2
GAO
H2O2
CHOO
Figure 27 GAO-catalyzed oxidation of D-galactose in the presence ofdioxygen to give D-galactohexodialdose and hydrogen peroxide.
Tyr495
H2OTyr-Cys
His
His
Figure 28 GAO active-site x-ray structure, highlighting the copper(II)coordinated Tyr–Cys cofactor (PDB code: 1GOG).
Copper Enzymes 163
Research aimed at themodeling of GAO active site has led to
the synthesis and characterization of a large number of copper
(II)–phenoxide complexes, to probe their chemistry and the
possibility of forming copper(II)–phenoxyl radical species;
the latter can be demonstrated to occur.106,111,114 Ghosh and
coworkers synthesized the copper complex, {[(3-tert-butyl-5-
methyl-2-hydoxybenzyl)(30-tert-butyl-50-methyl-20-oxobenzyl)(2-pyridylmethyl)]amine}Cu-(OAc), which is an excellent
structure and functional mimic of GAOs and is capable of cata-
lyzing the chemoselective oxidation of primary alcohols to the
corresponding aldehydes in high turnover numbers under
ambient conditions (Figure 30(a)).115 They also observed an
O
S(C228)
CuII
CuII CuII
CuII CuII CuII
ON N
H2O O· ·
·
·S(C228)
ON N
H2O O
S(C228)
N N
O
O
S(C228)
N N
O
Inactive Active
Reduced
e-RCH2OH
H2O
H2O
H2O2
O2
PCET
RCOH
(Y495) (Y495)
Y272 Y272
RH
H
(Y495)OH
Y272
O
S(C228)
N N
O
RH
(Y495)OH
Y272
H
O
S(C228)
CuIN N
(Y495)OH
Y272
HO· H
[CuII–OO·-]
(Y495)OH
Y272
O
S(C228)
N N
O
OH
(Y495)OH
Y272
[CuII–OOH]
Substrate oxidation: RCH2OH RCHO + 2e- + 2H+
Dioxygen reduction: O2 + 2e- + 2H+ H2O2
(oxidative half-reaction)
(reductive half-reaction)
or HAT
PCET
or HAT
Figure 29 Proposed galactose oxidase reaction mechanism.
N
N
OH
O CuII
CuII CuII
CuII
O
O
O O
Se
N
(a) (b) (c)
(d) (e)
CuII
OSe
ON
O
CuII
SeO
N
N N
O OtBu
tBu tBu
tBu tBu
tBu tBu
tBu
N N
O O
HH
CuSalred
[CuLSe(Et3N)] [CuLSe(PhCH2NH2)]2
CuSal
Figure 30 Model complexes for GAO/CAO.
164 Copper Enzymes
organic radical cofactor–Cu(II/I) redox couple analogous to
that thought to occur at the active site of GAO.115 Chaudhuri
and coworkers reported various copper complexes including
mono- and dinuclear Cu(II) compounds with the ligand 2,20-selenobis(4,6-di-tert-butylphenol) providing [O, Se, O]-donor
atoms, which serve as GAO and CAO model complexes.116
A mononuclear copper complex, [CuLSe(Et3N)], catalyzes the
oxidation of primary alcohols to the corresponding alde-
hydes (Figure 30(b)). A dinuclear copper complex, [CuL-Se(PhCH2NH2)]2 (Figure 30(c)), was found to be a catalyst
for the aerobic oxidation of benzylamine that occurs via a rate-
limiting substrate C–H hydrogen-atom abstraction pathway.116
Stack and coworkers synthesized two square-planar copper
complexes with tetradentate salen ligands (N2O2 system),
Copper Enzymes 165
[CuSal] and [CuSalred], which are structurally similar to the
active site of GAOs (Figure 30(d) and 30(e)).117 Both one-
electron oxidized forms, [CuSal]þ and [CuSalred]þ, react withbenzyl alcohol to yield 0.5 equiv. of benzaldehyde per com-
plex. Initial kinetic studies show that [CuSalred]þ reacts faster
with benzyl alcohol than [CuSal]þ.118 In order to gain insight
into the differential reactivity, they investigated the electronic
and geometric structure of [CuSal]þ both in the solid and
solution states. The locus of oxidation in [CuSal]þ is metal-
based, resulting in a Cu(III) complex formation in the solid
state and in the solution; however, a temperature-dependent
spin equilibrium between high-valent metal ([Cu(III)Sal]þ)and ligand-radical ([Cu(II)Sal•]þ) forms was observed, the
first example for copper (Figure 31). The researchers con-
cluded that the electronic structures of [CuSal]þand
N N
O OtBu ·+
tBu
tBu
tBu
CuII
Ligand radical or hig
Figure 31 GAO model compound investigation of a Cu(II)–phenoxyl radical
T2
1.7 ´ 106M-1s-1
2.2 ´ 106M-1s-1
e-, 3H+
CuICuI
CuI
CuI
CuI CuI
CuI
T1
T3
Metal subsitutionof T1 Cu site
HgIIHgII
His
t
TNC
Cu
Cu
O2
O2, H+
+ O2
+ O2
H2O
H2O
MII
OH2
OH2
Figure 32 The human ceruloplasmin active site and proposed reaction mec
[CuSalred]þ provide for the observed different reactivity with
benzyl alcohol.117
3.07.2.6.3 Multicopper oxidasesMCOs are a unique class of copper enzymes that couple the
one-electron oxidation of substrates, organics (e.g., ascorbate,
phenols, catechols, and diamines) or metal ions (e.g., iron(II)
and copper(I)) with the four-electron reduction of molecular
oxygen to water.10,11 MCOs contain at least four copper metal
ions per functional subunit, which include one of each tradi-
tional copper type: a T1 (blue copper site) that facilitates
electron transfer over 13 A via the peptide backbone (and
Cys–His electron-transfer pathway; Figure 32) to the separate
trinuclear copper site (TNC) where dioxygen reduction occurs.
The TNC includes one T2 (normal copper) and one T3
tBu
+
tBu
tBu
tBu
N N
O O
CuIII
h-valent metal?
versus Cu(III)–phenoxide electronic structure description.
10-3s-1
2H+
CuI
NI
PI
Cys
e-
ransfer
T1 site
13 Å
CuII
II CuII
CuIIII
CuIICuII
CuII CuII
H
OH2
H2O
OH2
OH2
O
OO
HO
hanism for MCOs. See text.
166 Copper Enzymes
(coupled dinuclear copper) site (Figure 32). MCOs that oxi-
dize metal-ion substrates possess an additional binding site
where substrates ligate and are oxidized. For example, Fe2þ
binds to a separate six-coordinate site in Fet3P.119 Other
MCOs have been suggested to be used for the detoxification
of reduced metals including Cu(I)120 and Mn(II)121. The fully
oxidized form of these enzymes is blue, hence their designa-
tion as ‘blue copper oxidases’ due to the presence of the
intensely colored T1 copper center. The TNC contains an
EPR-silent, hydroxo-bridged T3 copper site.
The proposed mechanism for dioxygen reduction by
MCOs is shown in Figure 32. When the native fully reduced
enzyme is exposed to dioxygen, a new species forms with
k�1.7�106 M�1 s�1.122,123 This complex is designated as the
native intermediate (NI), which has unique physical and spec-
troscopic properties including an EPR signal significantly lower
than g¼2.0. The NI can convert to the resting oxidized hydrox-
ide bridged compound, Figure 23.124 During catalytic turnover
(blue arrows, Figure 32), fast four-electron reduction of theNI to
the fully reduced enzymeoccurs. Experimentswhere the T1 site is
replaced with a redox inactive Hg2þ ion reveal a new intermedi-
ate, a peroxy species, PI.125 The peroxy fragment is thought to
bind to the TNC in a m3-Z1:Z1:Z2 fashion; PI slowly converts
to NI. A crystal structure on human ceruloplasmin demonstrates
a dioxygen-derived fragment that is bound side-on to both T3
coppers and end-on to the T2 copper ion (Figure 32).126
3.07.2.6.4 Heme–copper oxidases3.07.2.6.4.1 Cytochrome oxidase
CcOs or heme–copper oxidases (HCOs) are trans-membrane
enzymes that play crucial roles in aerobic respiration by acting
as the terminal electron accepter in the electron transport
chain.3,4,127–135 Molecular oxygen is reduced to water at a
bimetallic heme–copper active site. In addition to acting as
oxidases, they act as proton pumps resulting in the formation
of an osmotic/charge gradient spanning the lipid bilayer that
can in turn be used for cellular energy production in the form
of ATP. The net reduction of one molecule of dioxygen results a
net usage and/or movement of eight protons over the mem-
brane space with four protons consumed in the formation of
two water molecules and the translocation of four protons
across the membrane (eqn [1]).
4e� þ 8Hþ inþO2 ���!CcO4Hþ out þ 2H2O [1]
There are twomajor HCO superfamilies that include quinol
oxidases and CcOs. These differ in the number of metal sites
within the complex and the identity of the cofactor that sup-
plies electrons to the enzyme. Quinol oxidases are typically
found in prokaryotes and are comprised of three or four sub-
units. They contain three metal ions found at two sites within
subunit 1: one site contains a conserved bimetallic site consist-
ing of a high-spin (HS) heme (hemea3) and a proximal copper
ion (CuB) and this is where O2 reduction to water occurs. The
second metal site is a low-spin (LS) six-coordinate heme site
(hemea) that serves as an electron-transfer mediator from the
quinol electron-donor cofactor to the bimetallic heme–copper
site, where dioxygen binding occurs. The second major class of
HCOs is CcOs, which are found spanning the inner-membrane
space of eukaryotic mitochondria as well as in some aerobic
prokaryotes; these are typically multisubunit enzymes. Relative
to quinol oxidases, CcOs host two additional metal ions at a
dinuclear so-called CuA electron transfer site.
One electron at a time, the cysteine bridged mixed-valent
Cu(I)Cu(II) moiety accepts an electron from reduced cyt c, and
passes it on to the LS hemea site of subunit 1. The following
discussion on the active site and mechanism of HCOs will
focus on CcO.
There are several three-dimensional x-ray structures of CcO
derivatives, coming from both eukaryotic and prokaryotic
sources. These include structures from bovine heart,136 aa3-
type oxidases from Parracoccus denitrificans137–139 and from
Rhodobacter sphaeroides,140 and the ba3-type oxidase from the
thermophylic bacterium Thermus thermophillus.141 The spatial
arrangement of the three metal sites in reduced bovine CcO
is shown in Figure 33 (see below for more description). In
addition to the fully reduced and oxidized forms of the
enzyme, there are several derivatives of CcO including
a dioxygen-derived species (bound presumably as a peroxide
O22� moiety, see Figure 33), which have been crystallograph-
ically characterized,142,143 as well as CO136 and NO144 adducts
(bound to the heme or copper, respectively). These provide
valuable insights into local structure and thus mechanism of
reaction for CcOs.
The CuA site is the direct acceptor of electrons from the
reduced cytochrome c electron carrier (reductant) and it con-
tains two copper ions that are bridged by two cysteine thiolates
with a Cu–Cu distance of 2.58 A in the fully reduced state. The
mixed-valent Cu(I)Cu(II) oxidized form, best described as a
delocalized Cu1.5Cu1.5 moiety, accepts one electron at a time
from reduced cyt c, and passes it on to the six-coordinate LS
hemea site of subunit 1, which is �19.5 A distant. One of the
hemea axial histidine ligands is connected via a His–Phe–His
linker to the bimetallic hemea3–copper site. The hemea–
hemea3 angle is �104� and the Fe–Fe distance is �13.4 A.
The hemea3 site is high spin and has a proximal copper ion
positioned �5 A over the Fe center. The Cu ion is coordinated
by three histidine side chains, one of which has a unique Ne2–Ce2 linkage to a nearby tyrosine residue. This unique modifi-
cation has generated considerable interest into the role of this
Tyr during catalysis.4,145–147
The mechanism of catalytic dioxygen reduction at the
hemea3/CuB site is of considerable interest in terms of harnes-
sing the oxidative power of molecular oxygen for global
energy-transfer processes. HCOs are highly efficient and do
not release reactive partially reduced oxygen species (ROS)
into the surrounding environment. Several spectroscopic–
kinetic methods including time-resolved UV–Vis and rR
spectroscopy techniques applied to the fully reduced (Cu2Ia/
FeaII/Fea3
II–CuBI) as well as mixed-valent (Cu2
I/IIa/Fea
III/Fea3II–
CuBI) enzyme forms have revealed the identity of several
intermediates along the 4e� dioxygen reduction pathway.
However, certain details concerning O–O scission have
remained elusive and are still under debate. Below, we discuss
in more detail what are the generally accepted intermediates
pertinent to catalysis.
The first spectroscopic intermediate upon dioxygen expo-
sure to the fully reduced Fea3II� � �CuBI state affords a hemea3
ferric superoxo (A/Oxy) intermediate (Figure 34). This Oxy
species has spectroscopic features similar to what is found in
X-ray structure
His240-Tyr244Cross-linkHis290
His376
13.4 Å
His-Phe-
His
His291
CuB
Cua site
~22 Å
19.5 Å
Hemea3
Hemea3Hemea
Cu–FeCu–O
4.9 Å2.2 Å
1.7 Å2.2 Å
O–OFe–O
Figure 33 Arrangement of copper and heme centers in the bovine heart cytochrome c oxidase enzyme in the fully reduced state (left; PDB 1OCR)and the structure of a putative peroxo bridged heme–Cu active site (right; PDB 3ABL), also bovine heart enzyme.
HN
HN
HN
HN
HO
2H2O
2H+
H+
H+
FeIII
FeIV
FeIV
PM
F
Oxidized
2H+, 2e-
H+, e-
H+, e-
HOO
O
O·O
O
O-·
O
HO
HO
HO
HO
HO
HO(H)O
OH
HN
HN
FeII
FeIII
FeIII
A/Oxy
(Hydro)peroxo
O2+ 8H+(in)+ 4e-
(cyt. c reduced)
2H2O + 4H+(out)+ 4(cyt. c oxidized)
Overall reaction
Reduced
HN
HN
HN
HN
N N N
N
N
N N
N CuI
CuII
CuIICuII
CuII
CuI
NNN
N
N N
N N N
N
HN
HNN N N
N
N
N
O
Figure 34 Cytochrome c oxidase consensus mechanism for the catalytic reduction of molecular oxygen. Steps suggested to result in protontranslocation are indicated.
Copper Enzymes 167
168 Copper Enzymes
the end-on bound heme ferric superoxo species of hemoglobin
and myoglobin with rR n(FeIII–O)¼571 cm�1 and a UV–Vis
absorbance profile at l¼595 nm.148–151 At earlier times, there
is also some evidence that the incoming O2 molecule binds
initially to CuB, as the latter is sometimes referred to the active-
site ‘gateway’.152–154 It should be noted that a heme–Cu ‘bridg-
ing’ peroxo species can be generated as a stable entity and this
derivative has been crystallographically characterized by sev-
eral research groups (see Figures 33 and 37) although there is
recent questioning about the exact coordination described155;
rR spectroscopic experiments reveal that this dioxygen frag-
ment is in the peroxide oxidation state.156 However, this spe-
cies may or may not represent a catalytically relevant
intermediate.155
The second catalytic intermediate described has an absorp-
tion band at l¼607 nmandwas first postulated to be a ‘peroxy’
species; thus, it was coined the ‘P’ intermediate. However, later
isotopic labeling studies using molecular oxygen in conjunc-
tion with mass spectrometry demonstrated that the O–O bond
is already cleaved at this stage.157 An additional insight is that
one of the oxygen atoms derived from molecular oxygen is
exchangeable with bulk water. rR studies on this P (or PM, if
the mix-valent enzyme form is employed in the experiments)
intermediate reveals an isotope-sensitive band for the hemea3FeIV–oxo species with a n(FeIV]O) stretch at 803 cm�1, suggest-
ing that the moiety responsible for solvent exchange is located
at the CuB site and is most likely a CuIIB–OH/OH2 species
(Figure 34).158
Many questions arise when discussing the P intermediate.
A total of 4e� are required to fully reduce molecular oxygen to
the oxidation state level of water (oxide, if deprotonated);
however, only three electrons can be supplied by the dinuclear
metal site (1e� from Cu(I)!Cu(II); 2e� Fe(II)!Fe(VI)). This
has led to assignment of the origin of the fourth electron to
be the proximal tyrosine residue involved in the His–Tyr cross-
link found at the CuB site. This Tyr residue supplies the electron
in the form of a hydrogen atom (H•¼Hþ and e�), leavingbehind an organic tyrosine radical (Figure 34). Other protein
residues near to the CuB site have been suggested as elec-
tron donors and include a highly conserved tryptophan resi-
due, which is involved in p-stacking to one of the coordinated
histidines at the CuB ion.159,160 Postulations for hole migration
between these key Tyr and Trp residues have also appeared.
At the point of the formation of intermediate P, the
metal centers and proximal protein residues surrounding the
hemea3� � �CuB site have efficiently supplied the four electrons
required for the full reduction of dioxygen to water. However,
formally, only two reducing equivalents have been supplied to
the bimetallic site by the exogenous cyt c reductant, i.e., the
chemistry begins with O2-interaction with a reduced FeII–CuI
active site. The incorporation of a non-metal-based redox-
active moiety at the bimetallic site, i.e., H-atom donation by
the Tyr moiety, allows for efficient O–O bond cleavage without
the release of ROS.
The following steps of catalysis involve a series of sequential
electron- and proton-transfer events to replenish the active site.
The first equivalent of electrons/protons quench the organic-
based tyrosine radical leaving the persistent F intermediate,
which is a hemea3 FeIV–oxo complex. This species has a char-
acteristic broad absorbance at l�580 nm and rR spectroscopy
reveals that n(FeIV]O)¼785 cm�1.161,162 The fourth and final
reducing equivalent derived from cyt c reduces F, leading to the
formation of a ferric hydroxide species, n(FeIII–OH)¼450 cm�1 (Figure 34).154,161,163,164 Further protonation and
reduction lead to the release of water and regeneration of the
reduced dioxygen-sensitive hemea3� � �CuB site.
3.07.2.6.4.2 Models
CcO has served as the inspiration for the synthesis and study of
the intrinsic chemical properties of heme/copper complexes.
Highlighted in this chapter are several important findings per-
taining to dioxygen coordination and related chemistries of
small-molecule mimics of the bimetallic hemea3� � �CuB do-
main of CcO. For more information on small-molecule model-
ing of the Cua and hemeA sites, the reader is directed to the
following reviews: Kim et al.,4 Collman et al.,132 Collman and
Ghosh.133
One aspect of CcO modeling that received increased atten-
tion pertains to the details of the chemistry leading to the forma-
tion of the P intermediate. Considerable efforts have been made
to provide a better understanding and mechanistic details, that
is, those factors or requirements that lead to dioxygen reductive
bond cleavage. Several synthetic methodologies have been used
to synthesize and characterize 1:1:1 heme:copper:dioxygen
adducts/assemblies and such species have been derived via the
oxygenation of solutions containing stoichiometric amounts
of a reduced heme and copper(I) salt as well through the synthe-
sis of ornate hemea3 analogs containing an appended copper
chelate and/or an axial base moiety.4,132–134
Several dioxygen adducts have been observed using copper-
tethered heme systems. Collman and coworkers provided evi-
dence for an ‘Oxy’- like intermediate using a heme model
system based on the picket fence heme scaffold incorporating
a tridentate copper chelate with all imidazole donors in com-
bination with a tethered imidazole axial base.165 This room
temperature-stable, six-coordinate, ferric superoxo species
(Figure 35) complex exhibits diamagnetic properties as indi-
cated by NMR experiments. rR spectroscopic experiments
on the dioxygen-exposed species did not reveal an O–O
stretch in the �800 cm�1 region but instead only a vibration
assigned as a Fe–O stretch was observed at 570 (D ¼ 26 cm�1
upon 18O2 isotope labeling); this however, was suggested to be
similar to oxy-hemoglobin or oxy-myoglobin. Based on these
observations, Collman and coworkers suggested that the oxi-
dation state of the copper ion remains as cuprous and the O2
fragment is bound as a superoxide species.
Similar ‘Oxy’ intermediates have been observed by others. In
an elegant set of experiments carried out on a different heme–
copper complex, Naruta and coworkers demonstrated that
oxygenation of a fully reduced species leads to the formation
of a heterometalic, bridging peroxo intermediate (FeIII–O22�–
CuII), which slowly rearranges to the ferric–superoxo–copper
(I) (FeIII–O2•�–CuI) complex nFe-O¼ 574 cm�1 (D¼ 26 cm�1
upon 18O2 isotope labeling) (see Figure 35).166 This model
complex contains a histidine–tyrosine crosslink mimic in the
copper-chelating moiety, which is thought to aid in this rear-
rangement via stabilization of the ferric superoxo (FeIII–O2•�)
entity due to hydrogen bonding induced by the proximal
phenolic OH of the imidazole–phenol crosslink. Water was
also thought to be involved in the H-bonding network based
N
NN
1+
nFe–O= 570 cm−1
Δ(18O2) = −26 cm−1
nFe–O= 574 cm−1
Δ(18O2) = −26 cm−1
1+
N
N
HN
NN
N N
N
NH
NN
HN
NN
HN
CuI O
O
O
R
R
R
R�R�R�
F3C O
NFeIII
(O2•−)
O
N
NHN N
N
Mes
CuI
O
O
NFeIII
NH
N
N
NN
O
MesMes
tBu
−•O OHH
H
1+1+
Figure 35 Two examples of oxy heme–copper model complexes.
CuII
O O
FeIII FeIII FeIII
O O OO
CuII CuII
B(a) (b) (c)
Figure 36 Bridging peroxo binding motifs found for heme–Cumodel complexes, (a) m-Z2:Z1-peroxo, (b) m-Z2:Z2-peroxo, (c) end-onm-1,2 -peroxo species.
Cu–FeCu–Fe 3.9 Å O–O 1.46 ÅCu–O 1.91 Fe–O 1.89 Å
Fe–O(Cu) 2.03 Å
X-ray structure DFT calculated structure
4.01 Å 1.90 Å
O–O 1.40 ÅFe–O 1.82 ÅCu–O
Figure 37 Crystallographic and DFT-calculated structures of bridgingperoxo for heme–Cu model complexes. Left: Crystal structure seen byNaruta;167 Right: DFT-calculated structure of a low-spin heme–FeIII–peroxo–CuII synthetic complex.
Copper Enzymes 169
on the observation that solutions spiked with water led to
accelerated rates of ferric superoxo (FeIII–O2•�) production.
Although bimetallic peroxy-bridged intermediates have not
yet been directly observed in HCOs, chemical intuition suggests
that theymay form as fleeting intermediates prior to O–O bond
cleavage. Several examples of such heme/Cu complexes with
various electronic configurations and structures have been now
described (Figure 36) and they serve as useful starting points
for probing the sequence of events necessary for initiating O–O
bond scission. In general, the ligand environment enforced at
the copper site dictates the electronic-bonding description of
the bridging peroxo complex. Tetradentate copper ligands tend
to form m-Z2:Z1-peroxo species and observed peroxo O–O
stretches at n(O–O) �800 cm�1. Naruta and coworkers success-
fully crystallized a peroxy complex with this binding motif (see
Figure 37) using a tethered tetradentate TMPA-based copper
ligand.167 However, when tridentate copper ligands are
employed a m-Z2:Z2 peroxo complex is observed as indicated
by lower n(O–O) stretching frequencies<760 cm�1, which is a
hallmark of significant back bonding of the metal centers into
the p* peroxide molecular orbital.168
In both cases described above, the iron center of the heme is
HS and six-coordinate with two Fe–O bonds to the peroxy
moiety. However, recent work by Karlin and coworkers155
has shown that the spin state of the heme-iron center can
alter the electronic properties of the peroxo intermediate lead-
ing to a unique geometric bonding description similar to what
is observed in the CcO peroxo derivatives that have been
crystallographically characterized, possessing a proximal His
base.142,143 The oxygenation of a 1:1 mixture of the F8-Fe(II)
heme and the Cu(I) salt of the tridentate ligand AN results in
the formation of a (HS) heme m-Z2:Z2 dinuclear peroxy inter-
mediate with a n(O–O)¼756 cm�1 (Figure 38). However,
the addition of 1 equiv. of the axial base DCHIm (1,2-
dicyclohexylimidazole) leads to the formation of a new LS
species with a higher energy n(O–O) intra-peroxide stretching
frequency of 796 cm�1.155 DFT calculations in conjunction
with XAS studies suggest that this increase in O–O stretch is
the result of switching to an end-on m-1,2 binding geometry
(Figures 36 and 37). Axial coordination of the base leads to the
iron atom receding into the heme plane, resulting in a loss of
p* back bonding by the Femetal center to the peroxo fragment.
In addition, the change at the iron site is accompanied by a
switch from side-on to end-on binding of oxygen to the
Cu site. DFT calculations also suggest that this switch is
accompanied by an increase in both Fe–O and O–O bond
covalency, which is opposite to what is found in nonheme
iron systems where a strengthening of the Fe–O bond leads to
a weakening of the O–O bond. This unique binding mode to
the bimetallic site that is a result of a HS/LS spin switch may
have implications concerning the O–O bond-cleavage event
170 Copper Enzymes
by facilitating the formation of either a S¼1 or S¼2 iron
(IV)–oxo (FeIV]O) species.
A recent advance in the catalytic reduction of oxygen by a
heme–copper model complex comes from the Karlin labora-
tory using the heme–Cu model complex with the L6 binucleat-
ing ligand (Figure 39).169 Efficient solution phase oxidase
activity was observed in using this compound with exogenous
sources of protons (trifluoroacetic acid, TFA) and electrons
(decamethylferrocene (Fc*)). The rate-limiting step for reduc-
tion of oxygen appeared to change as a function of temperature
and provided evidence for the role of copper in the catalytic
cycle. At room temperature, the steady-state absorption spec-
trum was that of the fully reduced bimetallic FeII� � �CuI statesuggesting that the rate-limiting step for catalysis involved the
event in which molecular oxygen binds to the model complex.
In the absence of a copper ion (‘empty-tether complex’), the
rate of catalytic reduction was reduced around twofold from
27.3 (Cu) to 14 (no copper) M�1 s�1. This information sug-
gests that the role of copper is to assist in dioxygen capture and/
or directing this diatomic to the heme center.
By reducing the temperature to �60 �C, a new intermediate
was observed. At this temperature, the kinetics of both the
‘empty tether’ and copper-containing model systems for the
reduction of dioxygen are zero order with essentially the same
activation enthalpy (9.4 (with Cu) vs. 9.9 (‘empty tether’)
kcal mol�1). The steady-state absorption spectrum in the low-
temperature catalytic cycle is the same as that for which an
High spin μ-η2:
H
F ArFArF
ArF F
F
F
F
F
N
CuI
FeIII
N
H +
F8-FeII
AN-CuI
N
NN
N
NFeII
CuI
N
N
N
N
O
O2
O
Figure 38 Formation of a low-spin heme–peroxo–copper complex by imida
CuII
C
CuII
FeIII FeIII
L6 = Fc*
Fc*+
F
H+
excess
Steady stateat LT
2H2O
H2O
2Fc*+3H+
2Fc+
ArF
FAr
F
439, 557 nm
415
409, 505 nm
NN
NN
N N
F
(O2−)
(
NN
O
Figure 39 Catalytic mechanism for the four-electron four-proton reduction
excess of TFA is added to the bridging m-Z2:Z1-peroxo complex
formed upon dioxygen exposure to the fully reduced complex.
This new intermediate is postulated as a ferric heme hydroper-
oxo (FeIII–OOH)� � �CuII species. Thus, at low temperature,
O–O bond cleavage of the FeIII–OOH species is now rate
limiting, giving rise to the detailed mechanism for catalytic
dioxygen reduction by the L6-containing model complex
(Figure 39).169
3.07.2.7 Superoxide Dismutase
SODs are ubiquitous in aerobic organisms and are responsible
for the catalytic disproportionation of the deleterious radical
anion superoxide to the relatively benign products of dioxygen
and hydrogen peroxide.170,171 In higher eukaryotes, superoxide
forms primarily through incomplete O2-reduction occurring
during respiration,172 and xanthine oxidase action.173 In addi-
tion, superoxide generation is enhanced as part of the immune
response toward invading microorganisms.174 There are four
distinct classes of SODs divided according to the metal ion
held at the active site. These are the copper–zinc (Cu–Zn) SOD
(Figure 40) and iron, manganese, and nickel SODs; the latter
occur only in prokaryotic organisms. SODs do not require ex-
ternal reductants; the metal ion can act as both a one-electron
oxidant and reductant of superoxide. This is in contrast to
superoxide reductases which are only capable of reducing
superoxide and require a sacrificial electron donor.175–177
η2 peroxo Low spin μ-1:2 peroxo
ArF
ArFArF
FBase =
DCHImF
F
F
F
F
I CuIIN
H ++ N
N
NFeIIIN
N
N
N
N
NN
N
OO
zole derivative addition to a high-spin analog. See text.
CuII CuII
uII CuI
FeIII FeIII
Fc* Fc*+
eII FeII
Steady stateat RT
H+
422, 553 nm 422, 553 nm
, 538 nm
kobs= k(O2) = 26.4 M−1 s−1
418, 540, 558 nm
O2H−) (O22−)
O2
of dioxygen by a heme–copper dinuclear complex.
ZnII
His
HN NCuI
Arg+Arg+....O2· −
O2.−
ZnII
His
HNCuI
N
Arg+....−O2H
ZnII
His
CuII
−N N
H2O2
H+, H2O
Arg+
ZnII
His
HO
CuII
H
−N N
H2OO2·−
SOD2O2
· − H2O2 + O2
Arg+
H+
ZnII
His
O2
O2·−
CuII
−N N
OxidizedSOD
ReducedSOD
Figure 40 Proposed mechanism of SOD and crystallographically derived active-site diagrams. Note the neighboring Arg residue (PDB code:oxidized 1SDY, reduced 2C9U). See text.
Copper Enzymes 171
Cu–Zn SOD is commonly found in the cytosol, nucleus,
and peroxisomes of mammals as a homo dimer of 16 kDa
subunits. Many point mutations in Cu–Zn SOD have been
associated with the neurodegenerate disease amyotrophic lat-
eral sclerosis (ALS), ‘Lou Gehrig’s disease.’171,178–180 This
Cu–Zn SOD link with ALS is an important topic and bears a
large recent research literature.180,181
Cu–Zn SOD operates via a ping-pong mechanism where
superoxide is either oxidized or reduced depending on the
redox state of the copper ion at the enzyme’s active site (see
Figure 40). The zinc metal ion, which is separated by �6.6 A
from the copper site, is thought to play an active-site structural
role and it remains in a tetrahedral state in both the oxidized and
reduced forms of the enzyme. Metal substitution of the Zn site
with Cu(II), Co(II), or Cd(II) results in little loss of catalytic
activity.182 In the oxidized cupric state of Cu–Zn SOD, the
metal-ion center is pentacoordinate and is found in a square-
pyramidal coordination geometry with one water ligand, three
bound histidine imidazole groups, along with a fifth deproto-
nated histidine (i.e., as an imidazolate) ligand spanning the two
metal centers. The zinc(II) ion completes its overall tetradentate
coordination spherewith three additional ligands, which include
two histidines and an aspartate amino acid ligand. The aqua
ligand and the bridging histidines are lost in the reduced cuprous
state of the enzyme,making the copper ionoverall tridentatewith
the three histidines bound in a trigonal geometry. In this state, the
Znmetal site remains tetradentate with the once bridging histidi-
nate ligand now in the neutral protonated (i.e., with H–N(imid-
azole)) state and bound exclusively to the Zn ion.
Over and above the primary coordination sphere in Cu–Zn
SOD, a proximal arginine is found in the secondary coordina-
tion sphere. Mutagenesis of this residue to a hydrophobic
isoleucine residue results in a 90% loss of enzyme catalytic
activity.183 It is thought that the positively charged arginine
provides a means for the electrostatic attraction of superoxide
anion to the active site and/or it participates in hydrogen
bonding to superoxide, then facilitating docking to the copper
ion active site. The generally accepted reaction mechanism for
Cu–Zn SOD is given in Figure 40.184
There have been many structural and functional model
complexes for Cu–Zn SOD, including those emphasizing the
Cu–Zn bridging imidazolate moiety.185 We mention here only
one recent example. Moa and coworkers186 wished to provide
insight into the effects that a positively charged moiety located
in the secondary coordination sphere has on SOD activity of
synthetic copper complexes through the use of substituted
supramolecular cycloheptaamylose guest–host complexes
(Figure 25). In comparison to the SOD activity when there
was an appended alcohol versus a cationic guanidinium moi-
ety, they observed a 40% activity improvement for the latter.
The authors attributed this elevated SOD activity to the in-
creased ability of the guanidinium moiety to guide/attract the
anionic superoxide substrate to and from the copper active site,
in a manner that is analogous to what is attributed to the role
of the enzyme active-site Arg residue (Figures 40 and 41).
3.07.3 NOX Processing
3.07.3.1 Nitrite Reductase
Bacterial denitrification involves a multistep process where
nitrate (NO3�) is reduced to dinitrogen (N2).
187 Copper-
containing nitrite reductases (NiRs) catalyze the one-electron
reduction of nitrite to nitric oxide (NO) in the second step of
bacterial denitrification (Figure 42).187–189
NiR is a homotrimeric enzyme, in which each subunit con-
tains T1 and a T2 copper site.189,190 The T1 copper site, located
close to the surface of protein, accepts electrons from its natural
biological donor and transfers them one at a time to the T2
copper site where nitrite binds and is reduced. The T1 copper
OH
NH2
NH2
N CuII
NH2
NH2NH
NH2
NH2
+
N CuII
Figure 41 Illustration of a supramolecular copper complex catalyst model system for SOD activity, highlighting the importance of a nearbyguanidinium group. See text.
NO3− NO2
− NO N2O N2
2H+, 2e−2H+, 2e−2H+, e− H2O2H+, 2e− H2O H2OH2O
NAR N2ORNORNiR
Figure 42 The reaction of nitrate to dinitrogen in dissimilatory denitrification.
T2 site T1 site
His306
12.9 A
His255
His100 Asp98
His135
Cys136
e- transferHis-Cys link
Met150
His95
His145
H2O/OH-
Figure 43 T1 and T2 copper sites of NiR (PDB code: 2AFN).
His255
His306
His135
His100Asp98
Figure 44 Computational model for NO2-bound to the T2 copper site inNiR (PDB code: 1AS6).
172 Copper Enzymes
ion is coordinated by two histidines, one cysteine, and one
methionine. A LMCT from the sulfur of a cysteine to the oxidized
copper gives rise to the distinct greenor blue color ofNiRs,which
disappears when the copper becomes reduced. The T2 copper
center is tetrahedrally ligated by three histidines with a fourth
positionoccupiedbyoneof the following ligands:H2O,OH�, orNO2
�.189,190 The T1 is connected to the T2 site through a Cys–
His link for the facilitation of fast electron transfer (Figure 43).
In the last decade, the catalytic cycle of NiR has been the
subject of intense scrutiny and discussion, where a central
question has been, “what happens first at the T2 copper site,
nitrite binding or reduction of metal?”187,188,191,192 Crystallo-
graphic, ligand binding, electrochemical, and kinetic data sug-
gested the possibility of both routes.192,193 Further, the nature
of the NO2� substrate in NiR has been focus of a number of
biochemical, spectroscopic, and crystallographic studies. Re-
cently, based on DFT calculations, Solomon and coworkers
suggested that nitrite binds to the T2 copper ion via its two
oxygen atoms in an asymmetric bidentate fashion (Z2-O,O0)and that the enzyme reaction proceeds by initial protonation
which triggers electron transfer from the T1 copper center
(Figure 44).194
To understand the nature of NO2� substrate-binding
modes and its reductive cleavage, several biomimetic mono-
nuclear CuI–NO2� complexes have been synthesized leading
to the observation of a number of nitrite coordination modes.
Tolman and coworkers synthesized the first reduced copper
ion CuI–NO2� moiety, Li-Pr3CuI(NO2) (L
i-Pr3¼1,4,7-triisopro-
pyl-1,4,7-triazacyclononane),which contains an Z1-N bonding
feature.195 It was shown to undergo a stoichiometric conver-
sion to NO under acidic condition (Figure 45(a)). Hsu and
coworkers196 more recently synthesized a copper(I) nitrito
complex [Cu(Ph2PC6H4(o-OMe))2(ONO)], which contains
phosphane–ether ligands and is the first model compound
example with nitrite found in the asymmetric Z2-O,O0 binding
Copper Enzymes 173
fashion (Figure 45(b)). The Yeh laboratory reported a CuI–
NO2� complex with a pentadentate ligand with nitrite bound
in a unique Z2-O,O coordination mode.197 Protonation
of copper(I) nitrito complexes leads to the release of NO
(Figure 45(c)). Thus, all of the above complexes are good
functional models of the chemistry at T2 sites in NiRs.
3.07.3.2 Nitrous Oxide Reductase
Nitrous oxide (N2O) is a kinetically inert, colorless gas. In
biology, the reduction of N2O to dinitrogen (N2) is the last
step in the denitrification process of the bacterial nitrogen cycle
and is catalyzed by the copper-dependent enzyme nitrous
oxide reductase (N2OR).198,199 This reaction is thermodynam-
ically favorable (see equation, Figure 46); however it is kinet-
ically a difficult reaction. N2ORs have been characterized from
a variety of bacteria. The x-ray crystal structures of Pseudomonas
nautica (PnN2OR)200 and P. denitrificans (PdN2OR)201
revealed the presence of two multicopper centers per mono-
mer, CuA and CuZ, with distinctive spectroscopic properties
(a) (b)η1-N mode η2-O,O� m
N
N
N
CuI NO
O
OMe
Ph2POMe
C
O
LCu+ CH3CO2H
LCuI(NO2)
Figure 45 Copper(I)-nitrite model complexes showing bonding modes of c
CuA site CuZ site
N2O + 2e− + 2H+ N2 + H2O
Figure 46 Representation of the x-ray structure of the CuA and CuZ site of
(Figure 46). The CuA site is a delocalized mixed-valent
(Cuþ1.5Cuþ1.5) center proposed to mediate electron transfer
to the second multinuclear catalytic CuZ center. The latter
center consists of a novel m4-sulfide-bridged [4Cu:S] center
ligated by seven histidine imidazoles.202 In CuZ from
P. nautica (PnN2OR), all Cu atoms are bridged by a sulfur
atom. CuI and CuII are coordinated by two histidine residues
(His270/His325 and His79/His128, respectively), CuIII is
bound to His80, His376 and CuIV is coordinated to only one
ligand, His437. The CuIV site that is the most accessible because
of its single histidine ligand is believed to be where N2O binds
and is reduced, but so far no direct evidence exists. An extra
nonprotein ligand is present between CuI and CuIV and this is
believed to be an oxide (O2�), OH�, or most probably a water
molecule.201,203,204 The enzyme can exist in a variety of redox
states, depending on whether it is isolated aerobically or an-
aerobically. Spectroscopic studies revealed that the active form
of the enzyme is in a fully reduced state, Cu(I)4(S).205 Com-
putational investigations205,206 suggest that the N2O binds
through the terminal N-atom to CuI of the cluster and the
(c)ode η2-O,O mode
PPh2
uI
N
O
N N
P PCuI
Ph2Ph2
O ON
II(O2CCH3)2 + NO(g) + H2O
opper-nitrite and copper-nitrito complexes. See text.
His 325
His 270
His 376
His 80
His 128His 79
His 437
S
CuIII
CuII
CuIV
CuI
H2O/OH-
ΔG� = -340 KJ mol-1
N2OR from P. nautica (PDB code: 1QNI).
174 Copper Enzymes
terminal O-atom to CuIV, giving rise to a bent m-1,3-bridgedspecies, resulting in a reasonably low-energy pathway for N2O
reduction (Figure 47).207
An intriguing synthetic model system recently described
by Tolman and coworkers208 involves a complex copper–
sulfur cluster, [L3Cu3(m3-S2)]2þ (L¼1,4,7-trimethyl-triazacy-
clononane), which possesses a localized mixed-valent Cu(II)
Cu(I)2 core bridged in a m-Z2:Z1:Z1 fashion by a disulfide
moiety. This exhibits spectroscopic features similar to those of
the active site in N2OR and at low temperature reacts with N2O,
releasing N2 gas. Further investigations of this or other systems
should provide mechanistic insights and even suggestions for
alternative pathways for N2O reduction by N2OR.
Quite recently, a new crystal structure of N2OR from
P. stutzeri was obtained by Einsle and coworkers.209 The en-
zyme is a homodimer, found to be in a head-to-tail orientation
with the tetranuclear CuZ active site located in the N-terminal
domain and the dinuclear CuA site in the C-terminal cupre-
doxin domain. The distance of CuA–CuZ in one monomer is
around 40 A and thus too large for efficient electron transfer.
Instead, the CuA site of one monomer is at a distance of 10 A
from CuZ in the other monomer (Figure 48). Overall, the
S
CuI
CuIV Cu
II
CuIII
O
NN
S
CuI
CuIV
CuII
CuIII
N2O
2+
S
CuI
CuIV Cu
II
CuIII
O
2+
N2
H+ N2
S
CuI
CuIV Cu
II
CuIII
3+
HO
CuI4 state CuI
2CuII2 state
+ H+
2e−, H+
H2O
N2ORN2O + 2e- + 2H+ N2 + H2OOverall reaction:
Figure 47 Possible pathways for N2O reaction at the tetracopper CuZ.
M629
H583
H494H129
H178
F621
S2S1
IVII
IIIIH130
H433
H326 H382
CuZ
CuZ
H626
C618
C622
CuA
CuA
Figure 48 The CuA and CuZ sites of N2OR from Pseudomonas stutzeri (PDB
structure of isolated P. stutzeri N2OR is similar to the earlier
structures, but a major difference is that two sulfur atoms
instead of one are found together with the tetranuclear CuZsite. Based on x-ray diffraction data obtained by pressurizing
the crystals under N2O gas, and freezing the sample, the posi-
tion of the bound nitrous oxide ligand was found to be side-on
to the CuIII, CuIV, and S2 face.
There are still many questions remaining concerning the
exact nature of the CuZ active site, the number of S-atoms, the
binding site, and the coordination mode of an N2O substrate.
Further, an understanding of the detailed mechanism of N2O
reductive cleavage will require considerable future investiga-
tion, both enzyme and coordination chemistry.
3.07.4 Conclusion
Copper proteins participate in critical biological functions in-
volving redox chemistry. They serve as sites involved in elec-
tron transfer between proteins and in the binding and
reduction of important gaseous small molecules. These include
binding/reduction of molecular oxygen for O2 transport, sub-
strate oxygenation, and substrate oxidation coupled to O2
reduction. Nitrogen-oxide processing includes the reduction
of nitrite to nitric oxide (NO) or N2O to N2. The active sites
involved in this broad array of enzyme functions are varied,
consisting of mono-, bi-, tri-, and tetranuclear assemblies of
copper ions in a variety of coordination environments.
Scientific investigations over the last 40 years have provided
considerable insights, especially concerning protein and active-
site structure, details concerning copper ligation and coordina-
tion geometry, along with redox behavior. Considerable efforts
will be required to fully understand detailed aspects of sub-
strate binding to or near the copper ion(s) and reaction mech-
anisms. Given the general importance of the chemistry of
dioxygen and its reduced derivatives, as well as that pertaining
to the biological and environmental chemistry of nitrogen
oxide species, future investigations into copper protein
CuZ
CuZ–CuA: 40 Å CuZ–CuA: 10 Å
CuA
code: 3SBR).
Copper Enzymes 175
structure and function are needed. These areas of bioinorganic
chemistry promise to be both exciting and fruitful.
Acknowledgment
The authors gratefully acknowledge the financial support of
the National Institutes of Health, USA, for research support in
this field.
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