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BIOLOGICAL INORGANIC CHEMISTRY

Fraser Armstrong

Lecture 3 Cu and Zn as Catalysts in Biology

Cu

Most commonly, Cu is coordinated in a protein by two or more imidazole-N

(histidine) ligands. Thiolate-S (cysteine) is also found, particularly where the role is to engage
in fast electron transfer. Biological Cu has not been found to be coordinated by macrocyclic
ligands as found for Fe and Co. Cu is exploited for its redox properties either in electron
transfer or catalysis..
Unlike Fe which tends to be found inside cells, many Cu-containing cells are extracellular, i.e.
they are secreted. Cu appeared in evolution far later than Fe, probably due to its greater availability in
an aerobic environment. (Note: Cu2+ salts are more soluble than Cu+ but Fe3+ salts are less soluble
than Fe2+ .

Electron transfer
The best characterised Cu coordination site in
proteins is the Blue Cu centre, which is found in
small electron-transfer proteins and some enzymes
(oxidases). It has unique properties, including its
intense blue colour and a capability for extremely
fast electron transfer.
The Cu is usually
coordinated in a rigid protein scaffold (-sheet will
do this).
Shown at the right is the structure of plastocyanin,
which is a photosynthetic electron-transfer protein
found in plant chloroplasts.
The intense blue colour of the oxidized form is due
to a ligand-to-metal charge transfer transition
(cysteine-S to Cu(II)).

The ability to undertake efficient, very fast electron transfer without a high driving force
(recalling we know about Marcus theory) results from a Cu geometry that is constrained by the
protein to remain virtually unchanged regardless of whether the Cu is Cu(II) or Cu(I). Even
when the Cu is removed, the ligands remain in similar positions ! In most blue Cu proteins,
the Cu is coordinated by three strong bonds (N,N,S) in a nearly planar manner, with one or two
longer bonds to ligands that are axially disposed. In plastocyanin, there is one very long axial
bond to a methionine thioether-S. Effectively, the rigid protein hold the Cu close to the
transition state for Cu(II)/Cu(I) interchange.

HN

HN

N
HN

HN

2.06
N
Cu 2.07S
1.91
2.82
S
CH2
H3C

N
2.3

HN

CH2

2.1

Cu

2.1
S

CH2

HN

HS

CH2

2.9
S
H3C

oxidised

CH2

H3C

CH2

apo

reduced

In a few enzymes (the most important is cytochrome c oxidase) a binuclear Cu centre (known
as Cu-A) is used in electron transfer. Note that each Cu is coordinated by two cysteine
(thiolate) S and one imidazole-N, with a methionine (thioether-S) held in place at a longer
distance (too long for a proper bond). Note the similarity with the blue Cu centres. In the
oxidized form the unpaired electron is delocalized, again lowering reorganization energy (like
FeS clusters)
met

cys

his

his

met

cys

[Cu(II) Cu(I)]

+ e-

[Cu(I)Cu(I)]

Cu in O2 transport
Arthropods and molluscs (including lobsters, mussels and woodlice) carry O2 on a Cu protein
called hemocyanin (haemocyanin) which is contained in the blood plasma, i.e. it is not
contained in special cells (red blood cells) as hemoglobin is.

In the deoxy form, both Cu atoms are Cu(I). Note again the low coordination number typical
of late 3d elements, the three imidazole-N ligands, and the geometry which is a flattened
trigonal pyramidal at each Cu. The protein holds the two Cu(I) atoms apart at a separation
distance > 4 . The rapid coordination of O2 brings the two Cu atoms closer together. The
O2 binds 2 2, and is formally a peroxide, with each Cu now in oxidation state 2. The protein,
and the blood of these animals, is a blue colour.

Cytochrome c oxidase. How O2 is reduced to water in higher organisms.

Cytochrome c oxidase is located in the inner mitochondrial membrane.

Electrons (from cytochrome c)

4 H+ per O2 pumped across

membrane
Outer side
(intermembrane space)

mitochondrial
membrane

H2O
inner side
(matrix)
O2

The free energy of oxidation of cytochrome c by O2 ( > 50 kJ/mol cyt c) is used to produce a
proton (pH) gradient across the membrane (cytochrome c oxidase is a proton pump). The

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proton gradient is a way of storing energy and this energy is used to make ATP (using the
rotary motor enzyme ATP synthase). The O2-reducing site of cytochrome c oxidase contains
Fe porphyrin (like hemoglobin) and three-coordinate (tris-imidazole) Cu (like hemocyanin).

The atomic structure as revealed by x-ray diffraction is

shown at right, with an O2 molecule placed in position.
Proposed mechanism of reduction of O2.
Note the themes: O2 binding by Fe(II) porphyrin (like hemoglobin); then, assisted by the Cu, a
two-electron reduction to create a Fe(IV) ferryl intermediate (like peroxidase and P450); then
further reduction and release of water molecules. The energy drives conformational changes
that pump H+ across the membrane.
H-Y

O2

CuI

FeII

FeIIO

H-Y
CuI

2H2O

'oxy' (A)

2e, 2H+
HO
III OH

Fe

H-Y
II

Cu

HO

4H+

OUT

FeIV O

IN

O
e, H+

HO
FeIV O

CuII

H-Y

e, H+

CuII

H-Y

Cu centres involved in O2 reduction to water or O2 activation

to oxygenate substrates
Blue Cu oxidases.
Blue Cu oxidases catalyse one-electron oxidations (producing reactive radicals) of phenols,
bilirubin and ascorbic acid using atmospheric O2 as the oxidant in a four-electron reaction.
This has two very important applications in Biology: (i) formation of phenol radicals that
undergo further reactions, forming the skins of fruits; (ii) sequestration of O2 and its reduction
to harmless water (thus protecting the organism).

Laccase is an enzyme secreted by tree fungi to

oxidize lignin products (phenols) so that they can
be ingested as catechols. The active site for O2
reduction contains three Cu atoms, all
coordinated by imidazole-N from histidines.
Electrons flow to this site by long-range transfer
(12 ) from a blue Cu centre that is located
near to the enzymes surface. This blue Cu
centre converts phenols to phenol radicals. Here
is shown a sketch based on the crystal structure
in which a peroxido intermediate is coordinated
by two/three Cu. Another blue Cu oxidase
known as bilirubin oxidase, also found in fungi,
catalyses one of the reactions of Fe-porphyrin
degradation. Ascorbate oxidase is found in fruit
skins and protects the interior from O2.

Catechol oxidase
Catechol oxidase and tyrosinase catalyse the oxidation of phenols to produce the precursors for
melanin-type pigments (familiar consequences are the browning of apples, once the skin is
punctured and the aging of mushrooms).
O

OH
OH

O2

Their active sites contain two Cu atoms

coordinated in close proximity, each by three
imidazole-N from histidines, rather similar to
that observed in hemocyanin. Here we see the
structure of the active site of catechol oxidase,
with an inhibitor, phenylthiourea, coordinated.
It indicates how the aromatic ring can be held
close to an O2 molecule that is bound at the
same time between the two Cu atoms. We do
not discuss the mechanism here, but instead
leave a simple question.
Q. Next time you bite into an apple. How are two Cu enzymes responsible for the apple
tissue turning brown within a few minutes ?

Zn

Much progress has been made in understanding metalloenzymes that catalyze

reactions classed as being non-redox - transferases, hydrolases, lyases (dehydratases) and

isomerases. Catalytic activity of metal ions usually stems from their acidic properties.
Biological systems rarely have the extreme pH conditions under which catalysis by free H+ or
OH can occur. In any event, consider a hydrolase - the resulting effect of just having
extreme acid/base catalysis would be indiscriminate and all hydrolysable bonds would be
targets. Instead, Biology has harnessed properties (electron affinity) of certain metal ions, and
built them into protein structures designed to accomplish specific acid-base type reactions.

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Zinc is well suited for binding to proteins to hold them in a particular conformation: Zn2+ is
high in the IrvingWilliams series (and thus forms stable complexes, particularly to S and N
donors. It is also redox inactive, which is an important factor because it is crucial to avoid
oxidative damage in sensitive zones, particularly close to DNA.

The combination of strong

binding to protein ligands, rapid ligand exchange (coordinated H2O or substrate molecules)
good electron affinity, flexibility of coordination geometry, and no complicating redox
chemistry suits Zn(II) for its role in catalyzing specific acid-base reactions. Zn is also now
being discovered to have a crucial role in neurochemistry.
Note, however, that Biology has not used Zn to the exclusion of other metals for executing
acid-base catalysis. For example, numerous enzymes feature Fe(III or II) and Mn(II); Mg(II)
serves as the catalyst in ribulose 1,5-bisphosphate carboxylase (Rubisco the most abundant
enzyme on Earth) and Ni(II) is the active metal in urease, the enzyme that produces NH3 from
urea and allows the pathogen Helicobacter pylori to survive the acid environment of the
stomach, causing stomach ulcers and stomach cancer.

Acid-base catalytic mechanisms

The mechanisms of Zn enzymes are normally discussed in terms of two limiting cases. In the
'Zn-hydroxide mechanism', the Zn functions by promoting ionisation (deprotonation) of a
bound water molecule, thus creating a OH nucleophile that can now attack the carbonyl-C.
In the 'Zn-carbonyl mechanism', the Zn ion acts directly as a Lewis acid to accept an electron
pair from the carbonyl-O atom, and its role is therefore analogous to H+ in acid catalysis.
Analogous reactions occur with other X=O groups, particularly P=O.

There is an obvious advantage of achieving such catalysis at Zn or some other acid species that is
anchored in a stereoselective environment. In this lecture, we also see how catalysis depends on the
presence of other functionalities in the active site pocket, such as acids, bases, and hydrophobic
groups, each specifically positioned to carry out its task.

Some structures and reactions of zinc enzymes

Carbonic anhydrase; hydration / dehydration of CO2,

The high solubility of CO2 in water depends upon its hydration to form HCO3 .

However, the uncatalysed reaction at neutral pH is very slow, half-lives being measured in minutes.
Since turnover of CO2 by biological systems is very high, such a rate is far too slow to sustain

aerobic life in a complex organism. The CO2 / HCO3 equilibrium is important aside from CO2
transport since it provides a means for regulating tissue pH.

In 1932, Roughton and Meldrum at Cambridge University found an enzyme in erythrocytes that
catalyses this reaction with a remarkable rate enhancement. They called this enzyme carbonic
anhydrase (CA), but the enzyme is sometimes called carbon dioxide hydratase. In 1939, Keilin and
Mann showed that the enzyme contained one zinc per molecule. Although Zn had previously been
identified as an essential element, this was the first demonstration of an active role for the metal.

There are several forms of CA, all of which are monomers with MW ca. 30 000. CA II from red
blood cells has a turnover frequency for CO2 hydration of about 106 s1, making it one of the most
active of all enzymes.

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His

The crystal structure of human CA II shows that

a Zn atom is located in a conical cavity about 16
deep.

Thr
H2O

OH

The Zn is coordinated by three

Glu

histidine-N ligands and one H2O molecule. The

resulting geometry is that of a distorted

Zn

tetrahedron.
Triplet of His

Carbonic anhydrase reacts via the Zn-hydroxide mechanism. Limiting steps are Grotthus-type
proton transfer reactions (LHS of scheme) that re-set the Zn-OH site.

Bound OH
attacks C

H+ transfer steps
(Grotthus)

fast

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Carboxypeptidase (CPD); hydrolysis of peptide bonds

CPD is an exopeptidase which catalyses the hydrolysis of C-terminal amino-acids. There are two
types of Zn-containing enzyme (each synthesised as zymogens in the pancreas for secretion into the
digestive tract). CPD A is the best studied and acts upon terminal aromatic residues, whereas CPD
B acts upon basic residues.
CPD A is a monomer of MW 34 600. The x-ray structure shows that the Zn is located to one side of
a groove in which the substrate is bound.

It is coordinated by two histidine-N ligands, one

glutamate-COO- (bidentate) and one H2O.

The crystal structure of a complex formed with the inhibitor glycyl-L-tyrosine shows that C-terminal
aromatic group is in a -stacking interaction with the ring of tyrosine-248, the ligand water molecule
has moved away, and Zn is now bonded weakly to the peptide carbonyl-O atom and terminal aminoN. The water molecule, which is hydrogen-bonded to tyrosine-248 and glutamate-270, now attacks
the peptide carbonyl-C. This is therefore an example of the Zn-carbonyl mechanism.

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Alcohol dehydrogenase; oxidation of alcohols

Alcohol dehydrogenase (ADH) catalyses oxidation of various primary and secondary alcohols to the
corresponding aldehydes and ketones, using NAD+ as the e-/H+ acceptor. It is thus a redox enzyme
(an oxidoreductase). The role of the Zn is now to activate the substrate for reaction with the cofactor
(NADH is a hydride source).

RCH2OH

NAD+

RCHO

+ NADH

H+

ADH is a a dimer of MW 80 000. Each subunit contains two Zn atoms: one of these is the site of
catalysis and the other Zn stabilises the protein structure. The x-ray sructure shows that each subunit
has a deep cleft that separates two domains, one binding NAD(H) and the other binding the substrate
and providing ligands for the catalytic Zn. The ligands to the catalytic Zn are two cysteine-S, one
histidine-N and a H2O molecule. There are four protein ligands to the structural Zn, each is a
cysteine-S-.
View the mechanism from the point of view of reduction of the aldehyde: NADH is a hydride
donor, and the hydridic-H is the nucleophile.

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Alkaline phosphatase; hydrolysis of phosphate esters

Alkaline phosphatase (AP) catalyses the non-specific hydrolysis of phosphate monoesters.

R-OPO32- +

H2O

ROH

HOPO32-

Found in tissues as diverse as the intestine and bone, where it is located in the membranes of
osteoblasts - cells that form the sites of nucleation of hydroxyapatite crystals. Here, AP catalyzes the
general breakdown of organic phosphates, including ATP, to provide phosphate required for bone
growth. As its name implies, its pH optimum is in mild alkali region.
AP is a 2 dimer. Each subunit contains
two Zn atoms positioned in close
proximity.
The crystal structure of the
enzyme-phosphate complex reveals that the
phosphate ion (the product of the normal
reaction) bridges the two Zn ions, which
are only about 4.4 apart . The other
ligands to each Zn are histidines and
aspartate carboxylates.

glu

thr

Mg
asp

asp

his

ser
Zn

arg
Zn
phosphate
his

Simple models for Zn enzymes

There has been a lot of interest in producing catalysts that can
function as well as the Zn enzymes, but whereas they often
mimic structure and certain activity properties such as pH
effects, they are much less active. Examples include models
for carbonic anhydrase.

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The pattern of coordination of Zn centres

Catalytic Zn atoms (lower left) are coordinated by three protein ligands and one exchangeable
H2O. In most cases so far studied, the three protein ligands are arranged in the amino-acid sequence
as a closely spaced pair followed by a long gap before the third.
Structural Zn atoms (lower right) are coordinated by four protein ligands, usually cysteine and
histidine. There is no exchangeable site present because the object is to use the Zn atom to create a
particular fold of the protein and all Zn-coordination sites are needed.

Zn binding sites in proteins, which coordinate in these special amino-acid sequences, are
detectable in genes.

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Spectroscopic studies on Zn enzymes?

Illustration of the use of surrogate metals
Zn is silent to most common laboratory spectroscopic techniques. But it is often found that other
metals, particularly Co(II) or Cd(II) can replace Zn, giving substantial activity. The spectrum of
Co(II) substituted CA is shown here. A spectral change occurs between pH 5 and 8.

The spectral change is mainly due to ionisation of coordinated H2O. The spectrum is also perturbed
by other exogenous ligands, e.g. CN-, the substrate HCO3- and the inhibitor acetazolamide.