MercuryII binding to metallothioneinsVariables governing the formation and structural features of the mammalian Hg-MT species A`ngels Leiva-Presa, Merce` Capdevila and Pilar Gonza`lez-Du
Trang 1Mercury(II) binding to metallothioneins
Variables governing the formation and structural features of the mammalian
Hg-MT species
A`ngels Leiva-Presa, Merce` Capdevila and Pilar Gonza`lez-Duarte
Departament de Quı´mica, Facultat de Cie`ncies, Universitat Auto`noma de Barcelona, Spain
With the aim of extending our knowledge on the reaction
pathways of Zn-metallothionein (MT) and apo-MT species
in the presence of Hg(II), we monitored the titration of
Zn7-MT, Zn4-aMT and Zn3-bMT proteins, at pH 7 and 3,
with either HgCl2or Hg(ClO4)2by CD and UV-vis
spectr-oscopy Detailed analysis of the optical data revealed that
standard variables, such as the pH of the solution, the
binding ability of the counter-ion (chloride or perchlorate),
and the time elapsed between subsequent additions of Hg(II)
to the protein, play a determinant role in the stoichiometry,
stereochemistry and degree of folding of the Hg-MT species
Despite the fact that the effect of these variables is unques-tionable, it is difficult to generalize Overall, it can be con-cluded that the reaction conditions [pH, time elapsed between subsequent additions of Hg(II) to the protein] affect the structural properties more substantially than the stoi-chiometry of the Hg-MT species, and that the role of the counter-ion becomes particularly apparent on the structure
of overloaded Hg-MT
Keywords: mercury(II) binding; mercury-metallothionein; metallothionein; a-metallothionein; b-metallothionein
Mercury thiolates provide representative examples of the
structural diversity shown by the extensive family of metal
thiolates [1–4] The most striking features of mercury
thiolates in the solid phase are the different structures
obtained when Hg(II) is co-ordinated to very similar
thiolate ligands [5,6] and the distinctive behavior of Hg(II)
towards a particular thiolate compared with that of Zn(II)
or Cd(II) [7], which has been referred to as the zinc family
paradox [3] Moreover, correlations between solid-state and
solution complexes cannot be easily established Overall, the
diverse co-ordination preferences of Hg(II) ions (mainly
tetrahedral, trigonal-planar and digonal) and their
coexist-ence in polynuclear complex species, the various ligation
modes of the thiolate ligands (i.e terminal, l2-bridging or
l3-bridging) and the possibility of secondary Hg(II)–sulfur
interactions [8] make it difficult to anticipate the structure of
a particular mercury thiolate complex [1,3,9] This results
from the interplay of not only the above factors, but also the
reaction conditions Of these, the presence of additional
co-ordinating species, such as halide ions, make the bonding
situation for mercury even less straightforward than in the
case of homoleptic mercury thiolates [10,11]
The biological chemistry of mercury is dominated by co-ordination to cysteine thiolate groups in agreement with the preference of this metal ion for the soft sulfur ligands The high binding constants for binding of Hg(II) to cysteine residues account for the irreversible replacement of essential metals (Zn, Cu) in cysteine-containing metalloproteins and thus for the high toxicity of mercury to living systems Within the same context of the highly favored thermo-dynamically Hg-S bond, resistance to Hg(II) toxicity in several bacteria is based on an ensemble of proteins designated as Mer, most of which bind Hg(II) ions through cysteine residues ([3] and references therein) In mammals, detoxification of mercury by metallothioneins (MTs) occurs via cysteine complexation and sequestration [12] A major feature of this very large family of ubiquitous low molecular mass proteins is their extremely high content of cysteine residues, the binding of which to metal centers determines the 3D structure of the protein [13] Consideration of the high flexibility and multidentate ligand nature of the peptide chain in MTs together with the intrinsic complexity of mercury thiolate complexes suggests that elucidation of the stoichiometry and co-ordination geometries of mercury in solution Hg-MT species may be rather intricate
To date, optical spectroscopy (UV-vis and CD) has played a major role in the study of the mercury-binding properties of mammalian MTs, for which several Hg-MT stoichiometries have been reported [14] Thus, a detailed analysis of the electronic spectra of Hg(II)-reconstituted MT led Vasˇa´k et al [15] to propose that Hg(II) in Hg7-MT is co-ordinated at sites with tetrahedrally related geometry Subsequent studies by Johnson & Armitage [16] of the UV spectral data obtained in the titration of Cd(II)7-MT with Hg(II) showed that Hg(II) initially occupies tetrahedral sites but, above a Hg/MT stoichiometry of four, there is a shift
to linear co-ordination However, on the basis of X-ray
Correspondence to M Capdevila, Departament de Quı´mica, Facultat
de Cie`ncies, Universitat Auto`noma de Barcelona, E-08193 Bellaterra,
Barcelona, Spain Fax: + 34 935813 101, Tel.: + 34 935813 323,
E-mail: merce.capdevila@uab.es
Abbreviations: MT, metallothionein; TDPAC, time differential
per-turbed angular correlation of c-rays; UV-vis, ultraviolet-visible
elec-tronic absorption; t, stabilization time allowed for the co-ordination of
Hg(II) to the protein; X, counter-ion of the Hg(II) salt added as
titrating agent.
(Received 19 July 2004, revised 21 October 2004,
accepted 25 October 2004)
Trang 2absorption studies conducted on some of the species
observed in the titration of either apo-MT or Zn7-MT with
Hg(II), monitored by optical spectroscopy, Lu & Stillman
[17] proposed a distorted tetrahedral co-ordination for
Hg(II) in Hg7-MT with two short (2.33 A˚) and two long
(3.4 A˚) Hg-S distances [18] Previous extended X-ray
absorption fine structure (EXAFS) results for Hg7-MT
were consistent with a Hg-S bond length of 2.42 A˚ and
suggested that Hg(II) was in a three-co-ordinate thiolate
environment [19]
Although the protective role of MTs against Hg(II)
toxicity provides particular interest for the study of the
Hg(II)-MT system, most existing results are difficult to
reconcile With the aim of finding new strategies for this
study, we now report on the effect of two variables, the
reaction time and the presence of chloride ions, on
the stoichiometry, stereochemistry and degree of folding
of the Hg(II)-MT species formed by either the binding of
Hg(II) to apo-MT or Zn/Hg replacement in Zn7-MT
Materials and methods
Protein preparation and characterization
Fermentator-scale cultures, purification of the
glutathione-S-transferase-MT fusion proteins, and recovery and
ana-lysis of the recombinant mouse Zn7-MT1, Zn4-aMT1 and
Zn3-bMT1 domains were performed as previously described
[20,21] The Zn7-MT, Zn4-aMT and Zn3-bMT species were
obtained in both Tris/HCl and Tris/HClO4buffer (50 mM,
pH 7) [22] The protein concentration was 0.1 mMin the
six solutions, which were diluted to a final concentration of
10 lM (MT) or 20 lM (aMT and bMT fragments) with
MilliQ-purified and Ar-degassed water before being titrated
with Hg2+solutions at 25C
The apoproteins were prepared by acidification of the
recombinant material with 10 mMHCl or HClO4,
respect-ively, until pH 3 At pH values lower than 3.5 the Zn7-MT,
Zn4-aMT and Zn3-bMT species are entirely devoid of
metal, according to their respective CD spectra In contrast,
Hg(II) remains bound to SCys at this pH
Metal solutions
Glassware and solutions used in metal ion-binding studies
were prepared as described [20] A Riedel-de Hae¨n atomic
absorption spectrometry Hg2+standard of 1000 p.p.m was
used as the HgCl2 solution The Hg(ClO4)2 solution was
prepared from the corresponding salt in MilliQ-purified
water, and the Hg(II) concentration was quantified by
atomic absorption spectrometry using a Perkin–Elmer 2100
atomic absorption spectrometer In both cases the Hg(II)
concentration of the titrating agents was in the 1–10 mM
range
Metal ion-binding reactions
Metal-binding experiments were carried out by sequentially
adding molar-ratio aliquots of concentrated Hg(II) stock
solutions to single solutions of either the holoproteins or
apoproteins and followed spectropolarimetrically (CD) and
which differ in the time elapsed between subsequent additions of Hg(II) to the protein, were carried out In one set, the standard titration procedure [22] was followed, whereas in the other consecutive additions of Hg(II) were made every 24 h The electronic absorption and CD measurements were performed and corrected as already described [22]
All manipulations involving the protein and metal ion solutions were performed in Ar atmosphere, and the titrations were carried out at least in duplicate to ensure the reproducibility of each point
The pH (7 or 3) for all experiments remained constant throughout At pH 7, the acidity of the Hg(II) solutions required the addition of appropriate buffer solutions of Tris/HCl or Tris/HClO4 (50 or 70 mM at pH 7), but no buffering was required for the titrations carried out at pH 3
Results and Discussion
In view of the well-known complexity of Hg(II)–thiolate systems, the difficulties we encountered in analyzing the results obtained through preliminary titrations of the Zn-MT proteins with Hg(II) were not a surprise They indicate that the nature of the counter-ion (X) and the time elapsed between subsequent additions of the Hg(II) solution (t) have a significant effect on the stoichiometry, stereo-chemistry and degree of folding of the species formed Thus,
to understand the reaction pathways followed by Zn-MT and apo-MT species in the presence of Hg(II), the effect of each of the previous variables was analyzed separately To this end, the titration of Zn7-MT, Zn4-aMT and Zn3-bMT proteins, at pH 7 and 3, with either HgCl2 or Hg(ClO4)2
were spectroscopically monitored
The CD and UV-vis spectroscopic techniques used in this work are currently used to study metal-binding features of
MT as they provide information on the co-ordinative features of the predominant metal-MT species present in solution at each titration point and on the number of species formed during the titration Furthermore, titration of the separate fragments provides information on the depend-ence/independence relationship between the two constitu-tive domains of the whole MT protein [21,23]
With regard to the two pH values, titrations at pH 7 allow the subsequent substitution of Zn(II) and thus formation of heterometallic Zn,Hg-MT species, and titrations at acidic
pH values provide information on the binding of Hg(II) to the corresponding apo-MT form [23] In addition, compar-ison of the two sets of data gives an indication of the role of Zn(II) in the Hg(II)-containing species formed at physiolo-gical pH The use of two different Hg(II) salts allowed analysis of the possible role of the physiologically relevant chloride anion, which has a strong tendency to co-ordinate and bridge Hg(II) ions, in the degree of folding and 3D structure of the Hg-MT species The perchlorate anion is well known for its low co-ordinating ability towards metal centers
As regards the time variable, the spectroscopic changes observed in the titrations of Zn7-MT, Zn4-aMT and
Zn3-bMT with Hg(II), after different times were allowed for the reaction between the MT protein and the added Hg(II) ions, were indicative of a strong dependence of the
Trang 3with HgCl2were carried out at two different times, t¼ 0 h
and t¼ 24 h, whereas those with Hg(ClO4)2 were only
performed at t¼ 24 h The t ¼ 0 h label denotes that the
titration was performed under kinetic control conditions,
which means that, for each addition, the protein sample was
allowed to react with the metal ion until subsequent CD
spectra were essentially coincident [22] However, for most
samples, if the CD spectrum was recorded again after 24 h,
it showed significant differences from that recorded at t¼
0 h For this reason, titrations labeled t¼ 24 h denote those
carried out under thermodynamic control conditions, where
each molar-ratio aliquot of Hg(II) was added every 24 h, as
longer time intervals showed no further changes in the
spectroscopic features
Overall, evaluation of all the variables in the Hg-MT
system required the performance and analysis of 18
titrations and the corresponding duplicates The detailed and comparative analysis of the set of CD, UV-vis and difference electronic absorption spectra recorded for each titration (provided as Supplementary Material) provides information on the species formed by the Zn-MT peptides in the presence of Hg(II) under the different experimental conditions assayed and has allowed us to propose the reaction pathways (Schemes 1–3) for Zn/Hg replacement in Zn-MT species (pH 7) and for the binding of Hg(II) to apo-MT (pH 3) that are discussed below
Mercury content in the Hg(II)-MT species at each titration point has traditionally been established by assu-ming that, in solution, only one species is present, the metal content of which coincides with the number of Hg(II) equivalents (eq) added To validate the previous assumptions as well as to quantify the Zn content in the Zn,Hg-MT species observed at pH 7 (Schemes 1A, 2A and 3A), we unsuccessfully devoted much effort to obtaining ESI-MS data Thus, information on the Zn(II) content was retrieved from CD data and it is mainly of a qualitative nature
Reaction of recombinant mouse Zn7-MT with Hg(II) Analysis of the CD, UV-vis and UV-vis difference spectra obtained in the titration of Zn7-MT with Hg(II) at pH 7 (Fig 2, S1 and S2) and pH 3 (Figs S3–S5) for each set
of X and t values led to the reaction pathways shown
in Scheme 1
Comparative analysis of the three sets of data indicates that the stoichiometry of the species formed along the three titrations at pH 7 depends on neither the stabilization time,
t, nor the nature of the counter-ion The unique exceptions
Fig 1 Evolution with time of the CD spectra corresponding to the
addition of the tenth Hg(II) to Zn 7 -MT at pH 7.
Scheme 1 Proposed reaction pathways for Hg(II) binding to recombinant Zn 7 -MT at pH 7 (A) and at pH 3 (B), under thermodynamic (t ¼ 24 h)
or kinetic (t ¼ 0 h) control conditions, using HgCl 2 or Hg(ClO 4 ) 2 as titrating agents The and „ symbols denote similarity and difference, respectively, between the structure of two species compared.
Trang 4to this rule are: (a) Zn,Hg2-MT, observed as an intermediate
species only at t¼ 24 h; (b) the stoichiometries of the fully
loaded species, Hg15-MT and Hg16-MT Conversely, the
chirality of the species is highly dependent on the previous
variables, t¼ 24 h and X ¼ Cl–affording the most chiral
species, as shown by the intensity of the CD bands of the
Hg(II)-MT species formed under these conditions (Fig 2)
Similarly, t and X have a significant effect on the structure
of the Hg-MT aggregates, with a Hg to MT ratio equal or
higher than 7, as evidenced by the comparison of the CD
spectra of isostoichiometric species obtained under different
conditions The contribution of the counter-ion to the 3D
structure of the Hg-MT aggregates is demonstrated by the
outstanding example of Hg11-MT, which becomes one of
both kinetic and thermodynamic control conditions (Fig 3)
Another relevant feature is the formation of hetero-metallic Zn,Hg5-MT and Zn,Hg7-MT, both present in the three titrations The former shows a very specific CD fingerprint The significance of the latter lies in the Hg(II) stoichiometry, as previous studies proposed formation of homometallic Hg7-MT species [17,24] Under the experi-mental conditions used, the evolution of the CD spectra is fully consistent with the presence of heterometallic Zn,Hg7-MT as an intermediate species between Zn,Hg5
-MT and Hg9-MT Overall, the information obtained using the optical techniques allows Zn,Hg5-MT and Hg11-MT to
be considered the hallmark species formed in the Zn/Hg
Fig 2 (A) CD, (B) absorption UV-vis, and (C) difference absorption UV-vis spectra obtained by subtracting the successive spectra of (B), corresponding
to the titration of recombinant mouse Zn 7 -MT1 with HgCl 2 at pH 7 and t ¼ 24 h The Hg(II) to MT molar ratios are indicated within each frame.
Trang 5Data obtained at pH 3 show a strong influence of
tand X on the stoichiometry and structure of the species
formed, as shown in Scheme 1B, and thus, the three
reaction pathways followed at this pH are remarkably
different Notwithstanding this, there is a minor effect of
tand X at the beginning and end of the titration Thus,
the addition of the first 4–6 of Hg(II) to apo-MT gives
rise to Hg-MT species of comparable stoichiometry and
structure, i.e Hg4-MT and Hg5)6-MT, and also the
presence of an excess of Hg(II) cation leads invariably to
Hg18-MT Furthermore, within the previous range [from
4–6 to 18 Hg(II)], subsequent additions of Hg(II) led to
low-chirality Hg-MT species under all conditions The
only exception is Hg13-MT, formed at t¼ 0 h and X ¼
Cl–, which shows a well-defined CD fingerprint, also
indicative of a highly chiral species Concerning the role
of the counter-ion, the differences observed in the CD
spectra of overloaded Hg-MT species, such as Hg10-MT
and Hg18-MT, formed at t¼ 24 h, provide evidence for the interaction of the chloride anion with Hg(II), as already found at pH 7
Fig 3 Role of the chloride anion in the degree of folding of Hg-MT
species observed by comparing the CD spectra of the Hg 11 -MT species
obtained in the titration of Zn 7 -MT with either HgCl 2 (in black) or
Hg(ClO 4 ) 2 (in grey), both at pH 7 and t ¼ 24 h.
Scheme 2 Proposed reaction pathways for Hg(II) binding to recombinant Zn 4 -aMT at pH 7 (A) and at pH 3 (B), under thermodynamic (t ¼ 24 h)
or kinetic (t ¼ 0 h) control conditions, using HgCl 2 or Hg(ClO 4 ) 2 as titrating agents The and „ symbols denote similarity and difference, respectively, between the structure of two species compared.
Fig 4 CD spectra of (A) the Zn 2 Hg 4 -aMT (in black) and Hg 5 -aMT (in grey), and Zn,Hg 4 -aMT (in black) and Zn,Hg 5 -aMT (in grey) species, respectively, obtained in the titrations of Zn 4 -aMT with HgCl 2 (solid lines) or Hg(ClO 4 ) 2 (dashed lines), both at pH 7 and t ¼ 24 h and (B) the
Hg 11 -aMT species obtained in the titrations of Zn 4 -aMT with HgCl 2 (in black) or Hg(ClO 4 ) 2 (in grey), both at pH 3 and t ¼ 24 h.
Trang 6Reaction of recombinant mouse Zn4-aMT with Hg(II)
Consideration of the optical spectroscopic data obtained in
the titrations of Zn4-aMT with Hg(II) at pH 7 (Figs S6–S8)
and pH 3 (Figs S9–S11) allows the proposal of the reaction
pathways shown in Scheme 2
Analogously to Zn7-MT, the stoichiometry of the
Hg-aMT species formed at pH 7 (Scheme 2A) along the
three titrations does not depend on t and X
Notwith-standing this, the Hg7-aMT species is absent in the
presence of Cl–at t¼ 24 h, and the species containing the
highest Hg(II) content, Hg11-aMT, is only obtained if
t¼ 0 h and X ¼Cl– Conversely, the structure and
chirality of the various Hg-aMT species are significantly
influenced by t and X, as evidenced by their CD spectra
Thus, the species with a Hg to aMT molar ratio higher
than 6–7 became more chiral if formed in the presence of
Cl–, among which, those formed at t¼ 0 h show the
highest degree of chirality Exceptionally, only the
Zn,Hg4-aMT species are comparable with respect to their
chirality and structure under the three sets of experimental
conditions
Interestingly, concerning the Zn,Hg4-aMT species, the
244(+) nm CD band recorded after the addition of 4 Hg(II)
to Zn4-aMT under all sets of conditions not only gives a
clear indication of the presence of Zn(II) in the aggregate,
but its intensity also suggests that the highest Zn(II) content
is found when X¼ ClO4 (Fig 4A) A similar analysis
reveals the presence of Zn(II) in the Hg5-aMT species
formed with X¼ ClO4 but its absence for X¼ Cl–
Chelex-100 treatment [23] of an aliquot of the
correspond-ing sample and subsequent analysis of the Zn and Hg
content by inductively coupled plasma atomic emission spectroscopy and inductively coupled plasma mass spectro-metry allowed us to unequivocally establish the Zn2Hg4 -aMT and Hg5-aMT stoichiometries for the species formed
at t¼ 24 h and X ¼ Cl– Overall, all previous data indicate that the replacement of Zn(II) by Hg(II) in Zn4-aMT proceeds more efficiently in the presence of Cl–than in the presence of ClO4
At pH 3 (Scheme 2B) neither t nor X has a substantial effect on the stoichiometry of the species formed during the titrations, except for the formation of two additional species, Hg3-aMT and Hg7-aMT, at t¼ 24 h and X ¼ ClO4 Conversely, the nature of the counter-ion strongly affects the chirality of the species This effect is remarkable for those species with a Hg(II) stoichiometry equal to or higher than 6, X¼ Cl– and t¼ 24 h In contrast, the Hg-aMT species formed in the presence of ClO4 show a very low degree of folding, indicating that Cl–ions strongly participate in the acquisition of the 3D structure of the Hg-aMT species (Fig 4B)
Reaction of recombinant mouse Zn3-bMT with Hg(II) The spectroscopic data obtained in the titrations of
Zn3-bMT with Hg(II) at pH 7 (Figures S12–S14) and pH
3 (Figures S15–S17) are consistent with the reaction pathways shown in Scheme 3 Comparison of the three sets of data recorded at pH 7 (Scheme 3A) reveals that the Hg:bMT stoichiometry of the species does not depend on the nature of the counter-ion Conversely, the stabilization time determines the Hg-bMT stoichiometry of most of the species formed and becomes particularly evident as the
Scheme 3 Proposed reaction pathways for Hg(II) binding to recombinant Zn 3 -bMT at pH 7 (A) and at pH 3 (B), under thermodynamic (t ¼ 24 h)
or kinetic (t ¼ 0 h) control conditions, using HgCl or Hg(ClO ) as titrating agents The and „ symbols denote similarity and difference,
Trang 7nuclearity of the species increases Notwithstanding this,
saturation occurs in all cases for 10 Hg(II) On the other
hand, CD data indicate that the degree of chirality and the
structure of the species formed up to Zn,Hg3)4-bMT
depend on t and X, the most chiral species being those
obtained at t¼ 24 h and X ¼ Cl– As opposed to that
observed for the aMT fragment, the CD spectra reveal that
the presence of Cl–favors the Zn(II) ions remaining bound
to the bMT protein in the first stages of the titration
Titrations carried out at pH 3 (Scheme 3B) reveal that
the stoichiometries of the Hg-bMT species become
dependent on t and X after the formation of Hg7-bMT
Comparison of the three sets of CD data indicates that the
degree of chirality of the Hg-bMT species is generally
independent of t However, the chirality of the species
obtained in the presence of Cl– is much higher than that
achieved when X¼ ClO4, except for the Hg3-bMT
species, with a very low chirality in both cases, and the
Hg7-bMT species, which show comparable chirality for
X¼ Cl– and ClO4 (Fig 5) Comparison of the CD fingerprints of the Hg-bMT species formed along the three titrations shows that their 3D structure is strongly dependent on t and X, except for Hg3-bMT, which is poorly structured under all conditions
Co-ordination environments around Hg(II) in Hg-MT species
The complexity of the Hg(II)-MT system, which is mainly the result of its Hg-thiolate nature, makes it difficult to obtain information on the co-ordination geometry around Hg(II) in the Hg(II)-MT aggregates from optical techniques (CD and/or UV-vis spectra) by simple treatment of the data There are several reasons: (a) the presence of different chromophores in the same species including Zn and/or Hg
as metal ions and SCys and/or Cl– as ligands; (b) the absence of well-established relationships between most of the previous chromophores and the corresponding absorp-tion wavelengths [3]; (c) the overlapping of the absorpabsorp-tion bands corresponding to different chromophores, as shown
by the spectral envelopes in the difference UV-vis spectra Despite this, analysis of the difference UV-vis data, which discloses the effect of each Hg(II) addition, can give an insight into the evolution of the co-ordination geometry about Hg(II) in the MT species formed by either Zn/Hg replacement in Zn7-MT or the addition of Hg to apo-MT
By following this approach, comparison of the difference UV-vis spectra obtained in the titrations of Zn7-MT,
Zn4-aMT and Zn3-bMT with HgCl2 at pH 7 and t¼
24 h (Fig 2, S6 and S12) indicates a parallel evolution of the co-ordination geometry about Hg(II) in the three peptides These spectra evolve according to the following pattern: (a) the addition of the first 7 Hg(II) eq to Zn7-MT, or the first 4 Hg(II) eq to any of the aMT and bMT fragments, causes initially the appearance of an asymmetric broad band
Fig 5 CD spectra of the Hg 7 -bMT species obtained in the titrations
of Zn 3 -bMT at pH 3 with HgCl 2 at t = 24 h (solid black line) or
t = 0 (solid grey line), or with Hg(ClO 4 ) 2 at t = 24 h (dashed grey
line).
Scheme 4 An insight into the evolution of the coordination geometries about Hg(II) in the Hg-MT species formed during the titrations of Zn 7 -MT,
Zn 4 -aMT and Zn 3 -bMT with HgCl 2 at t ¼ 24 h and pH 7 (A) or pH 3 (B) The different coloured areas have been deduced from the difference UV-vis spectra Preliminary TDPAC measurements on the Hg-MT species within a square enable correlation of each area with an specific coordination geometry about Hg(II).
Trang 8(230–340 nm), which eventually transforms into two new
broad overlapping bands with absorption maxima at 230
and 320 nm; (b) the next Hg(II) eq added to the three
peptides gives rise to a negative broad band with absorption
minima at 260 and 310 nm, together with a positive
absorption with a maximum intensity in the range 220–
230 nm; (c) further Hg(II) additions to Hg11-MT, Hg6-aMT
and Hg5-bMT cause the former envelope to turn into a
positive broad band with an absorption maximum at
250 nm with a shoulder at 310 nm; (d) this profile
collapses in the last steps of the titrations to give rise to very
weak absorptions along the whole wavelength range This
common evolution of the three titrations gives force to
different scenarios (denoted differently in Scheme 4A),
which may be consistent with the presence of three different
sets of co-ordination environments around Hg(II) in MT
Although the UV-vis difference spectra also suggest the
existence of different scenarios in the binding of Hg(II) to
Zn7-MT, Zn4-aMT and Zn3-bMT at pH 3 and t¼ 24 h
(Figures S3, S9 and S15), their evolution for the three
peptides (Scheme 4B) does not show such good parallelism
as that found at pH 7 Thus, at the beginning and end of the
three titrations, the spectral envelopes compare well and
suggest two different scenarios The former includes all the
species formed up to Hg5-MT, Hg4-aMT and Hg4-bMT,
and consists of a positive very intense band with a maximum
at 220 nm and a shoulder at 290 nm The second
scenario, which includes the species with the highest Hg(II)
to MT ratios, is characterized by very low absorptions along
the whole wavelength range In addition, a broad band with
a maximum at 250 nm and a shoulder at 310 nm
denotes a third common feature apparent in different
intermediate stages of the three titrations However, only
MT and the aMT peptides give rise to a fourth common
profile showing negative absorptions at 260 and 310 nm
together with a positive absorption within the range 220–
230 nm
The evolution of the difference UV-vis spectra at pH 7
(Scheme 4A) and pH 3 (Scheme 4B) is consistent with
preliminary time differential perturbed angular correlation
of c-rays (TDPAC) measurements (A` Leiva-Presa, M
Capdevila, P Gonza`lez-Duarte & W Tro¨ger, unpublished
results) on several Hg-MT species These results not only
corroborate the proposals made from the difference UV-vis
spectra but also suggest the specific co-ordination
environ-ments about Hg(II) associated with each scenario The
correlation between optical and TDPAC data is summarized
in Scheme 4, where the influence of the pH on the
co-ordi-nation geometry about Hg(II) becomes apparent One main
difference is the predominance of tetrahedral geometry at pH
7 and digonal geometry at pH 3, both coexisting with other
co-ordination geometries at increasing Hg to MT molar
ratios Interestingly, TDPAC measurements disclose two
types of linear co-ordination environments about mercury:
[Hg(SCys)2] and [Hg(SCys)Cl] Further TDPAC studies,
now in progress, should provide definitive data on the
co-ordinative features of the Hg-MT species
Concluding remarks
The above results document the strong influence of standard
ability of the counter-ions) on the nature and structural features of the Hg(II)-MT species obtained by Zn/Hg replacement in recombinant Zn7-MT, Zn4-aMT and
Zn3-bMT Table 1 shows that this dependence is diverse and thus difficult to generalize However, it can be concluded that the reaction conditions (pH, t) affect the structural properties more substantially than the stoichiom-etry of the Hg-MT species, and that the effect of the counter-ion (X) is particularly apparent on the structure of overloaded Hg-MT Specific findings of this work are: (a) the high number of Hg-MT species observed (Schemes 1– 3); (b) the formation of heterometallic Zn,Hg-MT aggre-gates, which include species such as Zn,Hg7-MT and Zn,Hg4-aMT, where the Hg(II) content equals that tradi-tionally expected for bivalent metal ions; (c) the nonadditive behavior of the a and b fragments with respect to the whole
MT Moreover, the stoichiometry found for the Zn2Hg4 -aMT species indicates that the binding of one Hg(II) cation
to MT does not require the displacement of one Zn(II) from the protein No such findings have previously been reported Earlier reports including CD and UV-vis data for the titration of native apo-MT2 and Zn7-MT2 with Hg(II) at
pH 7 proposed formation of the same set of species, Hg7
-MT, Hg11-MT and Hg20-MT, along both titrations, the latter being replaced by Hg18-MT in the titration of apo-MT2 at pH 2 Similarly, the titration of both apo-apo-MT2 and
Zn4-aMT2 at pH 7 resulted in formation of Hg4-aMT and
Hg11-aMT exclusively [14,17] Possibly, the different source
of the protein and the different experimental conditions used account for the discrepancy between these results and those reported in this work Overall, the optical spectral data sets observed for Hg(II) binding to either Zn-MT or apo-MT confirm the requirement for accurate control of the experimental conditions
Particularly relevant is the time variable, which has been scarcely considered in previous metal-MT binding studies
On the one hand, it has often been considered that metal displacement reactions in MT are kinetically facile and are generally complete within a few seconds [25] Moreover, the kinetic lability and consequently continuous breaking and reforming of the metal-sulfur bonds are well documented for the group 12 metal thiolates in solution [26] On the
Table 1 Influence of the reaction time (t) and binding ability of the counter-ions (X) on the nature and structural features of the set of Hg(II)-MT species formed during the corresponding titration Variables
in bold denote that they have a strong influence on most of the Hg-MT species formed Variables underlined affect only a minority of the species Voids denote that no general conclusions can be drawn The effect of the pH can be deduced by comparing the data of the same protein at the two pH values.
Set of Hg-MT species
Set of Hg-aMT species
Set of Hg-bMT species
Trang 9Hg(II) to MTs, which would determine its reaction rate, is
unreported Remarkably, our results show that not only
do the reaction pathways at t¼ 0 h and t ¼ 24 h differ
considerably, but also that the CD features of a particular
species formed along the titration at t¼ 0 h do not evolve
with time to those found for the isostoichiometric species at
t¼ 24 h
Acknowledgements
This work was supported by a grant from the Spanish Ministerio de
Ciencia y Tecnologı´a (BQU2001-1976) Dr Sı´lvia Atrian, who kindly
provided us with the recombinant proteins used in this work,
acknowledges the Spanish Ministerio de Ciencia y Tecnologı´a for
financial support (BIO2003-03892) We also acknowledge the Servei
d’Ana`lisi Quı´mica, Universitat Auto`noma de Barcelona (CD, UV-vis)
and the Serveis Cientı´fico-Te`cnics, Universitat de Barcelona (inductively
coupled plasma-atomic emission spectroscopy and inductively coupled
plasma mass spectrometry) for allocating instrument time.
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Supplementary material
The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4456/EJB4456sm.htm
Figs S1–S17