Unusual structural features in the lysozyme derivative of the tetrakis(acetato)chloridodiruthenium(ii,iii) complex

Article abstract of DOI:10.1002/anie.201403337

The reaction between the paddle-wheel tetrakis(acetato) chloridodiruthenium(II,III) complex, [Ru₂(μ-O₂CCH ₃)₄Cl] and hen egg-white lysozyme (HEWL) was investigated through ESI-MS and UV/Vis spectroscopy and the formation of a stable metal-protein adduct was unambiguously demonstrated. Remarkably, the diruthenium core is conserved in the adduct while two of the four acetate ligands are released. The crystal structure of this diruthenium-protein derivative was subsequently solved through X-ray diffraction analysis to 2.1 A resolution. The structural data are in agreement with the solution results. It was found that HEWL binds two diruthenium moieties, at Asp101 and Asp119, respectively, with the concomitant release of two acetate ligands from each diruthenium center.

Full text of DOI:10.1002/anie.201403337

Angewandte
Chemie
DOI: 10.1002/anie.201403337
Metal–Protein Interactions
Unusual Structural Features in the Lysozyme Derivative of the
Tetrakis(acetato)chloridodiruthenium(II,III) Complex**
Luigi Messori,* Tiziano Marzo, Rute Nazarꢀ Fernandes Sanches, Hanif-Ur-Rehman,
Denise de Oliveira Silva,* and Antonello Merlino*
Abstract: The reaction between the paddle-wheel tetrakis-
(acetato)chloridodiruthenium(II,III)
complex,
[Ru2(m-
O2CCH3)4Cl] and hen egg-white lysozyme (HEWL) was
investigated through ESI-MS and UV/Vis spectroscopy and
the formation of a stable metal–protein adduct was unambig-
uously demonstrated. Remarkably, the diruthenium core is
conserved in the adduct while two of the four acetate ligands
are released. The crystal structure of this diruthenium–protein
derivative was subsequently solved through X-ray diffraction
analysis to 2.1 ꢁ resolution. The structural data are in agree-
ment with the solution results. It was found that HEWL binds
two diruthenium moieties, at Asp101 and Asp119, respectively,
with the concomitant release of two acetate ligands from each
diruthenium center.
Figure 1. The typical paddle-wheel structure of the [Ru2(m-O2CCH3)4]+
unit.
T
he tetrakis(acetato)chloridodiruthenium(II,III) complex
[Ru2(m-O2CCH3)4Cl] (1) was first prepared by Stephenson
and Wilkinson[1] and then structurally characterized by
Cotton and co-workers.[2] Notably, this compound bears
a dimetallic center in which two mixed-valence ruthenium
cal,[5] and magnetic[6] properties of this peculiar dimetallic
center were investigated and elucidated afterwards in a series
of physicochemical studies.[2a,7] Remarkably, compound 1 is
the precursor of a class of diruthenium complexes that have
found widespread application in a variety of fields.
ꢀ
ions are directly connected through a strong Ru Ru bond,
with a bond order of 2.5 and a short distance of 2.28 ꢀ
(Figure 1). In the crystal state, this compound exhibits
a polymeric structure in which the diruthenium centers,
coordinated to four bridging acetates, are linked by chloride
axial ligands into an infinite zig-zag chain.[2] The unique
structural,[2] electronic,[2–3] spectroscopic,[3–4] electrochemi-
For instance, diruthenium tetracarboxylates can act as
building blocks for cubic 3D network structured molecule-
based magnets[8] and as catalysts.[9] Early studies reported
their potential as catalysts for the hydrogenation of alkenes
and alkynes[9a,b] and for the competitive cyclopropanation of
alkenes.[9c] More recently, compound 1 and its derivatives
have been reported to act as catalysts for the oxidative
transformation of secondary amines into imines,[9d] the oxy-
genation of organic sulphides,[9e] the oxidation of alcohols,[9f]
and the skeletal reorganization of enynes.[9g]
[*] Prof. L. Messori, T. Marzo
Department of Chemistry, University of Florence
Via della Lastruccia 3, 50019 Sesto Fiorentino (FI) (Italy)
E-mail: luigi.messori@unina.it
Furthermore, diruthenium tetracarboxylate compounds
have been evaluated as prospective pharmaceutical agents.[10]
Compound 1 has been used as a precursor to prepare a novel
class of diruthenium(II,III) metallodrugs of the general
formula [Ru2(RCOO)4Cl], where RCOOꢀ is a carboxylate
anion derived from a therapeutic drug.[10c] In particular,
metallodrugs containing carboxylate ligands derived from the
nonsteroidal anti-inflammatory drug ibuprofen[10d,e] or from g-
linolenic acid[10f,g] were found to be active against glioma
tumor models and have been investigated for partial elucida-
tion of their mechanism of action. Moreover, the diruthe-
nium–ibuprofenate complex shows anti-inflammatory prop-
erties with reduced gastric ulceration in vivo compared to the
ibuprofen parent drug.[10h]
R. N. F. Sanches, Hanif-Ur-Rehman, Prof. D. de Oliveira Silva
Departamento de Quꢀmica Fundamental, Instituto de Quꢀmica
Universidade de S¼o Paulo
Av. Prof. Lineu Prestes, 748, B2T, 05508-000, S¼o Paulo, SP (Brazil)
E-mail: deosilva@iq.usp.br
Dr. A. Merlino
Department of Chemical Sciences, University of Naples Federico II
Complesso Universitario di Monte Sant’Angelo
Via cintia, 80126 Napoli (Italy)
E-mail: antonello.merlino@unina.it
[**] We thank G. Sorrentino and M. Amendola for technical assistance
and G. Di Martino for her help in the crystallization trials. L.M. and
T.M. acknowledge Beneficentia Stiftung (Vaduz, Liechtenstein) and
AIRC (IG-12085) for generous financial support and COST action
CM1105. D.d.O.S., R.N.F.S. and H.U.R. acknowledge Brazilian
agencies FAPESP (grant 2011/06592-1), CNPq and CAPES for
financial support.
As a result of the high degree of interest of this class of
compounds for bioinorganic chemistry and biomedical appli-
cations, compound 1 was recently investigated for reactivity
with some low-molecular-weight biomolecules such as amino
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201403337.
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acids and a few natural reducing agents (ascorbic acid and
glutathione).[11]
the occurrence of aggregation phenomena and the formation
of oligomeric species.
However, no detailed studies on the interactions of
compound 1 and its analogues with proteins have been
reported. This prompted us to analyze in depth the reaction of
tetrakis(acetato)chloridodiruthenium(II,III) with the model
protein hen egg-white lysozyme (HEWL) through independ-
ent biophysical methods to describe the resulting derivatives.
HEWL has already been used as a scaffold to characterize
a variety of metal adducts and thus unravel the molecular
basis for protein–metal complex recognition.[12]
At first, the behavior of compound 1 in a reference buffer
at pH 6.8 was investigated by UV/Vis spectroscopy (Fig-
ure 2a). The initial spectrum is characterized by a main band
at 423 nm that is assigned to the p(Ru-O)!p*(Ru2) elec-
Later, the same process was monitored by UV/Vis
spectroscopy in the presence of an equimolar amount of
HEWL (Figure 2b). In this case, a marked increase in
absorbance is detected in the 300–400 nm region and a less
pronounced increase is detected between 500 and 600 nm,
while the band at 423 nm is basically conserved. By contrast,
the generalized increase in absorbance over the whole
spectral window is no longer observed. At the end of the
process, ultrafiltration was carried out with a cut-off of 3 kDa,
and the two resulting fractions were analyzed by UV/Vis
spectroscopy. Notably, the band at 423 nm is associated with
the low-MW fraction while the band at 330 nm is mainly
associated with the protein fraction (see the Supporting
Information). These results imply that the presence of the
protein greatly hinders the aggregation/oligomerization pro-
cesses; ruthenium binding to the protein results in the
appearance of a characteristic broad band centered at 330 nm.
Electrospray ionization mass spectrometry (ESI-MS)
measurements were then carried out to better characterize
adduct formation between compound 1 and HEWL. ESI-MS
is a potent investigation tool that can be used to identify the
exact nature of protein-bound metal fragments and precisely
assess the metal/protein stoichiometry in this kind of
adduct.[13] The resulting ESI-MS spectra are shown in
Figure 3. Remarkably, a rather intense new peak is detected
at 14656.6 Da, which corresponds to a complex formed by
HEWL with a fragment of the type [Ru2(m-O2CCH3)2-
(H2O)2]3+. The intensity of this peak progressively increases
with time over 72 h.
Figure 2. Time-dependent UV/Vis spectra (up to 24 h) of compound 1
10ꢀ3 m dissolved in 20 mmolLꢀ1 ammonium acetate buffer, pH 6.8 (a),
and in the presence of HEWL (b). [compound 1]=5ꢁ10ꢀ4 mmolLꢀ1
complex/protein=1:1.
,
tronic transition.[3] The maximum wavelength of this band
indicates the predominance in solution of the neutral axially
substituted [Ru2(m-O2CCH3)4(H2O)2]+ species.[11a] Monitoring
the electronic spectra over 24 h shows specific and progressive
increases in the absorbance in the UV (300–370 nm) and
visible (500–700 nm) regions. The new bands appearing in
these regions might be related to replacement of the axial
ligands as previously reported for the reactions of [Ru2(m-
Figure 3. Deconvoluted ESI-MS of HEWL treated with 10ꢀ3 molLꢀ1
[Ru2(m-O2CCH3)4Cl] (complex/protein=10:1) in 20 mmolLꢀ1 ammoni-
um acetate buffer, pH 6.8 recorded after 6 h (A), 24 h (B), 48 h (C),
and 72 h (D) of incubation at RT.
O2CCH3)4(H2O)2]+ with amino acids.[11a]
A generalized
increase in absorbance is also detected over the whole 300–
800 nm spectral interval, a result that might be attributed to
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The observation of this new peak implies that the
dimetallic center is retained upon protein binding, while
two of the four acetate ligands are lost owing to ligand-
replacement reactions.
To further elucidate the structure of the metal–protein
adduct, we tried to crystallize the protein in the presence of
compound 1. Crystals suitable for X-ray diffraction studies
were grown (see the Supporting Information). The structure
was refined at 2.15 ꢀ resolution to an R-factor of 18.9 (R-free
25.4) and deposited in the Protein Data Bank (PDB ID
4OOO, see the Supporting Information). The overall protein
conformation is not significantly affected by the binding of
compound 1 (Figure 4): in fact, the HEWL structure in the
complex is almost identical to that of the native protein (PDB
code 193L), with root mean square deviation in the positions
of the backbone a-carbon atoms as low as 0.26 ꢀ.
Figure 4. Overall structure of HEWL–compound 1. The diruthenium
centers are covalently attached to the Asp101 or Asp119 side chains.
Figure 5. Details of the binding sites of the compound 1 moieties in
HEWL. The adduct contains diruthenium diacetate fragments coordi-
nated to Asp101 (a) or Asp119 (b) side chains. In both sites, the
binding of compound 1 to HEWL is accompanied by the loss of two
acetate groups. The 2FoꢀFc electron density maps are contoured at
the 3s (red) and 1s (grey) level.
Inspection of F0ꢀFc electron density maps led to the
identification of two distinct binding sites for the bis-
(acetato)diruthenium fragments on the HEWL surface
(Figure 5).
The side-chain oxygen atoms of Asp101 or Asp119 are
found to coordinate to the diruthenium center in a fashion
leading to a distorted lantern-like structure. In both sites, the
Ru atoms have occupancy equal to 0.5. Selected bond lengths
and angles for the diruthenium center in the adduct are given
in Table S1 in the Supporting Information.
The structural results provide valuable hints for the likely
mechanism of protein metalation. It may be postulated that
the aspartate group is able to displace one of the acetate
ligands coordinated to the diruthenium core in compound
1 and to replace it as a bidentate ligand; a second acetate is
then removed, possibly owing to steric hindrance with the
protein, and is replaced by two water molecules. Despite the
evident similarity of the metal–protein interactions at the two
distinct sites, it must be noted that in one case, the two
remaining acetate ligands are trans to each other, while in the
other case, they have a cis configuration.
In conclusion, we have shown that the [Ru2(m-O2CCH3)4]+
paddle-wheel complex is able to interact with the model
protein HEWL to form a stable adduct. The adduct was
characterized in detail based on largely consistent ESI-MS
and X-ray diffraction results. In particular, the peculiar
structural features of the adduct were elucidated through
high-resolution crystallographic data. Interestingly, the
HEWL derivative of compound 1 bears two diruthenium
centers tightly anchored to two distinct aspartate groups in
positions that are accessible to the solvent. In both cases,
association of the diruthenium moieties with the protein is
achieved through the formation of two coordination bonds
between the two oxygen atoms of the side-chain carboxylate
group of Asp and the two ruthenium atoms of the diruthe-
nium motif. To our knowledge, this peculiar mode of
association between the diruthenium motif and the protein
is unprecedented. However, it is worth noting that quite
similar behavior has emerged for dirhodium tetracarboxylate
complexes in their interactions with a variety of peptides as
described by Ball and co-workers.[14] Remarkably, association
of the dirhodium motif to two aspartate groups of the ZF
As noted above, the degree of occupancy of the two
aspartate binding sites by the diruthenium fragment is similar,
thus comparable affinities may be inferred. The resulting
adduct is apparently very stable as indicated by the time-
course absorption spectra.
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peptide after the release of two acetate ligands, in a fashion
that closely resembles the behavior of the diruthenium
complex with lysozyme, has been reported.[14a]
Upon consideration of the known and established cata-
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Received: March 14, 2014
Published online: && &&, &&&&
Keywords: bioinorganic chemistry · metallodrugs · protein–
.
metal adducts · ruthenium · X-ray diffraction
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Communications
Metal–Protein Interactions
Paddling through: The adduct formed
between the paddle-wheel tetrakis-
(acetato)chloridodiruthenium(II,III)
complex and hen egg-white lysozyme
(turquoise ribbon structure) was charac-
terized through ESI mass spectrometry
and X-ray crystallography. Unusual and
interesting features were revealed in the
binding mode of the diruthenium centers
(circled) to the protein side chains.
L. Messori,* T. Marzo,
R. N. F. Sanches, Hanif-Ur-Rehman,
D. de Oliveira Silva,*
A. Merlino*
&&&& — &&&&
Unusual Structural Features in the
Lysozyme Derivative of the
Tetrakis(acetato)chloridodiruthenium-
(II,III) Complex
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www.angewandte.org
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These are not the final page numbers!