Tài liệu Báo cáo khoa học: Neuronal growth-inhibitory factor (metallothionein-3): reactivity and structure of metal–thiolate clusters* doc

MINIREVIEW
Neuronal growth-inhibitory factor (metallothionein-3):
reactivity and structure of metal–thiolate clusters*
Peter Faller
1,2
1 CNRS, LCC (Laboratoire de Chimie de Coordination), Toulouse, France
2 Universite
´
de Toulouse, UPS, INPT; LCC; Toulouse, France
Introduction
Metallothionin-3 (MT3) was originally dubbed neuro-
nal growth-inhibitory factor (GIF) [1] because of the
discovery that it is a factor in brain extract with the
ability to inhibit neuronal outgrowth. Moreover, MT3
or GIF was reported to be down-regulated in extract
from Alzheimer’s disease (AD) brain. Later on it
became clear that GIF belongs to the metallothionein
(MT) family based on its high cysteine and metal con-
tents. In mammals, the MT family consists of four dif-
ferent subfamilies designated MT1 to MT4 [2–4].
Mammalian MTs are composed of a single polypep-
tide chain of 61–68 residues. They are characterized by
a conserved array of 20 cysteines and the absence of
His and aromatic amino acids. MT3 contains 68
amino acids with 70% sequence identity to the MT1
and MT2 (MT1 ⁄ 2) isoforms. The MT3 sequence
contains two inserts: an acidic hexapeptide in the C-
terminal region and a Thr in position 5. Moreover, a
conserved Cys-Pro-Cys-Pro motif between positions 6
and 9 is unique to MT3 [1,4,5].
All mammalian MTs can bind a variety of different

mono-, di- and trivalent metal ions via their cysteine
residues. Most relevant under normal conditions in
biology is the binding of Zn
2+
and Cu
+
. However,
MTs can also bind other metals (Cd
2+
,Hg
2+
,Ag
+
,
Pt
2+
,Pb
2+
and Bi
3+
) when they are administered to
animals. Classically, the metal ions studied in detail
[mainly Zn(II), Cu(I), Cd(II), Hg(II) and Ag(I)] are
Keywords
bioinorganic chemistry; copper; growth
inhibitory factor; metallothionein; metal–
thiolate clusters; protein structure; zinc
Correspondence
P. Faller, CNRS, LCC 205, route de
Narbonne, 31077 Toulouse, France

Fax: +33 5 61 55 30 03
Tel: +33 5 61 33 31 62
E-mail:
*This article is dedicated to Prof. M. Vasak
on the occasion of his retirement
(Received 3 December 2009, revised 4 May
2010, accepted 17 May 2010)
doi:10.1111/j.1742-4658.2010.07717.x
Metallothionein-3, also called neuronal growth-inhibitory factor, is one of
the four members of the mammalian metallothionein family, which in turn
belongs to the metallothionein, a class of ubiquitously occurring low-
molecular-weight cysteine- and metal-rich proteins containing metal–
thiolate clusters. Mammalian metallothioneins contain two metal–thiolate
clusters of the type M(II)
3
-Cys
9
and M(II)
4
-Cys
11
[or Cu(I)
4
-CysS
6-9
].
Although metallothionein-3 shares these metal clusters with the well-
characterized metallothionein-1 and metallothionein-2, it shows distinct bio-
logical, structural and chemical properties. This short review focuses on the
recent developments regarding the chemistry of the metal clusters in metal-

lothionein-3, in comparison to those in metallothionein-1 and metallothion-
ein-2, and discusses the possible biological and functional implications.
Abbreviations
Ab, b-amyloid; AD, Alzheimer’s disease; apo-T, apo-thionein; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid)); GIF, neuronal growth-inhibitory factor;
M(II), divalent metal ion; MT, metallothionein.
FEBS Journal 277 (2010) 2921–2930 Journal compilation ª 2010 FEBS. No claim to original French government works 2921
bound in mammalian MTs collectively in two metal–
thiolate clusters located in independent domains
(a b-domain for the N-terminal cluster and an
a-domain for the C-terminal cluster). In the absence of
metals, apo-thionein (apo-T) is predominantly unstruc-
tured and only upon metal coordination is a defined
3D structure formed. The cluster structures for the
divalent metals have been resolved for MT1 ⁄ 2 and they
were found to be highly unusual compared with other
proteins (Fig. 1). The N-terminal b-domain (amino
acids 1-30) binds three divalent metal ions by nine
deprotonated cysteines in a hexane-like cluster [M(II)
3
-
Cys
9
], including three bridging and six terminal cyste-
ines. The C-terminal a-domain (amino acids 31-61)
binds four divalent metal ions by 11 deprotonated
cysteines in an adamantane-like cluster [M(II)
4
-Cys
11
],

including five bridging and six terminal cysteines. All
seven divalent metals are bound in tetrahedral coordi-
nation (Fig. 1) [4]. The precise cluster structure of the
Cu(I)
X
-CysS
Y
moieties in mammalian MTs has not yet
been determined, and the only structure available is
from the Cu(I)
8
-Cys
10
cluster in yeast MT (Fig. 2) [6].
It is unlikely, however, that mammalian MTs contain
such a cluster. Spectroscopic experiments on mamma-
lian MT1 ⁄ 2 indicate that they contain Cu(I)
4
-CysS
6-9
,
Cu(I)
6
-CysS
9-11
or Cu(I)
3 ⁄ 4
-Zn
x
CysS

x
clusters and
hence have a distinctly different cluster organization. In
all cases, Cu(I) shows a preference (at least partial) for
binding to the N-terminal b-domain [4,7].
These metal–thiolate cluster structures are unusual
for metal-binding proteins and are responsible for the
typical reactivity properties of MTs. First, the metal
binding is thermodynamically relatively stable, with
the following order Cu(I)>Cd(II)>Zn(II). The cluster
structure raises the possibility of a cooperative binding
of the metal ions. Cooperative binding has been
reported but is not always found, being dependent on
the type of metal, the type of cluster and the pH
[2,5,8]. In contrast to Zn(II)-MT and Cd(II)-MT, apo-
T is is oxidatively unstable under aerobic conditions as
a result of the formation of disulfide bridges [2]. Sec-
ond, in contrast to the high thermodynamic stability,
the binding is kinetically labile, allowing rapid intra-
molecular and intermolecular metal transfer. This is a
direct consequence of the relatively high structural
dynamics and flexibility typical of all MTs [5]. Third,
the deprotonated cysteines bound to the metal ions
(i.e. thiolates) are good nucleophiles, conferring a high
reactivity with radical species (HO·, O
2
·
)
, NO·) as well
as with alkylating and oxidizing agents. Such reactions

result in oxidation or derivatization of the cysteines
with subsequent metal release [4].
Although MT3 belongs to the MT family and hence
shares the unusual properties of their metal–thiolate
clusters, there are important differences between MT3
and the well-characterized MT1 ⁄ 2 [4]. Such chemical
and structural differences are probably important for the
biological roles of MT3, such as its growth-inhibitory
activity, the non-inducibility of its gene by diverse metal
ions and other compounds known to elicit the formation
of MT1 ⁄ 2, its predominant localization in the central
nervous system with an accumulation in zinc-enriched
neurons, and its possibility of being excreted into the
extracellular space [4,9,10]. Note, that the latter may not
be restricted to MT3 as evidence is accumulating that
MT1 ⁄ 2 also occurs extracellularly [4]. This is summa-
rized in Table 1 and discussed in more detail below.
Metal content of MT3
Initially, MT3 was isolated from human brain with a
metal content of four Cu(I) and three Zn(II) per MT3
Fig. 1. Scheme of the two metal–thiolate clusters containing
Zn ⁄ Cd in mammalian MT1 ⁄ 2. Left: four-metal cluster [M(II)
4
-Cys
11
]
localized in the C-terminal a-domain. Right: three-metal cluster
[M(II)
3
-Cys

9
] localized in the N-terminal b-domain. The scheme is
based on structures obtained by X-ray and NMR for MT1 ⁄ 2. MT3
contains the same type of four-metal cluster. M, divalent metal, S,
cysteine thiolate. Adapted from a previous publication [4].
Fig. 2. Scheme of the Cu(I)
8
(Cys)
10
cluster from yeast CuMT. Cu,
copper; S, cysteine thiolate. Adapted from a previous publication [6].
Metal–thiolate clusters in metallothionein-3 P. Faller
2922 FEBS Journal 277 (2010) 2921–2930 Journal compilation ª 2010 FEBS. No claim to original French government works
Table 1. Comparison between MT3 and MT1 ⁄ 2. NOS, nitric oxide synthase; ROS, reactive oxygen species.
Feature
Similarities Differences
MT1 ⁄ 2 and MT3 MT3 MT1 ⁄ 2
Putative
biological
function
Involved in Zn and Cu metabolism Synthesis not inducible Synthesis induced by Zn, Cu, Cd
Growth-inhibitory activity
ROS and NOS scavengers
(antioxidant)
Protects cultured neurons against
Ab toxicity [42]
Does not protect [42]
Localization Can occur extracellularly [14] Predominantly in the brain,
neurons and ⁄ or astrocytes? [3]
Ubiquitous; in the brain mostly in

astrocytes [3]
Primary structure 70% sequence identity 68 amino acids 61 amino acids
20 cysteines, same arrangements Acidic 6-amino-acid insert in the
C-terminal domain
Absence of 6-amino-acid and Thr
insert
Thr insert at position 4
Cys(6)-Pro-Cys-Pro(9) Cys(5)-Ser-Cys-Ala(8)
Metal content Binds without metal exposure Zn
and perhaps Cu. No
heterometallic Zn ⁄ Cu clusters
Isolated as a mixture of Cu and Zn
[1,11], but perhaps in vivo
predominantly Zn [13]
Isolated predominantly as Zn only
Zn binding 7 Zn(II) bound to 20 thiolates Additional specific (eighth)
Zn-binding site [30,31]
Similar overall apparent K
d
of Zn Cu
4
Zn
4
: 4.2 · 10
)12
M[32] Zn7:
1.6 · 10
)11
M [17]
Zn7: 3.2 · 10

)12
M [17]
Individual K
d
at
pH 7.4
Zn ⁄ Cd binding stronger in 4-metal
cluster than in 3-metal cluster
[18] Zn
3
and Zn
4
cluster:
non-cooperative? [21,22,30]
Not known Additional eighth site:
1 · 10
)4
M
7th Zn: 2.0 · 10
)8
M 6th Zn: 1.1 ·
10
)10
M 5th Zn: 3.5 · 10
)11
M
First 4 Zn: 1.6 · 10
)12
M
Cd binding 7 Cd(II) bound to 20 thiolates Additional specific eighth binding

site [30]
No specific additional sites [30]
Cd binding similar to Zn Cd
7
form less compact than Zn7
[26]
Similar compactness [26]
Similar overall apparent K
d
of Cd Cd
7
: 5.0 · 10
)15
M [17] Cd
7
: 1.4 · 10
)15
M [17]
More non-cooperative Cd binding
[17]
More cooperative Cd binding (at pH
7.4) [17]
Zn ⁄ Cd–thiolate
clusters
Cd ⁄ Zn
3
-CysS
9
in b-domain and
Cd ⁄ Zn

4
-CysS
11
in a-domain Same
connectivities in Cd form in Cd
4
cluster [5,23,24]
Dynamics Cd
3
-CysS
9
more dynamic than
Cd
4
-CysS
11
[5]
Cd
3
-CysS
9
very dynamic (precluded
structure determination by NMR)
[24,27,39]
Cd
3
-CysS
9
less dynamic, NMR
structure available [5]

Reaction with NO Cysteine oxidation and Zn release Zn release is faster [38] Zn release is slower [38]
Reaction with
ROS
Cysteine-oxidation and Zn-release
rates relatively similar [38]
Reaction with Pt
compounds
Reaction with Cys, Pt bound to
Cys
Cisplatin and transplatin react
faster [36]
Cisplatin and transplatin react
slower [36]
Cu(I) binding Cooperative formation of Cu
4
-
CysS
x
[33,43] Further forms:
Cu
8
MT (two Cu
4
-CysS
x
clusters in
each domain) Cu
12
MT (Cu
6

-CysS
9
and Cu
6
-CysS
11
)[33]
Cu
4
-CysS
8-9
[32,33] Cu
4
-CysS
6-7
[44]
Established that first Cu
4
-CysS
8-9
is
in the N-terminal domain [33]
Not established, but evidence
provided
Cluster stable in air [12,32] Cluster not stable in air [44]
Redox-labile site in Zn
4
cluster of
Cu
4

Zn
4
MT-3 [32] formation of
Cu
4
Zn
3
MT-3 with disulfide
Not studied
K
d
of Cu(I) K
d
estimated to be 1 · 10
)19
M Stronger Cu(I) affinity? [35] Weaker Cu(I) affinity? [35]
Cu(II) binding to
Zn
7
-MT
MT binds Cu(II) after reduction to
Cu(I) through cysteine oxidation
Formation of Cu(I)
4
Zn
4
MT-3 with
two disulfide bridges [37]
Zn form not studied
P. Faller Metal–thiolate clusters in metallothionein-3

FEBS Journal 277 (2010) 2921–2930 Journal compilation ª 2010 FEBS. No claim to original French government works 2923
[1]. A mixture of Zn(II) and Cu(I) has also been found
in MT3 isolated from bovine and equine brain [11,12]
as well as from mice with disrupted Mt1 and Mt2
genes [13]. In all cases only homometallic clusters have
been reported and the predominant Zn ⁄ Cu form might
thus be Cu(I)
4
Zn(II)
4
-MT3[4]. The physiological accu-
mulation of Cu in MT3 has been questioned as it was
speculated that in vivo MT3 binds Zn almost exclu-
sively [13]. This draws support from the demonstration
that during the purification of Zn(II)-MT3, metal
exchange reactions do occur. It could allow picking up
the more firmly binding Cu
+
during purification, but,
of course, yields no proof that MT3 functions solely as
a Zn protein(II) [13]. Clearly, further investigations are
needed to clarify how much, and under what condi-
tions, Cu(I) is bound to MT3. The binding of Cu to
MT3 has to be considered to occur not only intracellu-
larly because it is now known that MT3 also occurs
extracellularly [14] and evidence is accumulating that
Cu can be released into the synaptic cleft [15]. More-
over, there is evidence that MT3 can bind Cu(I) when
Cu homeostasis breaks down, such as in AD [16].
Binding of Zn and Cd to MT3

MT3 binds predominantly seven Zn(II) or Cd(II) ions
with overall apparent dissociation constants, at pH
7.4, of 1.6 · 10
)11
m and 5.0 · 10
)15
m, respectively
[17]. The three-metal cluster was less stable than the
four-metal cluster for Zn(II) and Cd(II) [18]. Initially,
information about the presence of two separate metal–
thiolate clusters came from spectroscopic studies and
comparison with other MTs of Zn(II)- and Cd(II)-
MT3, as well as their individual domains [12,19–22].
Precise structural data were obtained by NMR show-
ing a Cd(II)
4
-CysS
11
cluster in the C-terminal domain
with Cd(II)-Cys connectivities identical to those found
in the structure of human MT2 [23,24]. Such informa-
tion is still lacking for the N-terminal cluster, but
molecular dynamics simulation proposed a Cd(II)
3
-
CysS
9
cluster structure essentially identical to that of
MT2 [9,25]. Thus, apart from determining the Cd(II)
3

-
CysS
9
structure in MT3, the confirmation that Cd
replaces Zn isostructurally (as shown for MT2) is still
required. This seems important because the isostructur-
al replacement has been challenged for MT3 (but
not MT2) based on the observed difference in signal
intensity of the charge states of Zn(II)-MT3 and
Cd(II)-MT3 in ESI-MS, suggesting that with Cd(II)
the N-terminal domain [the M(II)
3
-CysS
9
cluster] has a
less compact structure with Cd than with Zn [26]. No
comparable difference was seen in MT2, indicating
that the more open structure in MT3 with Cd(II) is
not just the result of the larger ionic radius of Cd(II)
over Zn(II) and that the difference in amino acid
sequence plays a role.
One of the most important aspects is the greater
dynamics of the Cd(II)
3
-CysS
9
cluster of MT3. It was
observed that the resonances of Cd(II)
3
-CysS

9
in the
113
Cd(II) NMR were much less intense than those of
Cd(II)
4
-CysS
11
. Increasing the temperature did sharpen
them but their intensity was not enhanced [27].
Recently, Wang et al. [24] confirmed the low intensity
of the resonances of Cd(II)
3
-CysS
9
, although their dif-
ference from those of Cd(II)
4
-CysS
11
was smaller.
Moreover, NMR measurements of mouse MT3 and
human MT confirmed a high dynamical structure
caused by rapid internal motion (mostly of the first 12
amino acids) [24] and this was viewed as conforma-
tional exchange broadening. This dynamic structure,
only observed in the Cd(II)
3
-CysS
9

of MT3, is intrigu-
ing because it correlates with the growth-inhibitory
activity [9,28] as well with the higher chemical reactiv-
ity of the metal cluster (e.g. with NO·, see later).
Moreover, it is also the reason why the determination
of the spatial structure of the N-terminal domain con-
taining the Cd(II)
3
-CysS
9
cluster was thus precluded.
For a more detailed discussion of the dynamics of the
protein structure and its implication for the biological
function, see the minireview by Huang et al. in this
minireview series [29].
What is the reason for the higher dynamics of the
MT3 structure? Initially it was proposed that slow
exchange occurs between alternative configurations
involving CysS-Cd(II) bond breaking ⁄ formation in the
conversion, which may include a cis–trans isomeriza-
tion of the Cys-Pro amide bonds in the Cys
6
-Pro-Cys-
Pro(6–9) motif [17]. Only one configuration is detected
by
113
Cd(II)-NMR, whereas the other is broadened
beyond detection by fast exchange processes. However,
another possibility has been proposed by Palumaa
et al. [30]. They found, by ESI-MS, that Zn(II) and

Cd(II) do not bind cooperatively to MT3 at pH 7.3.
Upon adding seven equivalents of Cd(II) or Zn(II) to
MT3, distributions of metal loading were detected,
ranging from five to nine for Cd(II) and from six to
eight for Zn(II) (note that identical experiments with
MT1A showed more homogeneous M(II)-binding of
seven metals per MT [30]). This would mean
that Cd(II)
7
-MT3, and, to a lesser extent, also Zn(II)
7
-
MT3, are heterogeneous in their metal content,
and that metal-exchange reactions between different
metal-loaded forms could be the reason for the higher
dynamics detected in NMR. However, this conclusion
has its limits, first because the MS analysis is not
quantitative and, second, because more recent MS
Metal–thiolate clusters in metallothionein-3 P. Faller
2924 FEBS Journal 277 (2010) 2921–2930 Journal compilation ª 2010 FEBS. No claim to original French government works
measurements did not confirm binding of more than
eight equivalents of Zn(II) [31]. Importantly, the bind-
ing of the eighth metal ion was confirmed by other
methods and studied in more detail. [31] An additional
equivalent of Zn(II) can bind to Zn(II)
7
-MT3 [but not
to Zn(II)
7
-MT2] with an apparent K

d
of 0.1 mm. Simi-
larly, Cd binds to Cd(II)
7
-MT3 but with an even stron-
ger affinity (exact K
d
not reported). Binding of either
additional ion disturbs the CD signatures of the thio-
late clusters, indicating interference with the cluster
structure. Moreover, a decrease in the Stokes radius
was observed, suggesting a mutual approach of the
two domains. Also, the additional binding of M(II)
induced slow, but appreciable, non-covalent dimeriza-
tion of MT3 [40% for Zn(II) and 80% for Cd(II)].
Subsequent analysis of
113
Cd(II) NMR revealed com-
plete loss of the Cd
3
-CysS
9
resonances. The reported
data indicate that the b-domain provides the binding
site for the eighth equivalent of Zn(II) [and Cd(II)].
Moreover, the occurrence of a weaker eighth binding
site in Cd(II)
7
-MT could be involved in metal-
exchange reactions and hence contribute to the flexibil-

ity of the Cd(II)
3
-CysS
9
cluster. This is supported by
the almost complete loss of resonance intensity upon
binding of an additional Cd(II).
Disulfide bond formation might be another way to
produce heterogeneity and ⁄ or increased structural
dynamics in the b-domain, leading to partial disulfide
bonds and ⁄ or to disulfide exchange reactions, respec-
tively. No evidence for disulfide bond formation in
Zn(II)
7
-MT3 has been reported. Nevertheless, it might
be worthwhile to re-investigate this point because
oxidation of cysteine was noted in the Zn(II)
4
-CysS
11
cluster of freshly prepared Cu(I)
4
Zn(II)
4
-MT3 [32].
Definitive studies will be important to explore the
isostuctural replacement of Zn(II) with Cd(II), by
monitoring the peptide structure and the flexibility of
Zn(II) and Cd(II) by NMR spectroscopy. Another
interesting (and to my knowledge not yet reported)

experiment would be assessment of the growth-inhibi-
tory activity of the Cd-containing form of MT3. If
affirmed, this would support true isostructural replace-
ment, as this MT3-specific activity is believed to be a
structure-dependent feature. A problem with such a
measurement could be the toxicity of Cd(II). Neverthe-
less it might work as Cd(II) is tightly bound to MT-3
and Cd(II)-MT1 ⁄ 2 could be used as a control, the
issue of Cd toxicity may be overcome. So far the struc-
ture determination of the b-domain of Cd7-MT-3 by
NMR was hampered by the high structural dynamics
and exchange reactions. The recently gained insights
discussed above (dimerization, disulfide formation,
additional Zn(II) ⁄ Cd(II) binding and Mg ⁄ Ca effects)
might be used to slow down or increase the time
regime of exchange reactions as such that they are
more favorable for NMR studies [30–32].
It seems clear now that Zn
7
-MT3 and Cd
7
-MT3 can
bind specifically an additional eighth equivalent of
Zn(II) or Cd(II). Binding of more Zn(II) ⁄ Cd(II) is very
likely to be non-specific. Whether the binding of seven
equivalents of Zn(II) ⁄ Cd(II) in MT3 is more heteroge-
neous than the binding of seven equivalents of
Zn(II) ⁄ Cd(II) to MT1 ⁄ 2 is still not known. There are
indications that binding of Cd is less cooperative in
MT3 compared with MT2 [17]. For Zn this is less clear.

Binding of Cu(I) to MT-3
The spectroscopic characterization of MT3 isolated
from bovine and equine brain showed that Cu is
bound in the oxidation state I in a four Cu(I)–thiolate
cluster, Cu(I)
4
-CysS
X
[12,20]. Furthermore, reconstitu-
tion experiments with human apo-T3 and its separate
domains reproduced closely the features of the isolated
native MT3 forms (bovine, equine) [32,33]. Moreover,
titration experiments of Cu(I) to apo-T3 (or to apo-
a- and apo-b-domains) showed cooperative formation
of the Cu–thiolate cluster involving eight or nine cyste-
ines [i.e. Cu(I)
4
-CysS
8-9
] [21,22,33]. In apo-T3 the first
cluster formed, Cu(I)
4
-CysS
8-9
, was localized in the
N-terminal domain [33]. The structure of this Cu(I)
4
-
CysS
8-9

cluster is not known, but extended X-ray
absorption spectroscopy data yield a Cu-Cu distance
of 2.67 A
˚
and a Cu-S distance of 2.26 A
˚
. The latter
points to mainly trigonal coordination of Cu(I) [the
correlation would predict one or two digonal bound
Cu(I)]. This clearly suggests that Cu(I) and Zn(II)⁄
Cd(II) bind preferentially to the b- and a-domains,
respectively, and that no heterometallic clusters con-
taining both Cu(I) and Zn(II) ⁄ Cd(II) are formed.
Addition of Cu(I) beyond four equivalents results in
the cooperative formation of a Cu(I)
4
-CysS
8-9
cluster
in the C-terminal domain. After completion of the two
Cu(I)
4
-CysS
8-9
clusters, further addition of Cu(I)
results in the formation of Cu(I)
6
-CysS
9
clusters and

Cu(I)
6
-CysS
11
in the N-terminal and C-terminal
domains, respectively [33].
The dissociation constant of Cu(I) to MT3 has not
been measured, but by analogy with other MTs it can
be estimated to be around 10
)19
m [34]. Judged from
the higher reactivity of Cu(I)-MT1 with 5,5¢-dithio-
bis(2-nitrobenzoic acid) (DTNB) compared with Cu(I)-
MT3, it has been proposed that the affinity of MT3
for Cu(I) is higher than that of MT1 ⁄ 2 [35]. One of
the remarkable features of Cu(I)
4
-CysS
8-9
clusters in
the isolated Cu(I)
4
-Zn
3-4
MT-3 is its stability in air.
P. Faller Metal–thiolate clusters in metallothionein-3
FEBS Journal 277 (2010) 2921–2930 Journal compilation ª 2010 FEBS. No claim to original French government works 2925
The spectroscopic features of isolated Cu(I)
4
-Zn

3-4
MT-
3 did not change over time in air [12]. The Cu(I)
4
clus-
ter in freshly reconstituted Cu(I)
4
-Zn
4
MT-3 also
seemed to be stable, although oxidation occurred in
the Zn cluster [32]. Indeed, exposure of Cu(I)
4
-
Zn
4
MT3 to air resulted in the slow formation of a
disulfide linkage by cysteine oxidation of the a-domain
and a concominant release of one Zn ion, but the spec-
troscopic features of the Cu(I)
4
cluster did not change.
This might indicate that in the isolated Cu(I)
4
-Zn
4
MT3
this oxidation had already occurred (for further discus-
sion see below). The molecular basis of the air stability
of the Cu(I)

4
cluster is not known. Cu(I)–thiolates are
normally oxidized by molecular oxygen, resulting in
the formation of disulfide bonds (Eqn 1). Thiyl radi-
cals have been observed as an intermediate. The mech-
anism might be oxidation of Cu(I) by oxygen (Eqn 2).
The formed superoxide might oxidize a further equiva-
lent of Cu(I) (Eqn 3). A two-electron reaction of Cu(I)
with oxygen yielding H
2
O
2
is probably favored (Eqn
4) as the reduction of O
2
to O
2
•)
is thermodynamically
unfavored, but the two-electron oxidation of O
2
to
H
2
O
2
is favored.
2CuðIÞþ2S
À
þ O

2
þ 2H
þ
! 2CuðIÞþS À S þ H
2
O
2
ð1Þ
CuðIÞþO
2
! CuðIIÞþO
À
2
ð2Þ
CuðIÞþO
2
À
þ 2H
þ
! CuðIIÞþH
2
O
2
ð3Þ
ðEqn1þ Eqn3Þ2CuðIÞþO
2
þ2H
þ
! 2CuðIIÞþH
2

O
2
ð4Þ
CuðIIÞÀS
À
! CuðIÞÀS

ð5Þ
2S

! S À S ð6Þ
Then, Cu(II) can oxidize thiolate to thiyl (Eqn 5)
and two thiyls form a disulfide bridge (Eqn 6). [It is
also possible for S
•
to react with a neighboring thiolate
to yield a disulfide radical anion (S-S
•)
), which can be
further oxidized to disulfide]. In the framework of this
mechanism several possibilities can be envisaged to
explain the air-stability of Cu(I)
4
-Zn
4
MT. The first
possibility is no accessibility of oxygen to the cluster.
This is unlikely because MT3 is more dynamic and
hence more exposed to the solvent. The second possi-
bility is steric hindrance to form disulfide bridges, and

the third is that the redox potential of Cu(I) [or the
Cu(I)–thiolate moiety] is high enough that it is not oxi-
dized by molecular oxygen. In this context it is note-
worthy that absorption data indicate that a different
number of cysteines is bound to Cu(I) (i.e. eight or
nine in MT3 and six or seven in MT1 ⁄ 2). This indi-
cates that the cluster structure is different. More cyste-
ines involved means either fewer bridging cysteines or
a higher coordination number of MT3 compared with
MT1 ⁄ 2, features that might be responsible for the sta-
bility in air.
Reactivity of the metal–thiolate
clusters in MT3
First, some general considerations about the reactivity
of metal–thiolate clusters, normally concerning all
metallothioneins, are given. In the case of Zn (and
Cd) the metal is bound by thiolates (i.e. deprotonated
thiol groups of cysteine). Thiolate is a soft ligand and
therefore shows a preference for soft metals. More-
over the structures of metallothioneins are not rigid
and hence there is little selectivity concerning the size
of the ion. Thus, the affinity of the different metal
ions in MTs is governed primarily by the thermody-
namic stability of the thiolate–metal bond. Therefore,
soft metals such as Cu(I), Cd(II), Hg(II), Pb(II) and
Pt(II) bind more strongly than Zn(II), leading to
Zn(II) release.
The cysteine side chains are also very reactive. At
physiological pH, free cysteine is predominantly pro-
tonated. The availability of Zn(II) leads to their depro-

tonation at physiological pH, yielding Zn–thiolate
complexes. This renders thiolates more nucleophilic
than thiols. However, their binding to Zn(II) generally
also inhibits the formation of disulfides under aerobic
conditions. The latter occurs with uncoordinated thio-
lates. Thus, Zn(II) binding elicits the formation of
thiolates, which are more reactive than thiols, but less
reactive than uncoordinated thiolates. This can be con-
sidered as the sulfur reactivity of MT on a biological
time scale is controled by the Zn-binding state. Thiols
would react too slowly, whereas free thiolates would
react too fast and be difficult to control.
Metal-centered reactions of MT3
In the framework of considering thiolates as simple
metal ligands, metal-exchange reactions such as Cd(II)
or Hg(II) with Zn(II)-MT have been studied with
MT1 ⁄ 2. However, as MT3 synthesis is not inducible
by exposure to metal ions, a role in detoxification is
less likely, which is probably the reason why metal-
exchange reactions with Cd(II), Hg(II), Pb(II), etc.,
have not been studied so far. Moreover, the interac-
tion of Zn(II)
7
MT3 with biologically relevant Cu(I)
has not been reported. By contrast, the reaction of
MT3 with cis-amminedichloridoplatinum(II) (cisplatin)
and trans-amminedichloridoplatinum(II) (transplatin)
has been studied. These reactions are of interest
because MTs play an important role in the acquired
Metal–thiolate clusters in metallothionein-3 P. Faller

2926 FEBS Journal 277 (2010) 2921–2930 Journal compilation ª 2010 FEBS. No claim to original French government works
resistance of platinum-based anticancer drugs. Here,
MT3 is important because its gene is overexpressed in
a number of cancer tissues ([36] and references
therein). It was shown that cisplatin and transplatin
react with the cysteines in Zn(II)
7
MT3, causing the
stoichiometric release of Zn(II). The reactions were
much faster than with MT2. Transplatin reacted more
quickly, but retained the two ligands. By contrast, the
slow-reacting cisplatin had all ligands replaced
with thiolates. Cisplatin binds preferentially to the
b-domain (but binding with transplatin has not been
determined).
A special case of metal-exchange reaction is Cu(II),
because it involves, in addition to its exchange with
Zn, also the reduction to Cu(I) by the oxidation of
cysteine. Meloni et al. [37] showed that Zn(II)
7
MT3
scavenges free Cu
2+
ions through reduction to Cu(I)
and binding to the protein. In this reaction, thiolate
ligands are oxidized to disulfides concomitant with
Zn
2+
release. The binding of the first four Cu
2+

is
cooperative, forming a Cu(I)
4
–thiolate cluster in the
N-terminal domain of Cu(I)
4
,Zn(II)
4
MT3 together
with two disulfide bonds. Because four zinc ions
remain bound, it seems likely that the four-metal Zn
4
–
thiolate cluster in the a-domain stayed intact. As a
consequence the two disulfides would be localized in
the b-domain with the Cu(I)
4
–thiolate cluster. The
formed Cu(I)
4
–thiolate cluster has spectroscopic prop-
erties similar to the isolated and the Cu(I)-reconsti-
tuted Cu(I)
4
Zn(II)
4
MT3 described above, including
stability in air. The reaction of Zn(II)
7
MT3 with

Cu(II) has been proposed to be the underlying mecha-
nism for the protective effect of MT3 against b-amy-
loid (Ab) neurotoxicity linked to AD. It was shown
that a metal swap between Zn(II)
7
MT3 and soluble
and aggregated Cu(II)–Ab abolishes the production of
reactive oxygen species and the related cellular toxicity
[16]. Thus, MT3 might have a role in protecting Ab
from aberrant Cu binding.
Thiolate-centered reactions (NO
•
,
reactive oxygen species, DTNB)
Metals in MTs are relatively deeply buried in the pro-
tein. The modification of the surface-accessible sulfur
of cysteine ligands is thought to be the key that
unlocks the metals from the protein [38]. Thus, in gen-
eral, reactions of the cysteine thiolates result in a con-
comitant release of Zn(II). A variety of reagents
reacting with thiolate have been studied in MTs [5],
but such reactions are limited for MT3. The reaction
with NO
•
has attracted particular attention [38,39].
Most interest in the reaction with NO
•
comes from the
possible role of MTs as NO
•

scavengers and in the
conversion of a NO
•
signal to a Zn(II) signal. The
reaction with NO
•
providing S-nitrosothiols is sug-
gested to be a transnitrosation (i.e. translocation of
NO
+
from the S-nitrosothiols to the cysteine of MT),
which then releases NO
)
during the formation of disul-
fides [38]. This means that NO
)
is not stable in the
MTs and a storage function of MTs for NO· is less
likely. By comparing the reaction of NO and S-nitros-
othiols in MT3 and MT1 ⁄ 2, Chen et al. [38] found
that MT3 was much more reactive, whereas the activi-
ties with reactive oxygen species (H
2
O
2
, OCl
)
,O
2
•)

)
were comparable. In line with this, MT3 was also more
potent in protecting rat embryonic cortical neurons
against S-nitrosothiols. The b-domain showed greatest
reactivity in Zn(II) release and cell protection. The
higher reactivity of the b-domain in MT3 versus NO
has been confirmed by NMR studies on Cd(II)
7
MT3
[39]. Moreover, the NMR data suggest a non-selective
release of the metals from the b-domain first, followed
by a partial release of two Cd(II) ions from the
a-domain, without a significant change in the poly-
peptide structure. Further addition of NO resulted in a
complete loss of protein structure.
DTNB has often been used to probe the nucleophilic
reactivity of thiolates in MTs. DTNB contains an intra-
molecular disulfide bond and, upon nucleophilic attack
from MT, disulfide exchange occurs resulting in an in-
termolecular disulfide bond between MT and 5-thio-2-
nitrobenzoic acid. It has been shown that Cd(II)
7
MT3
reacts faster with DTNB than MT1 ⁄ 2 does [35,40]. For
Zn(II)
7
MT3, similar kinetics have been reported for
human MT3 and rat MT1, but this was monitored in
the presence of EDTA and hence is more likely to reflect
the reactivity of the unstructured apo-T than that of the

metal-loaded form [19].
In general, it can be suggested that MT3 is more
reactive than MT1 ⁄ 2, which is probably related to its
more flexible b-domain and hence to a better access of
compounds to the metal–thiolate cluster. This is also
in line with the general (but not exclusive) observation
that the difference in reactivity is more pronounced for
larger molecules (DTNB, providing S-nitrosothiols)
than for small molecules (H
2
O
2
, OCl
)
,O
2
•)
).
Conclusions and Perspectives
MT3 is clearly a member of the MT family as it shares
with them several biological and chemical properties;
however, there are also very distinct chemical features
that might be directly relevant to the particular biolog-
ical properties of MT3. Therefore, comparison of the
properties of MT3 with those of the well-studied
P. Faller Metal–thiolate clusters in metallothionein-3
FEBS Journal 277 (2010) 2921–2930 Journal compilation ª 2010 FEBS. No claim to original French government works 2927
MT1 ⁄ 2 forms could yield information on the structural
and chemical features responsible for the biological
peculiarities of MT3, such as its growth-inhibitory

activity.
The fact that synthesis of MT3, in contrast to that
of MT1 ⁄ 2, is not inducible by metal ions (Zn, Cu, Cd,
etc.) suggests that it has no essential role in sequester-
ing toxic or an overload of essential metals. This is in
line with a lower binding affinity to Cd(II) for MT3
compared with MT1 ⁄ 2.
One of the most striking features is the greater struc-
tural dynamics and flexibility of MT3 and in particular
of the N-terminal b-domain. This seems to be connected
with the lower metal-binding affinity and with the
higher reactivity towards nucleophilic reagents (NO
•
,Pt
compounds, DTNB) and the ease of Zn release. One
could speculate that higher structural dynamics should
also result in a faster metal transfer from and to MT3
and hence is in line with an involvement in the traffick-
ing of Zn(II) in particular in zinc-containing neurons
[41]. However, metal-exchange rates have not yet been
measured experimentally.
Importantly, MT3 shows increased Zn(II) release
compared with MT1 ⁄ 2 upon reacting with NO
•
and
other compounds, supporting a function in Zn meta-
bolism. It was suggested that MT3 could be involved
in turning a NO
•
signal into a Zn signal [38].

Although the question of whether Cu(I) is physio-
logically bound to MT3 is not as yet resolved (see ear-
lier), it is clear that MT3 has the capacity of binding
Cu(I) under conditions of Cu-homeostasis breakdown.
There are several unanswered questions concerning
Cu(I)MT3. First the exact cluster structures of the dif-
ferent forms are not known [i.e. Cu(I)
4
,Zn(II)
4
MT3
(with or without disulfides), Cu(I)
4
,Cu(I)
4
MT3 and
Cu(I)
6
,Cu(I)
6
MT3]. The determination of a 3D struc-
ture seems crucial for understanding the differences
between Cu–MT3 and Cu–MT1 ⁄ 2 (structure also not
known) and the presence and role of the disulfide
bonds. This would also give insight into the stability
of the Cu(I)
4
-CysS
X
cluster in the b-domain of MT3

towards oxidation by molecular oxygen. The data seem
to be contradictory (see above) as the Cu(I)
4
cluster is
stable in air, but freshly reconstituted Cu(I)
4
,Zn(II)
4
MT3 shows an oxidation reaction forming disulfide
bonds in the Zn-loaded a-cluster. One could ask why
this reaction seems not to occur in the Zn(II)
7
-MT-3,
as in Cu(I)
4
-Zn(II)
4
-MT-3 the Cu(I)
4
-cluster seems not
to be involved? Moreover, in the reaction of Cu(II)
with Zn(II)
7
MT3, a Cu(I)
4
cluster and two disulfide
bridges are formed in the b-domain, while the remain-
ing Zn cluster seems stable. The spectroscopic features
of this Cu(I)
4

cluster are very similar to those of the
reconstituted forms. One partial explanation would be
that the spectroscopic features of the Cu(I)
4
cluster are
not affected by the presence of disulfide bridges and
this cluster is only stable in air in the presence of one
or two disulfide bonds. This could explain the sensitiv-
ity to oxidation of freshly reconstituted Cu(I)
4
,
Zn(II)
4
MT3 and the stability of Cu(I)
4
,
Zn(II)
4
MT3 [isolated, incubated or generated upon
Cu(II) binding] in air. However, this does not explain
why the disulfide bridge is formed in the a-domain
(instead of the b-domain) upon oxidation of freshly
reconstituted Cu(I)
4
,Zn(II)
4
MT3. To shed more light
on this issue it might be worthwhile investigating the
number and localization of disulfide bridges in diverse
preparations of MT3.

With regard to the putative role of MT3 in Cu traf-
ficking, it would be important to determine the binding
constants. Very little is known about Cu(I) affinity in
the MTs, and the values in the literature are mostly
estimates. This is mainly because of the very low disso-
ciation constants, with K
d
estimated to be about
10
)19
m, and the lack of suitable competing ligands
with well-known binding constants. However, even rel-
ative affinities could give important insights, such as
comparison of MT3 with other MTs. The comparison
reported in the literature, based on reactivity with
DTNB, is indirect (see above) [35]. Relative Cu(I)
affinities between MT3 and other MTs should be mea-
sured by a competition assay using MS, as accom-
plished previously for Zn(II) and Cd(II) [18], or by
NMR analysis.
The high affinity of Cu(I) to MT3 (or to MTs in
general) means that Cu(I) release into solvent, as a
result of thiolate bonding, is too slow to be biologi-
cally relevant. Therefore, transfer of Cu(I) is impossi-
ble via ‘free’ Cu(I). One way to transfer Cu(I) to
another protein on a biologically relevant time scale is
through the formation of a ternary complex [MT–
Cu(I)–protein] (i.e. by a coordination bridge forming
an interaction between MT and the acceptor protein).
Other possibilities are that the transfer is assisted by

cysteine oxidation ⁄ modification, by protonation or by
protein breakdown. If the Cu(I) transfer did not taking
place, MT would just be a sink for Cu(I). This could
be sufficient for a redox-silencing role of MT3 for Cu.
In this context it might be interesting to search for
possible binding partners of Cu(I)
4
MT3.
Since the discovery of MT3 [1] almost 20 years ago,
it has been discovered that this member of the family
has unusual biological and chemical properties, clearly
distinct from the widely expressed MT1 ⁄ 2. This holds
also for structure and reactivity of the metal–thiolate
clusters, in particular for the cluster in the b-domain.
Metal–thiolate clusters in metallothionein-3 P. Faller
2928 FEBS Journal 277 (2010) 2921–2930 Journal compilation ª 2010 FEBS. No claim to original French government works
Several intriguing facets have been observed, such as
high dynamics, formation of disulfides, high reactivity,
stability of the Cu(I)
4
-cluster, etc. A better understand-
ing of these features will help to shed light on the
specific biological roles of MT3.
Acknowledgement
Gabriele Meloni (Caltech, USA) and Milan Vasak
(Univ. Zu
¨
rich) are acknowledged for very helpful
discussion.
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