Solution structure of Cu
6
metallothionein from the fungus
Neurospora crassa
Paul A. Cobine
1
, Ryan T. McKay
2,
*, Klaus Zangger
2,
†, Charles T. Dameron
3
and Ian M. Armitage
2
1
Health Science Center, University of Utah, Salt Lake City, UT, USA;
2
Department of Biochemistry, Molecular Biology and
Biophysics, University of Minnesota, Minneapolis, MN, USA;
3
Department of Chemistry and Biochemistry, Duquesne University,
Pittsburgh, PA, USA
The 3 D-solution structu re o f Neu rospora crassa Cu
6
-metal-
lothionein (NcMT) polypeptide backbone was determined
using homonuclear, multidimensional
1
H-NMR spectros-
copy. It r epresents a new metallothionein (MT) fold with a
protein chain where the N-terminal half is left-handed and

lower field, less sensitive
1
H-NMR spectral data. The accu-
racy of the structure calculated without these constraints is,
however, supported by the similarities of the 800 MHz
structures of the a-domain of mouse MT1 compared to the
one recalculated without metal–cysteine connectivities.
Keywords: copper; metallothionein; Neurospora crassa;
NMR; solution s tructure.
Metallothioneins (MTs) are a ubiquitous class of proteins
occurring in both prokaryotes and eukaryotes [1]. MTs a re
known for their small size (< 7 kDa), the ability to
coordinate a diverse range of metals, a lack of definable
secondary structure, high cysteine content ( 30%), and
degeneracy in the remaining residues (e.g. predominance of
cysteine, serine, lysine and no aromatic residues). The high
cysteine content and their spacing give the MTs a high
affinity for m etals (e.g. K
a
of Zn–MT  1 · 10
12
M
)[2,3].
While an essential physiological role has yet to be ascribed
to MT, there is no question that MTs are involved in the
protection of cells against metal intoxication through t he
sequestration of the excess essential metal ions like copper
and zinc, as well as nonessential metal ions, like cadmium,
mercury and silver [4]. Although the essential metals have
critical structural, catalytic and regulatory r oles in proteins,

c
, correlation time.
Note: The PDB file has been assigned the Brookhaven Protein Data
Bank Accession no. 1T2Y and the chemical shifts have been deposited
in the BMRB data bank under accession number 62 90.
*Present address: 101 NANUC, University of Alberta, Ed monton,
AB Canada T6G 2E1.
Present address: Institute of Chemistry/Organic and Bioorganic
Chemistry, University of Gra z, Heinrichstrasse 28, A-8010 Graz,
Austria.
(Received 3 0 June 2004, revised 2 September 2004,
accepted 7 September 2004)
Eur. J. Biochem. 271, 4213–4221 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04361.x
protein stoichiometry and Cu(I) with a 6 : 1 stoichiometry
[12]. The NcMT cysteine arrangement is identical to the
first seven amino-terminal cysteines of the b-domain of
human MT (Fig. 1), thou gh the human MT has t wo
additional cysteines in this domain. Similar cysteine
spacing is seen in the metal binding motifs of the
metalloregulatory proteins from Enterococcus hirae
(CopY), Saccharomyces cerevisiae (Ace1 and Mac1) and
Candida albicans (AMT1) though these proteins do not
share a ny other significant homology, Fig. 1. Along with
the metal coordination number, the positioning of the
cysteines in t he primary s tructure has been determined to
be the critical f actor in determining the global fold of t he
MTs [14]. The structures of several MT isoforms have
been determined including that from yeast [15,16], crab
[17], sea urchin [14], an antarctic fish [18] and various
mammals [19–23]. With the exception of the yeast Cu

6
MT and
potentially useful for other copper(I)-regulated proteins
that contain t he - CxCxxxxCxC- and - CxCxxxxxCxC-
motifs, F ig. 1.
The
1
H NMR resonance assignments f or the C u
6
NcMT
purified from N. crassa have been reported [ 27] and these
were used as a template to compare and confirm a similar
overall fold for the in vitro reco nstituted, synthesized protein
used in this study. W e report here the 3D NMR solution
structure o f the Cu
6
NcMT, obtained without establishing
any metal–cysteine restraints, and discuss the formation
of the Z n
3
NcMT precursor m olecule t hat was needed to
obtain the Cu
6
NcMT structure.
Materials and methods
Proteins and peptides
The NcMT w as synthesized chemically to avoid its prob-
lematic induction and purification. As noted previously by
another group [28], N. crassa can use several or a mixture of
copper resistance mechanisms to detoxify excess copper. In

pyridine assay [30]. All subsequent manipulations of the
apo- a nd metallated N cMT were p erformed under anaer-
obic conditions, 5% H
2
and 95% N
2
(v/v), in a glove box
(Atomspure Protector Glove Box, Labconco, Kansas City,
MO, USA).
Metal titrations
Copper(I) titrations were performed as d escribed by Byrd
et al. [31]. Protein samples with reduction state > 95%
were used for all titrations. Sequential additions of
copper(I), as [Cu(CH
3
CN)
4
]ClO
4
in 200 m
M
ammonium
acetatepH7.9,weremadetotheapo-NcMT. Identical
titrations were performed with Zn(II)
3
NcMT instead of the
apo-NcMT. The Zn
3
NcMT was prepared from ap o-NcMT
by adding 3.5 molar equivalents of Zn(II) (added as Z n(II)-

The final Cu
6
NcMT co ntained 1 .8 lmol N cMT and
10.9 lmol Cu(I).
NMR sample preparation
Lyophilized Cu
6
NcMT (handled under an argon atmo-
sphere) was dissolved in 500 lL 90% H
2
O, 10% D
2
O,
pH 6.5, 0.1 m
M
2,2-dimethyl-2-silapentane-5-sulfonate (as
an internal reference) [32], and 0.02% NaN
3
that was
degassed prior to protein addition under high vacuum and
re-pressurized to 1 atm under argon to avoid oxidation/
disproportionation of the C u(I)–protein. T he sample was
loaded into a 5 mm NMR tube, c apped a nd sealed with
parafilm.
NMR spectroscopy and assignments of NcMT
A2D
1
H,
1
H-TOCSY [33,34] and 2D

a
) were obtained from 1D-proton and 2D-COSY
spectra using d econvolution. Due to t he different temper-
atures used during the NMR experiments (10 °Cinthe
present s tudy and 25 °C i n [ 27]) t here are expected t o b e
small differences in chemical shifts, typically larger for NH
protons. However, the overall very good agreement in
these shifts can be confidently attributed to the protein
forming the same structure. Gradient NOESY spectra
were acquired w ith WaterGate solvent s uppression taken
from the ÔgnoesywgÕ pulse sequence in ÔProtein PackÕ as
supplied b y Varian I nc.
Structure calculations for NcMT
Distance restraints (50 kcalÆmol
)1
ÆA
˚
)2
) for NcMT structure
calculations were classified as short (1.8–2.7 A
˚
), medium
(1.8–3.3 A
˚
), and long (1.8–5.0 A
˚
) based on their N OE
intensities. The upper bound of an NOE restraint was
extended by 0.5 A
˚

and r.m.s.d.
for bond and angle deviations from ideality of less than
0.01 A
˚
and 5°, respec tively.
Structure calculations for mouse aMT
All structure calculations involving the a-domain of the
mouse MT, aMT-1, were performed as previously reported
[23] with the exception that the metal–cysteine restraints
were not used and therefore all metals w ere absent in the
calculations. To determine the precision [how well the
individual structures calculated with a limited set of l ong-
range N OEs (d
ij
, j > i + 4) compare to each other ] and
accuracy (the simila rity of the calculated structures with a
reduced set o f long-range NOEs to the structure calculated
with all NOEs) of the calculated structures, all 22 long-range
NOEs were first removed and then, by a random selection
process reintroduced one by one for the structure calcula-
tions. T hereby, for each number of long-range NOEs, 1 0
random selections of reintroduced long-range NOE sets
were made and 10 structures calculated for each set, giving a
total of 100 structures calculated for each point in Fig. 4.
Results
Metal binding stoichiometry of NcMT
N. crassa can express MT in response to excessive concen-
trations of copper in the growth media. NcMT purified
from the mycelium of t he fungus contains 6 Cu(I) ions per
mole of protein [9]. The copper(I) ions are bound to the

containing a mixture of inter converting NcMT structures
and/or structural instabilities. In an attempt to restrict the
family of conformers formed during the copper(I) titrations,
we first titrated zinc into the apo-protein to form a
Zn
3
NcMT precursor. The excess zinc was removed by
treatment of the sample with Chelex-100. The zinc stoi-
chiometry of the resultant protein, 3-Zn(II):1 NcMT, was as
expected from previous studies of NcMT and by analogy to
the well s tudied b-domains of the mammalian MTs [12].
Sequential titration of the Zn
3
NcMT with Cu(I) salts as
before yielded a luminescent core but in this case, the
stoichiometry w as 6 Cu(I):1 NcMT, Fig. 2C,D. Most
importantly, t he 1D
1
H-NMR p attern for the Cu
6
NcMT
is equivalent to the native Cu(I) proton spectrum [27].
Attempts to prepare the Ag(I) derivative of N cMT d id not
result in a stoichiometry of 6 metals p er mole of protein.
Attempts to displace all of the z inc from the Zn
3
NcMT by
silver were unsuccessful and always resulted in the forma-
tion of a mixed metal s pecies inappropriate for s tructural
studies. The structure determination of Zn

contacts for precision in structure calculations. This can be
quantitatively evaluated by recalculating the mouse MT1
a-d omain structure in the absence of all long-range NOE
restraints and
113
Cd–Cys connectivities and then syste-
matically and randomly reintroducing long-range NMR
Fig. 3. Ribbon backbone diagrams of the a-domain of mouse MT-1
calculated with (dark) and without (grey)
113
Cd–Cys restraints. The Cd
atoms (rendered to van der Waals radii), cysteine sulfur, and Cd-S
bonds are indicated by the large spheres, sma ll sphere s and black lines,
respectively. The N and C termini are labelled for orientation. Dia-
grams were generat ed using t he program
INSIGHTII
(Molecular Simu-
lations, Inc.).
Fig. 2. In vitro copper reconstitutions o f
N. crassa MT monitoring Cu(I)-S luminescence
at 580 nm after excitation at 295 nm. (A) The
reconstitutions at 10 nmolÆmL
)1
of apo-
NcMT with copper(I) the increasing additions
of copper the luminescence makes uniform
steps (B). (C) Titratio ns of 50 nm olÆmL
)1
Zn
3

provide interproton distance restraints, r espectively, on
NcMT. The chemical shifts of NcMT determined here at
10 °C a re similar t o t hose of a previous study perfo rmed at
25 °C [27]. A lmost a ll the
1
H chemical shifts were v isible in
the T OCSY with the exception of the side c hain of Cys5
(assigned from the N OESY), and a ll resonances of Gly1
(not identified i n either experiment). Assignment of Gly1
was not possible; this was most likely due to rapid exchange
of the amide proton of Gly1 with the s olvent, coupled with
chemical shift overlap reported previously [27]. The chem-
ical shifts have been deposited in the BMRB data bank
under accession number 6290. Examination of the chemical
shifts showed no indication of secondary structure [42,43].
Line widths of 1D-
1
H NMR resonances have been used as
an efficient method for determining the aggregation state of
a protein [44,45]. The average line width of NcMT amide
resonances from the 1D-
1
H spectrum was determined to be
5.6 Hz. When six Cu(I) atoms are considered bound to
NcMT, the correlation time (s
c
) and line width for the
monomer are expected to be 2.6–3.7 n s and  4.5–6 Hz,
respectively [34]. The observed line widths for the reconsti-
tuted Cu

(i,i+1)
G
20
10 20
10
Residue Number
Number of NOEs
20
15
10
5
0
DCGC SGASS CNCGSGCSCSNCGS K
d
βN
(i,i+1)
d
αN
(i,i+1)
d
αN
(i,i+2)
A
B
Fig. 5. NOE map for NcMT. (A) Summary of inter-residue N OEs
determined for NcMT that are typically used to ind icate secondary
structure. Strong, medium a nd weak intensity NOE cross-peaks are
indicated by tall dark, medium grey, and small white boxes, respect-
ively. Th e primary sequence is shown at the b ottom in the o ne letter
code. (B) T he total number of NO Es assigned for NcMT displayed on

less than 0.01 A
˚
and an r.m.s.d. for angle deviations from
ideality of less than 5 °) with the lowest energy were selected
which yielded a backbone r.m.s.d. to the average minimized
structure of 0.79 A
˚
for the well-defined region (residues
5–20) and 1.59 A
˚
for the entire length of the protein. The
final family of 10 NcMT structures is shown in Fig. 6 with
the N - and C-termini labelled and the c ysteine sulfur atoms
in the Ôclosest to meanÕ structure drawn as spheres. The
structure of NcMT has been deposited in the Protein Data
Bank under accession number 1T2Y. The structural statis-
tics for NcMT are presented in Table 1. The program
PROCHECK
-
NMR
[46] showed that > 90% of the ø, w angles
for the 10 structures fell into the core or a llowed regions.
Despite the complete absence of elements of r egular
secondary structure, which is a quite common situation
for MTs, the backbone global fold of the 25-residue peptide
is well defined if one excludes the N and C termini. It shows
a new polypeptide structure with t he backbone being
wrapped around an empty space containing the copper–
sulfur cluster, on going from the N to C terminus, in a left
handed form for the first half of the molecule then in a right

structure are shown as yellow spheres.
Table 1. Structural statistics for NcMT, for the 10 lowest overall energy
structures out of 12 without a ny NOE or d ihedral violations.
NOE restraints
Total 152
Intraresidue 63
Sequential (|i-j| ¼ 1) 53
Medium (2 6 |i-j| 6 4) 25
Long range (|i-j| P 5) 11
Dihedral restraints (ø) 13
r.m.s.d. to average structure (A
˚
)
Well-defined regions (N,C
a
,C)
a
0.79
All regions (N,C
a
,C) 1.59
All heavy atoms 1.97
Energies (kcalÆmol
)1
)
E
overall
82.40 ± 7.95
E
bonds

b
[46]
In most favoured regions 44.4% (80)
In additional allowed 44.4% (80)
In generously allowed 5.6% (10)
In disallowed regions 5.6% (10)
a
Residues 5–20.
b
Number of residues out of all 10 structures.
Total non-glycine and non-proline is 180. Number of glycines is 60,
with 10 end-residues, for a total of 250 residues.
4218 P. A. Cobine et al. ( Eur. J. Biochem. 271) Ó FEBS 2004
residue position r.m.s.d. between the structurally related
b-domain of mouse MT1 and human MT2 a re 0.57 and
2.10 A
˚
for the backbone heavy atoms and the sulfur atoms,
respectively.
Discussion
The solution structure of the Cu
6
NcMT, which was solved
without the acquisition and inclusion of specific metal–
cysteine NMR r estraints, shows a novel polypeptide fold
and represents only the second copper MT s tructure to be
elucidated [15,16]. Although other MTs show a strong
sequence similarity to NcMT (e.g. 32% with the b-domain
of human MT2) the 3D structure of the polypep tide
backbone i s completely different. The unique fold of this

3
–NcMT might constitute a scaffold where the zinc can
be replaced by Cu(I) without the need for large structural
rearrangements. In other words the less favourable enthalpy
changes by binding 6 Cu (I) atoms to only seven cysteines in
NcMT are compensated by the smaller differences in
entropy when copper substitutes zinc i n Zn
3
–NcMT, rather
than binding to apo-NcMT.
The variable coordination number of c opper, which can
adopt a linear two-coordinate (digonal) or a distorted
planar trigonal three-coordinate geometry with sulfur
ligands [49] adds an addition al level of complexity in solving
the copper MT structures. These coordination possibilities
of the copp er(I) ions are perhaps responsible for the titration
of the apo-NcMT to a non-native (4 : 1) stoichiometry. In
thecaseoftheNcMT,itseemsthatthecoordinationofzinc
in Zn
3
NcMT was necessary to conform/stabilize a structure
such that the Cu(I) titration produced the native Cu
6
Cys
7
metal–thiolate cluster rather than the Cu
4
Cys
7
made from

1DME
e
5.22 5.31
1M0G
f
3.49 4.45
1M0J
g
3.90 4.98
1QJK
h
4.47 5.29
1QJL
i
3.85 4.77
a
b-domain of lobster Homarus americanus MT.
b
Cu(I)-yeast
Saccharomyces cerevisiae MT.
c
b-domain of mouse Mus musculus
MT1.
d
a-domain of blue crab Callinectes sapidus MT.
e
b-domain
of blue crab Callinectes sapidus MT.
f
a-domain of the fish Noto-

angles to direct the sul fur atoms into the core o f the protein.
Ó FEBS 2004 NMR structure of N. crassa Cu6MT (Eur. J. Biochem. 271) 4219
lived and hard to i solate species a s copper i ons would b e
expected to readily displace the Zn(II). However, there are
no in vivo data t o indicate that the o rgan ism has to form a
zinc precursor. The requirement of Zn(II) in the f ormation
of Cu(I)MT clusters, in particular the b-domain, has been
observed for mouse MT [50]. Additionally, the same spacing
of cys teine residues i s also f ound in the repressor protein
CopY from Enterococcus hirae and it is apparent that the
metal-binding properties of this protein may require
the binding of Zn(II) in this site [51,52]. The stability of
the Cu(I)-S core and the DNA-binding activity are
dependent o n zinc binding. In addition, the copper chap-
erone CopZ is a specific source of copper for the Zn-form
of CopY [51]. However, apo-CopY does not demonstrate
this specificity suggesting a potential loss of structure that
confers this property. A combination of the data suggests
that Zn(II)-binding to these cysteine-rich, copper-binding
sites orders a structure that is required for activity.
A model structure of Cu
6
NcMT demonstrating the
shielding of the copper core, using one model with copper
atoms satisfying the Cu–S bond distances and minimizing
any interactions within the NMR structure, is shown in
Fig. 7. While there is probably a multitude of models that
would fit the NMR data, there is no way to co nfirm any
from NMR s tudies using t he NMR inactive Cu(I) metal
form of the protein. The NcMT backbone structure

6
MT which consists of a half left- and half
right-handedly polypeptide backbone wrapped around the
copper(I)-cysteine cluster. No direct information about the
metal–sulfur connectivities could be obtained b y using an
isomorphic, NMR active metal substitute for Cu
+
,suchas
Ag
+
, because a stable homogenous Ag substituted NcMT
could not be prepared. Nevertheless, the use of data at
800 MHz on th e reconstituted Cu
6
NcMT was sufficient to
allow for an accurate backbone fold to be determined.
Acknowledgements
This work was supported by NIH grant DK18778 to I.M.A. NMR
instrumentation was provided with funds from the NS F (BIR-961477)
and t he University of Minnesota Medic al School. K.Z. thanks the
Austrian Science Foundation FWF for financial support under project
number P15289. We would like to thank Dr David Live and Dr Be verly
G. Ostrowski for maintenance of the spectrometer facility and
computers, Matt Vetting for help with software in modeling of the
Cu(I)-NcMT an d expertise in use of the A rgon Chamber for NMR
sample preparation, and Gu
¨
lin O
¨
z for critical reading of the

containing redox drug, releases zinc from metallothionein. Bio-
chem. Biophys. Res. Commun. 248, 569–573.
8. Faller, P., Hasler, D.W., Zerbe, O., Klauser, S., Winge, D.R. &
Vas
ˇ
a
´
k, M. (1999) Evidence for a dynamic structure of hum an
neuronal gro wth inhibitory fac tor and for major rearran gements
of its metal-thiolate clus ters. Biochem ist ry 38, 10158–10167.
9. Lerch, K. (1980) Copper metallothionein, a copper-binding pro-
tein from Neurospora crassa. Nature 284, 368–370.
10. Smith, T.A., Lerch, K. & Hodgson, K.O. (1986) Structural study
of the Cu sites in metallothionein from N eur os por a crassa.
Inorganic C hem. 25, 4677–4680.
11. Beltramini, M. & Lerch, K. (1983) Spectroscopic studies on
Neurospora co pp er metallothonein . Biochemistry 22 , 2043–2048.
12. Beltramini, M., Lerch, K. & Vasak, M. (1984) Metal substitution
of Neurospora copper metallothionein. Biochemistry 23, 3422–
3427.
13. Munger, K., Germann, U.A. & Lerch, K. (1987) The Neurospora
crassa metallothionein gene. JBiolChem262, 7363–7367.
14.Riek,R.,Precheur,B.,Wang,Y.,Mackay,E.A.,Wider,G.,
Gu
¨
ntert,P.,Liu,A.,Ka
¨
gi, J .H.R. & Wu
¨
thrich, K . (1999) NMR

20. Robbins, A.H., McRee, D.E., Williamson, M., Collett, S.A.,
Xuong, N.H., Furey, W.F., Wang, B .C. & Stout, C.D. (1991)
Refined crystal structure of Cd, Zn metallothionein at 2.0 A
˚
resolution. J. Mol. Biol. 221, 1 269–1293.
21. Messerle, B.A., Scha
¨
ffer, A., Vas
ˇ
a
´
k, M., Ka
¨
gi, J .H. & Wu
¨
thrich,
K. (1990) Three-dimensional structure of human [113Cd7]metal-
lothionein-2 in solution determined by nuclear magnetic resonance
spectroscopy. J. Mol. Biol. 214, 765–769.
22. Schultze, P., Wo
¨
rgo
¨
tter, E., B ruan, W., Wagner, G., Vas
ˇ
a
´
k, M.,
Ka
¨

Saccharomyces cerevisiae and Ne urospora crassa c ontain heavy
metal sequestering phytochelatin. Arch. Microbiol. 157, 305–310.
29. Schnolzer, M., Ale wood , P., Jo nes, A., Alewood , D. & Kent,
S.B.H. (1992) In situ neutralization in Bo c-chemistry solid phase
synthesis. Int. J. Peptide Protein Res. 40, 180–193.
30. Grassetti, D.R. & Murray, J.F. Jr (1967) Determination of sulf-
hydryl groups with 2,2¢-or4,4¢-dith iod ip yri din e. Arch . B iochem .
Biophys. 119, 41–49.
31. Byrd, J., Berger, R .M., McMill in , D.R., Wright, C.F., H ame r, D.
& Winge, D .R. (1988) Charac terization of the copper-thiolate
cluster in yeast metallothionein and two truncated mutants.
J. Biol. Chem. 263, 668 8–6694.
32. Markley, J.L., Bax, A., Arata, Y., Hilbers, C.W., Kaptein, R.,
Sykes, B .D., Wright, P.E. & Wu
¨
thrich, K . (1998) R ecomm enda-
tions for t he presentation of N MR structures of pr oteins and
nucleic acids. J. Mol. Biol. 280, 933–952.
33. Braunschweiger, L. & Ernst, R.R. (1983) Coherence transfer by
isotropic mixing: application to proton correlation spectroscopy.
J. Magn. Reson. 53, 521–528.
34. Cavanagh, J., Fairbrother, W.J., Palmer, A.G. III & Skelton, N.J.
(1996) Pro tein NMR Spectroscopy: P rinciples and Practice,1st
edn. Academic Press, San Diego.
35. Wu
¨
thrich, K. (1986) NMR of Proteins and Nucleic Acids.John
Wiley & Sons Inc , New York.
36. Delaglio, F., Grzesiek, S., Vuister,G.W.,Zhu,G.,Pfeifer,J.&
Bax, A. (1995) NMRPipe: a multidimensional spectral processing

domains of cardiac and E 41A skeletal m uscle troponin C re veal
the importance of site I to energetics of the induc ed structu ral
changes. Bioch emi stry 36, 12519–12525.
45. Anglister,J.,Grzesiek,S.,Ren,H.,Klee,C.B.&Bax,A.(1993)
Isotope-edited multidimensional NM R o f calcineurin B in the
presence of the non-deuterated detergent CHAPS. J. Biomol.
NMR 3, 121–126.
46. Laskowski, R.A., M acArthur, M .W., Mo ss, D .S. & Thorton,
J.M. (1993) PROCH ECK: a program to c heck the sterochemical
quality of p rotein structures. J. Appl. Crystallogr. 26, 283–290.
47. Brouwer, M., Winge, D .R. & Gray, W.R. (1989) Structural and
functional diversity of copper-metallothioneins from th e Amer-
ican lobster Homarus americanus. J. Inorg. Biochem. 35, 289–303.
48. Ka
¨
gi, J.H.R. (1991) Overview of metallothionein. Methods Enz-
ymol. 205, 613–626.
49. Pickering, I.J., George, G.N., D ameron, C.T., Kurtz, B., Winge,
D.R. & Dance, I.G. (1993) X-ray absorption spectroscopy of
cuprous-thiolate multinuclear clusters in p rote ins and model sys-
tems. J. Am. Chem. Soc. 115, 9498–9505.
50. Bofill, R., Capdevila, M., Cols, N.,Atrian,S.&Gonzalez-Duarte,
P. (2001) Zinc (II) is required for the in vivo and in vitro folding of
mouse copper metallothionein in two domains. J. Biol. I norg.
Chem. 6, 405–417.
51. Cobine, P., Wickramasinghe, W.A., Harrison, M .D., Weber, T.,
Solioz, M. & Dameron, C.T. (1999) The Enterococcus hirae copp er
chaperone CopZ delivers copper (I) to the CopY repressor. FEBS
Lett. 445, 27–30.
52. Cobine, P .A., Jones, C.E. & Dameron, C.T. (2002) Role for zinc