comprehensive inorganic chemistry ii || copper enzymes

29
3.07 Copper Enzymes RL Peterson, S Kim, and KD Karlin, Johns Hopkins University, Baltimore, MD, USA ã 2013 Elsevier Ltd. All rights reserved. 3.07.1 Introduction 149 3.07.1.1 Scope of the Chapter 149 3.07.1.2 Copper Chemistry Basics 150 3.07.2 O 2 Utilization 152 3.07.2.1 Fundamental Properties of Dioxygen 152 3.07.2.2 Hemocyanin 152 3.07.2.3 Tyrosinase 153 3.07.2.4 Other Monooxygenases 156 3.07.2.4.1 Noncoupled dinuclear monooxygenases 156 3.07.2.4.2 Particulate methane monooxygenases 158 3.07.2.4.3 Cellulose monooxygenase 159 3.07.2.5 Dioxygenases (Quercetin 2,3-Dioxygenase) 160 3.07.2.6 Copper Oxidases 161 3.07.2.6.1 Mononuclear copper oxidases 161 3.07.2.6.2 Galactose oxidase 162 3.07.2.6.3 Multicopper oxidases 165 3.07.2.6.4 Heme–copper oxidases 166 3.07.2.7 Superoxide Dismutase 170 3.07.3 NO X Processing 171 3.07.3.1 Nitrite Reductase 171 3.07.3.2 Nitrous Oxide Reductase 173 3.07.4 Conclusion 174 Acknowledgment 175 References 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 (O 2 ) and nitrogen oxides (NO X )), 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 1 1–5 for a listing of copper proteins and their properties.) Much of the biochemistry of copper surrounds the rich one-electron (e ) redox chemistry of copper shuttling within the Cu I /Cu II oxidation states, which can be easily accessed under physiological con- ditions using biologically available oxidants (i.e., O 2 ) and reduc- tants such as ascorbate and glutathione. Through evolution, Nature tunes the driving force of these electron-transfer events through modulation 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 (O 2 ) and nitrogen oxide (NO X ) processing by copper proteins along with our presentation of bioinspired studies using synthetic copper ‘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, O 2 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 1 7 ). 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 from molecular oxygen. The copper monooxygenases dopamine-b-monooxygenase (DbM), peptidylglycine-a-hydroxylating monooxygenase (PHM), and Comprehensive Inorganic Chemistry II http://dx.doi.org/10.1016/B978-0-08-097774-4.00309-0 149

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Page 1: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 2: Comprehensive Inorganic Chemistry II || Copper Enzymes

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.

Page 3: Comprehensive Inorganic Chemistry II || Copper Enzymes

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.

Page 4: Comprehensive Inorganic Chemistry II || Copper Enzymes

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.

Page 5: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 6: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 7: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 8: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 9: Comprehensive Inorganic Chemistry II || Copper Enzymes

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,

Page 10: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 11: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 12: Comprehensive Inorganic Chemistry II || Copper Enzymes

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.

OHO

Quercetin 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

Page 13: Comprehensive Inorganic Chemistry II || Copper Enzymes

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).

Page 14: Comprehensive Inorganic Chemistry II || Copper Enzymes

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).

Page 15: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 16: Comprehensive Inorganic Chemistry II || Copper Enzymes

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),

Page 17: Comprehensive Inorganic Chemistry II || Copper Enzymes

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.

Page 18: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 19: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 20: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 21: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 22: Comprehensive Inorganic Chemistry II || Copper Enzymes

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.

Page 23: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 24: Comprehensive Inorganic Chemistry II || Copper Enzymes

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

Page 25: Comprehensive Inorganic Chemistry II || Copper Enzymes

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).

Page 26: Comprehensive Inorganic Chemistry II || Copper Enzymes

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).

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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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