Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domain

Article abstract of DOI:10.1016/j.jinorgbio.2011.01.011

Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties of MTs have been quite limited. Recently, we focused on the α-domain of MT (MTα) as a protein matrix and incorporated a tetranuclear metal cluster as a reductant. UV-visible, CD and MS data indicate the formation of the stable tetranuclear metal-cysteine cluster in the MTα matrix with Fe ^II₄-MTα and Co^II₄-MTα species existing in water. Furthermore, the Fe^II₄- MTα species was found to promote the reduction of met-myoglobin and azobenzene derivatives under mild conditions. Particularly, the stoichiometric reduction of methyl red with Fe^II₄-MTα (1:1) was found to proceed with a conversion of 98% over a period of 6 h at 25 °C. This indicates that all of the four Fe(II) cores contribute to the reduction. In this paper, we describe the preparation and reactivity of the tetranuclear iron cluster in the protein matrix.

Full text of DOI:10.1016/j.jinorgbio.2011.01.011

Journal of Inorganic Biochemistry 105 (2011) 702–708
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Preparation and reactivity of a tetranuclear Fe(II) core in the
metallothionein α-domain
Yohei Sano a, Akira Onoda a,b, Rie Sakurai a, Hiroaki Kitagishi a,1, Takashi Hayashi a,
⁎
a
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka 565–0871, Japan
b
Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, Suita, Osaka 565–0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions
and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties
of MTs have been quite limited. Recently, we focused on the α-domain of MT (MTα) as a protein matrix and
incorporated a tetranuclear metal cluster as a reductant. UV–visible, CD and MS data indicate the formation of
the stable tetranuclear metal–cysteine cluster in the MTα matrix with FeII4–MTα and CoII4–MTα species
existing in water. Furthermore, the FeII4–MTα species was found to promote the reduction of met-myoglobin
and azobenzene derivatives under mild conditions. Particularly, the stoichiometric reduction of methyl red
with FeII4–MTα (1:1) was found to proceed with a conversion of 98% over a period of 6 h at 25 °C. This
indicates that all of the four Fe(II) cores contribute to the reduction. In this paper, we describe the preparation
and reactivity of the tetranuclear iron cluster in the protein matrix.
Received 16 October 2010
Received in revised form 13 January 2011
Accepted 14 January 2011
Available online 26 January 2011
Keywords:
Metallothionein
Tetranuclear iron–sulfur cluster
Met-myoglobin
Azobenzene reduction
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
clusters were prepared by several research groups as models of the
active site of nitrogenase [2–5]. Moreover, a variety of de novo-designed
Iron–sulfur clusters in metalloenzymes contribute widely to a
variety of biological redox reactions including electron transfer
systems [1]. For example, the mononuclear site of rubredoxin, and
the di-, tri- and tetranuclear sites of ferredoxins function as redox
centers. One of the most sophisticated iron–sulfur cofactors is the
FeMo cofactor of the nitrogenase family, which reduces N2 to
ammonia under physiological conditions [1]. An important charac-
teristic of iron–sulfur clusters such as ferredoxins is their negative
reduction potential as a result of coordination of sulfide ions and/or
cysteine thiolate groups which have strong electron-donating ability.
Several iron–sulfur proteins are capable of mediating electron transfer
at low potential within the metabolic system or promoting difficult
chemical reactions.
To unravel the elegant chemical nature of iron–sulfur containing
metalloenzymes, studies of iron–sulfur cluster models using synthetic
thiolate ligands or well-designed peptide ligands including cysteine
residues have recently been conducted in the field of bioinorganic
chemistry [2–13]. These investigations have suggested that sterically-
hindered thiolate ligands stabilize the iron–sulfur core in organic
solvents [2–7]. As an excellent example, double-cubane type iron–sulfur
peptides were investigated with the aim of preparing iron–sulfur
clusters in aqueous media [8–13]. For example, Holm et al. reported the
construction of a tetranuclear iron–sulfur structure using artificially-
created helix–loop–helix peptides as a model of the A-cluster in carbon
monoxide dehydrogenase (CODH). Therefore, to exploit non-natural
iron–sulfur clusters which promote reduction of substrates in an
aqueous environment, a cysteine-rich protein with high affinity for
heavy metal ions would be expected to be a promising surrogate for a
synthetic ligand. However, to the best of our knowledge, a sophisticated
functional model with sufficient redox reactivity has not been reported.
Metallothioneins (MTs) are small cysteine-rich proteins, which
function in the storage of essential metal ions or detoxification of toxic
metal ions in living organisms [14–17]. MTs are known to bind a
variety of metal ions including the d10 metal ions (ZnII, CdII, HgII, CuI
and AgI), divalent transition metal ions (FeII, CoII and NiII), and
synthetic metal clusters [18–29]. Rat liver metallothionein-2 (MT2)
consists of two metal binding domains, an α-domain at the C-terminus
and a β-domain at the N-terminus. These two domains form the
tetranuclear M4(S-Cys)11 and trinuclear M3(S-Cys)9 clusters, respec-
tively [30,31]. The tetranuclear cluster which has an adamantane-type
structure (e.g. M=Zn, Cd) in the α-domain of the metallothioneins
(MTα) has been reported to be more stable than the trinuclear cluster
in the β-domain [21]. Because of their unique structures of these
clusters, redox chemistry of MTs containing d10 metal ions has been
studied in previous reports [32,33]. In contrast, the chemical reac-
tivity of the metal-substituted MTs with redox-active metal ions, such
as Co(II), Fe(II), and Ni(II), has not yet been explored although their
⁎
Corresponding author at: Osaka University, Department of Applied Chemistry, 2-1
Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 6 6879 7928; fax: +81 6 6879
7930.
E-mail address: thayashi@chem.eng.osaka-u.ac.jp (T. Hayashi).
Present Address: Department of Molecular Chemistry and Biology, Faculty of
1
Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610–0321, Japan.
0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.01.011

Y. Sano et al. / Journal of Inorganic Biochemistry 105 (2011) 702–708
703
binding properties have been extensively studied [22–27]. In this
paper, we present our finding that an Fe(II)-substituted MTα domain
which has a tetranuclear Fe(II) core can smoothly perform the one-
electron reduction of met-myoglobin (met-Mb) and multi-electron
reduction of azobenzene derivatives in water.
using N,N′-diisopropylcarbodiimide (DIPCI) and HOBt as coupling
reagents in N,N-dimethylformamide (DMF), where a preloaded Fmoc-
Ala Wang resin was used for the peptide syntheses. A tyrosine residue
was attached at the N-terminus of each peptide to easily quantify the
concentration of the peptide using characteristic absorbance at
280 nm. The resin was washed with DMF and dichloromethane
(DCM) after each coupling cycle. The peptides were cleaved from the
resins by treatment with trifluoroacetic acid (TFA)/1,2-ethanedithiol
(EDT)/H2O/triisopropylsilane (TIS)=94/2.5/2.5/1 for 30 min in an ice
bath, with standing for an additional 2 h at room temperature. A
solution containing the cleaved peptides was concentrated and crude
peptides were precipitated by addition of diethyl ether. The crude
peptides were purified on a reverse-phase HPLC column (YMC, YMC-
Pack Pro C18, 250 mm×20 mm I.D. S=20 μm) with MeCN and water
as eluents (gradient containing 0.1% TFA), and the purities of the
peptides were confirmed by analytical HPLC (N95%). Free SH groups
of the peptides were quantified by reaction with Ellman's reagent,
5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), in 10 mM KPi (pH 7.4).
The purified solutions of the peptides were lyophilized to give a white
powder. The peptides were identified by ESI-TOF MS with 50% MeCN
aqueous solution containing 1% acetic acid. The molar absorption
coefficient was determined using the tyrosine band in the peptide
(MTα; ε280 =1240 M−1cm−1).
2. Experimental
2.1. Instruments
UV–visible (UV–vis) spectra were recorded using an MCPD-3000
multi-channel photo detector and UV–vis light source (MC-2530)
using optical fibers (Otsuka Electronics Co., Ltd. Japan) in a UNILab
glove box (b1 ppm O2; MBraun, Germany). Circular dichroism (CD)
spectra were recorded using a JASCO J720S spectrometer. ESI-TOF MS
analyses were carried out using an Applied Biosystems Mariner API-TOF
Workstation or a JEOL JMS-T100CS spectrometer. High-performance
liquid chromatography (HPLC) analyses were performed using a
Shimadzu SCL-10Avp HPLC system with a reversed-phase analytical
column. pH values were monitored using a HORIBA F-52 pH meter.
Kinetic measurements were carried out using a stopped-flow system
constructed by Unisoku, Co., Ltd. (Osaka, Japan).
2.2. Materials
2.4. Preparation of MTα peptide complexes
Fmoc-Ala Wang resin (Novabiochem), Fmoc-protected amino
acids and hydroxybenzotriazole (HOBt) were purchased from Peptide
Institute, Inc. (Osaka, Japan). Met-myoglobin (met-Mb) was prepared
according to a previously reported procedure with some modification
[34]. The protein from horse heart (N95%, checked by SDS-PAGE) was
purchased from Sigma (100684-32-0) and purified by column chroma-
tography through CM-52 (Whatman) and Sephadex G-25 (GE health-
care) columns. Purified Mb was completely oxidized to met-Mb with
excess potassium ferricyanide. To remove the used oxidant, met-Mb
solution was passed twice through the Sephadex G-25 column.
The solution of purified fraction was lyophilized and brought into a
N2-purged glove box. After purification, all experiments were carried
out under N2 atmosphere. All other chemicals were obtained commer-
cially. Distilled water was demineralized using a Barnstead NANOpure
Diamond™ apparatus.
All of the sample solutions were prepared with degassed water
prior to use in a nitrogen-purged glove box (b1 ppm O2). MTα peptide
solution (500 μM) was mixed with a freshly prepared aqueous
solution of FeCl2 or CoCl2 (20 mM). Titration experiments for MTα
with Fe(II) or Co(II) ion were carried out by adding aqueous solutions
of 20 mM Fe(II) or Co(II) ion into a stock solution of the MTα peptide in
50 mM Tris·HCl (pH 8). The tetranuclear complexes, FeII4–MTα and
CoII4–MTα, were identified by ESI-TOF MS in a MeCN/ammonium
acetate buffer (50 mM, pH 8) co-solvent (1:1 v/v). Folding of the
peptides upon addition of Fe(II) or Co(II) ion was determined by
performing CD measurements.
2.5. Reduction of met-Mb by FeII4–MTα
The reduction of met-Mb was spectrophotometrically monitored
after mixing with an FeII4–MTα solution by a stopped-flow apparatus
under a N2 atmosphere at 25 °C. Met-Mb (1.5 μM) in 50 mM KPi buffer
(pH 8) and FeII4–MTα (12–48 μM) in 50 mM KPi buffer (pH 8) were
prepared. The absorbance change at 428 nm for the formation of
2.3. Syntheses of MTα and mutated peptides
MTα and mutated peptides (MTα-C30G and MTα-C31G) were
manually synthesized according to a standard solid-phase method
Fig. 1. (a) Tetranuclear metal cluster within the MTα domain (PDB: 4MT2). The metal ions are represented as balls and the coordinating Cys residues are shown as sticks. (b) Amino
acid sequences of MTα peptides used in this study.

704
Y. Sano et al. / Journal of Inorganic Biochemistry 105 (2011) 702–708
deoxy-Mb was monitored after rapid mixing of both solutions. Rate
constants of the reduction were analyzed by one-phase exponential
kinetics to yield a pseudo-first-order rate constant (k1′). Plotting the
rate constants against FeII4–MTα concentrations yielded a second-
order rate constant k2 from the slope.
2.6. Reduction of azo-derivatives by FeII4–MTα
Reduction of 2-carboxy-4′-dimethylaminoazobenzene (methyl
red) by FeII4–MTα was followed by UV–vis spectrometry. Two
solutions, 100 μM methyl red in 100 μL of KPi buffer (50 mM, pH 8)
and 100 μM FeII4–MTα in 100 μL of KPi buffer (50 mM, pH 8), were
mixed and the absorption decay of the starting substrate, methyl red,
was monitored at 430 nm over 6 h. The reduced products (the aniline
derivatives, 2-carboxyaniline and 4-dimethylaminoaniline) were
determined by HPLC analysis. Reduction of the other azo-derivatives,
4-sulfo-4′-dimethylaminoazobenzene (methyl orange) and 4-carboxy-
4′-dimethylaminoazobenzene (p-methyl red), by MTα complexes
(CoII4–MTα, FeII4–MTα–C30G and FeII4–MTα–C31G) were also per-
formed in a similar manner.
3. Results and discussion
3.1. Syntheses of MTα and its mutants
Amino acid sequences of MTα analogs used in this study are
shown in Fig. 1b. The mutant peptides, MTα–C30G and MTα–C31G,
lack one of the terminal or bridging thiolate groups of the cysteines,
which may provide a vacant site around the metal center which is
Fig. 2. (a) Analytical HPLC elution profile of isolated MTα peptide. (b) ESI-TOF mass
spectrum of isolated MTα. The peaks for the MTα peptide are labeled with the observed
mass numbers, the calculated mass numbers, and the charge states.
Fig. 3. (a) Fe(II) and (b) Co(II) titration experiments (0–8 equiv.) of MTα monitored by UV–vis spectroscopy. Conditions: 20 μM MTα peptide, 50 mM Tris·HCl (pH 8.0) under a N2
atmosphere at 25 °C. The inset shows the absorbance at 315 nm and 329 nm as a function of the molar ratio of Fe(II)/MTα and Co(II)/MTα, respectively. ESI-TOF mass spectra of
(c) Fe4–MTα and (d) Co4–MTα. The peaks for the MTα complexes are labeled with the observed mass numbers, the calculated mass numbers, and the charge states.

Y. Sano et al. / Journal of Inorganic Biochemistry 105 (2011) 702–708
705
Fig. 4. Fe(II) and Co(II) titration experiment of MTα by CD spectroscopy (20 μM MTα in 50 mM Tris·HCl (pH 8.0) at 25 °C). (a) 0–6 equiv. Fe(II), (b) 0–2 equiv. Fe(II), (c) 2–4 equiv. Fe(II),
(a) 0–6 equiv. Co(II), (b) 0–2 equiv. Co(II), (c) 2–4 equiv. Fe(II). The spectra were recorded after addition of each aliquot of 0.5 equiv.
accessible for substrates. The series of MTα peptides was synthesized
by a standard solid-phase method with Fmoc chemistry using DIPCI
and HOBt as the coupling reagents in DMF. Cleaved crude peptides
were purified on a reverse-phase HPLC column with MeCN and water
as eluents. The isolated peptides were characterized by ESI-TOF MS
(actual mass number for apo-MT, 3272.2182; found, 3272.3370
(Fig. 2)). The result obtained by Ellman's analysis was in good
accordance with expected numbers of free thiol groups of cysteine
residues.
similar conditions. As shown in Fig. 3b, we observed a gradual
increase in intensity of the LMCT band (329 nm) and the d–d
transition bands at 690 and 747 nm which originate from the
tetrahedral CoII(S-Cys)4 core. The titration profile showed a clear
inflection point at 4 equiv. of Co(II) with respect to the MTα peptide
[24]. The formation of CoII4–MTα was also determined by ESI-TOF MS
analysis (calcd for CoII4–MTα (3+) 1167.63; found 1167.75) (Fig. 3d).
In this case, only the Co4–MTα species is detectable, indicating that
the binding affinity of MTα for Co(II) ion may be higher than that for
Fe(II), although both metals can be bound within the MTα matrix at a
ratio of 4:1.
3.2. Preparation of metal complexes with MTα peptides
The metal-binding behavior in the MTα peptide was also
investigated by CD spectroscopy (Fig. 4a and d). In the index region
for a peptide conformation, the intensity at 222 nm gradually
increased up to approximately 4 equiv. of Fe(II) ion. This provides
evidence that the MTα peptide gains a folded structure upon addition
of Fe(II) ions. The titrimetric CD profile also shows changes in the
negative band at 314 nm and the positive band at 346 nm, which are
involved in a range of LMCT absorption originating from the
tetrahedral FeII(S-Cys)4 geometry. Interestingly, the increase of the
positive and negative band intensities up to 2 equiv. of Fe(II) turned
into a decrease with a clear inflection point at the ratio of around 1:2
(Fig. 4b and c). In the pathway for the formation of FeII4–MTα, the
Fe(II) ions would be bound to the cysteine thiolate groups of the
peptide as a mononuclear complex up to around 2 equiv. (Fig. 4b)
and then the folding of the MTα–Fe(II) complex would begin at the
higher Fe(II) ratio (Fig. 4c). Such metal binding behavior in con-
sistent with the non-cooperative two-stage mechanism can be ex-
plained in previous MT studies [35,36]. To monitor the formation of a
The stoichiometry of the complex between MTα and Fe(II) ions
was analyzed by titrimetric UV–vis experiments as illustrated in Fig. 3.
Upon addition of FeCl2 into the MTα peptide in 50 mM Tris·HCl (pH
8.0), the absorption at 315 nm gradually increased (Fig. 3a). This is
attributed to a ligand-to-metal charge transfer (LMCT) band charac-
teristic for tetrahedral FeII(S-Cys)4 coordination [22]. The intensity of
the increasing absorption was saturated at the [MTα]:[FeII] ratio of
1:4. The successful formation of this tetranuclear ferrous complex was
determined by ESI-TOF MS analysis (calcd for FeII4–MTα (3+)
1163.79; found 1163.64) (Fig. 3c), although the mass numbers of
the mono-, di-, and trinuclear ferrous complexes were also detected
due to the insufficient affinity of MTα for Fe(II) ions under the mass
spectroscopic conditions. The ferrous complexes of mutated peptides,
FeII4–MTα–C30G and FeII4–MTα–C31G, were also confirmed as
tetranuclear Fe(II) complexes under similar conditions. Furthermore,
to confirm the binding stoichiometry of the MTα peptide with other
metal ions, we performed titrimetric experiments of Co(II) ions under
Scheme 1. One electron reduction of met-Mb by FeII4–MTα.

706
Y. Sano et al. / Journal of Inorganic Biochemistry 105 (2011) 702–708
Fig. 6. Reduction of methyl red with FeII4–MTα monitored by UV–vis spectroscopy. An
example of a set of UV–vis spectra used to monitor the reaction. Conditions: 100 μM
methyl red, 100 μM FeII4–MTα, 50 mM KPi (pH 8.0) under a N2 atmosphere at 25 °C.
states (408 nm and 428 nm for met-Mb and deoxy-Mb, respectively
(Scheme 1)), because the absorption of FeII4–MTα is not strong
enough to follow the oxidation of one of the Fe(II) ions within the
tetranuclear cluster. After mixing the solution of met-Mb and FeII
–
4
MTα, the absorption changes occurring during conversion of met-Mb
to deoxy-Mb were monitored by performing stopped flow measure-
ments under anaerobic conditions (Fig. 5a). The 408 nm absorption
decreased with a concomitant increase of the 428 nm absorption,
indicating that met-Mb was smoothly converted to the deoxy-form.
Thus, the redox potential of FeII4–MTα/FeII3FeIII–MTα seems to be
lower than that of deoxy-Mb/met-Mb (46 mV vs NHS) [37]. The
single-phase first-order kinetics affords a rate constant for production
of the deoxy-form of 1.26×103 M−1 s−1 based upon the apparent rate
constant and the FeII4–MTα concentration (Fig. 5b). The
corresponding k2 constants for the MTα peptide without any metal
ions or only peptide-free FeCl2 were determined to be 171 M−1 s−1
and 23.9 M−1 s−1, respectively. The larger k2 constant obtained by
FeII4–MTα suggests that the one-electron reduction was promoted by
the tetranuclear Fe(II) cluster bound within the MTα matrix.
Fig. 5. Reduction of met-Mb with FeII4–MTα monitored by stopped-flow UV–vis spec-
troscopy. (a) Observed spectra; the inset shows an example of the time course for the
formation of the deoxy-form at 428 nm. Conditions: 1.5 μM met-Mb, 12 mM FeII4–MTα,
50 mM KPi (pH 8.0) under a N2 atmosphere at 25 °C. (b) FeII4–MTα dependence under
pseudo first-order conditions. The solid line is for a FeII4–MTα-dependent rate constant of
1260 M−1 s−1
.
tetranuclear Co(II) cluster, we performed titrimetric experiments using
CD spectroscopy under similar conditions (Fig. 4d). The titrimetric
CD spectra acquired during the formation of the CoII4–MTα complex
exhibited a positive maximum at 311 nm and a negative maximum at
370 nm originating from the tetrahedral CoII(S-Cys)4 moieties. The
intensity of both LMCT bands increased with addition of up to 2 equiv. of
Co(II) ion and subsequently decreased in accordance with the formation
of Co(II) cluster (Fig. 4e and f). These spectrometric measurements
support the proposal that the M4–MTα species (M=Fe(II) or Co(II)) are
found to be the tetranuclear metal clusters.
Next, we investigated multi-electron reduction using FeII4–MTα
in an aqueous buffered solution. As a result of this survey, we found
that azobenzene derivatives can be reduced by the FeII4–MTα com-
plex. In general, the azobenzene compounds can be converted to
corresponding hydrazobenzene derivatives. Subsequent reduction
leads to aniline derivatives as the final products. Each reduction step
requires two-electron reduction coupled with two protons
(Scheme 2). We first monitored the reduction of methyl red, which
is a common substrate in enzymatic studies of azo-moiety reduction
[38–40], in the presence of FeII4–MTα. A decrease of absorption at
430 nm originating from the starting azo compound indicates
conversion into the corresponding reduced derivatives (Fig. 6).
Intermediate hydrazobenzene derivatives containing strong electron
donating substituents, such as the dimethylamino group of methyl
red, were reported to be unstable because a disproportionation
3.3. Reductive activity of FeII4–MTα
To investigate the reactivity of FeII4–MTα as a reductant, we first
analyzed a one-electron redox reaction with ferric Mb, which has a
strong Soret-band absorption dependent upon the FeII/FeIII oxidation
Scheme 2. Reduction of methyl red by FeII4–MTα. Electrons and protons originate from FeII4–MTα and water, respectively.

Y. Sano et al. / Journal of Inorganic Biochemistry 105 (2011) 702–708
707
rigid core than that of FeII4–MTα, CoII4–MTα exhibited lower
reactivity than that of FeII4–MTα (Table 1, entry 9) probably because
of unmatched redox potentials. These results provide evidence that
the FeII4–MTα protein has reductive activity with respect to various
azobenzene derivatives.
4. Conclusions
We have described the preparation and reactivity of an artificially
created iron–sulfur cluster in apoprotein matrix. A tetranuclear Fe(II)
or Co(II) core ligated by cysteines was successfully formed in the
metallothionein α-domain. Furthermore, the tetranuclear FeII core in
the MTα peptide is surprisingly capable of not only one-electron
reduction of met-Mb but also multi-electron reduction of azobenzene
derivatives such as methyl red, in an aqueous solution. To the best of
our knowledge, this is the first example of an artificial metal–sulfur
core within a protein matrix which promotes substrate reductions in
water. Further investigations on modifications of metallothionein to
enhance its reactivity and/or to catalyze substrate reduction are now
in progress.
Abbreviations
met-Mb met-myoglobin
Fig. 7. Analytical HPLC chromatograms (322 nm) of a reaction mixture at (a) 0 h, (b) 2 h,
(c) 6 h.
MT
metallothionein
MTα
α-domain of metallothionein
Fmoc-Ala 9-fluorenylmethyloxycarbonyl protected alanine
reaction occurs to produce the corresponding reduced anilines and
oxidized azobenzene derivatives [41]. Thus, the products were also
analyzed by HPLC, indicating the conversion into the corresponding
aniline product, 2-carboxyaniline (Fig. 7). Interestingly, the conver-
sion of methyl red in the presence of FeII4–MTα was 98% after 6 h
(Table 1, entry 2). This finding indicates that all four of the Fe(II) ions
in the peptide can contribute to the reduction of the azobenzene
derivatives. In contrast, the reduction of methyl red was found to be
strikingly slow when either the MTα peptide without the Fe(II)
cluster or a peptide-free Fe(II) ion was added to the substrate solution.
Therefore, FeII4–MTα was confirmed as a reactive species for the
reduction of the azobenzene moiety (Table 1, entry 3 and 4).
HOBt
DIPCI
DTNB
KPi
hydroxybenzotriazole
N,N′-diisopropylcarbodiimide
5,5′-(dithiobis)(2-nitrobenzoic acid)
potassium phosphate
methyl red 2-carboxy-4′-dimethylaminoazobenzene
methyl orange 4-sulfo-4′-dimethylaminoazobenzene
p-methyl red 4-carboxy-4′-dimethylaminoazobenzene
LMCT
ligand-to-metal charge transfer
Acknowledgements
FeII4–MTα also reacted with other azobenzene analogs having
different substituents at the para position, such as the carboxyl group
(p-methyl red) and the sulfonate group (methyl orange) (Table 1,
entry 5 and 6). As a result, it is found that for methyl red, FeII4–MTα
shows the highest reactivity among the above three azobenzene
analogs. In addition, mutated FeII4–MTα–C30G and FeII4–MTα–C31G,
which presumably have more flexible coordination spheres relative to
that of the FeII4–MTα, were used for the reduction of methyl red. The
reduction reactions using FeII4–MTα–C30G and FeII4–MTα–C31G
were found to be slower than that of FeII4–MTα (Table 1, entry 7
and 8). Therefore, the designed tetranuclear core supported by the
native metal binding fragment seems to be more favorable for
promoting the reductive reaction. Although CoII4–MTα has a more
The authors thank Prof. Masahito Kodera (Doshisha University)
for helping the ESI measurements. This work was supported by
Grants-in-Aid for Scientific Research from MEXT. Y.S. acknowledges a
support from the Japan Society for the Promotion of Science (JSPS)
Research Fellowships for Young Scientists and Global COE Program
“Global Education and Research Center for Bio-Environmental
Chemistry” of Osaka University. A.O. acknowledges a support from
the Frontier Research Base for Global Young Researchers, Osaka
University, on the Program of MEXT, from the Advanced Technology
Institute, Tokyo, and from the Sasakawa Scientific Research Grant,
the Japan Science Society.
References
[1] B. Schmid, H.-J. Chiu, V. Ramakrishnan, J.B. Howard, D.C. Rees, in: A. Messerschmidt,
R. Huber, T. Poulos, K. Wieghardt (Eds.), Handbook of Metalloproteins, vol. 2, John
Wiley & Sons, Ltd, Chichester, 2001, pp. 1025–1036.
Table 1
Reduction of azo compounds by MTα complexesa.
[2] Y. Zhang, R.H. Holm, J. Am. Chem. Soc. 125 (2003) 3910–3920.
[3] Y. Zhang, J. Zuo, H. Zhou, R.H. Holm, J. Am. Chem. Soc. 124 (2002) 14292–14293.
[4] Y. Ohki, M. Imada, A. Murata, Y. Sunada, S. Ohta, M. Honda, T. Sasamori, N. Tokitoh,
M. Katada, K. Tatsumi, J. Am. Chem. Soc. 131 (2009) 13168–13178.
[5] Y. Ohki, Y. Ikagawa, K. Tatsumi, J. Am. Chem. Soc. 129 (2007) 10457–10465.
[6] S.C. Lee, R.H. Holm, Chem. Rev. 104 (2004) 1135–1158.
Entry
Azo compounds
MTα complexes
Time (h)
Conversion (%)
1
2
3
4
5
6
7
8
9
Methyl red
Methyl red
Methyl red
Methyl red
Methyl orange
p-Methyl red
Methyl red
Methyl red
Methyl red
FeII4–MTα
FeII4–MTα
MTα
2
6
2
2
2
2
2
2
2
81
98
8.0
1.2
33
30
60
62
1.2
FeCl2
[7] P.V. Rao, R.H. Holm, Chem. Rev. 104 (2004) 527–559.
FeII4–MTα
FeII4–MTα
FeII4–MTα–C30G
FeII4–MTα–C31G
CoII4–MTα
[8] C.E. Laplaza, R.H. Holm, J. Am. Chem. Soc. 123 (2001) 10255–10264.
[9] K.B. Musgrave, C.E. Laplaza, R.H. Holm, B. Hedman, K.O. Hodgson, J. Am. Chem. Soc.
124 (2002) 3083–3092.
[10] A.L. Nivorozhkin, B.M. Segal, K.B. Musgrave, S.A. Kates, B. Hedman, K.O. Hodgson,
R.H. Holm, Inorg. Chem. 39 (2000) 2306–2313.
[11] C.L. Hill, J. Renaud, R.H. Holm, L.E. Mortenson, J. Am. Chem. Soc. 99 (1977) 2549–2557.
[12] L. Que Jr., J.R. Anglin, M.A. Bobrik, A. Davison, R.H. Holm, J. Am. Chem. Soc. 96
(1974) 6042–6048.
a
Conditions: 100 μM azo compound, 100 μM FeII4–MTα, 50 mM KPi (pH 8.0) under
N2 atmosphere at 25 °C.

708
Y. Sano et al. / Journal of Inorganic Biochemistry 105 (2011) 702–708
[13] B.R. Gibney, S.E. Mulholland, F. Rabanal, P.L. Dutton, Proc. Natl Acad. Sci. USA 93
(1996) 15041–15046.
[28] S. Ariyasu, A. Onoda, R. Sakamoto, T. Yamamura, Bioconjug. Chem. 20 (2009)
2278–2285.
[14] H.-J. Hartmann, U. Weser, in: I. Bertini, H.B. Gray, E.I. Stiefel, J.S. Valentine (Eds.),
Biological Inorganic Chemistry, University Science Books, Sausalito, 2007, pp. 156–162.
[15] E. Babini, M.S. Viezzoli, in: I. Bertini, A. Siegl, H. Sigel (Eds.), Handbook on
Metalloproteins, Marcel Dekker, Inc, New York, 2001, pp. 1001–1021.
[16] D.M. Templeton, M.G. Cherian, Meth. Enzymol. 205 (1991) 11–24.
[17] J. Chan, Z. Huang, M.E. Merrifield, M.T. Salgado, M.J. Stillman, Coord. Chem. Rev.
233–234 (2002) 319–339.
[18] J.H.R. Kägi, A. Schäffer, Biochemistry 27 (1988) 8509–8515.
[19] H. Willner, M. Vašák, J.H.R. Kägi, Biochemistry 26 (1987) 6287–6292.
[20] K.B. Nielson, C.L. Atkin, D.R. Winge, J. Biol. Chem. 260 (1985) 5342–5350.
[21] M.J. Stillman, W. Cai, A.J. Zelazowski, J. Biol. Chem. 262 (1987) 4538–4548.
[22] M.T. Werth, M.K. Johnson, Biochemistry 28 (1989) 3982–3988.
[23] M. Good, M. Vašák, Biochemistry 25 (1986) 8353–8356.
[29] S. Ariyasu, A. Onoda, R. Sakamoto, T. Yamamura, Dalton Trans. 39 (2009) 3742–3747.
[30] A.H. Robbins, D.E. McRee, M. Williamson, S.A. Collett, N.H. Xuong, W.F. Furey, B.C.
Wang, C.D. Stout, J. Mol. Biol. 221 (1991) 1269–1293.
[31] W. Braun, M. Vašák, A.H. Robbins, C.D. Stout, G. Wagner, J.H.R. Kägi, K. Wüthrich,
Proc. Natl Acad. Sci. USA 89 (1992) 10124–10128.
[32] J. Limson, T. Nyokong, Electroanalysis 9 (1997) 255–260.
[33] A.R. Green, M.J. Stillman, Inorg. Chim. Acta 226 (1994) 275–283.
[34] T. Hayashi, H. Dejima, T. Matsuo, H. Sato, D. Murata, Y. Hisaeda, J. Am. Chem. Soc.
124 (2002) 11226–11227.
[35] K.E.R. Duncan, M.J. Stillman, FEBS J. 274 (2007) 2253–2261.
[36] A. Krężel, W. Maret, J. Am. Chem. Soc. 129 (2007) 10911–10921.
[37] J. Ye, R.P. Baldwin, Anal. Chem. 60 (1988) 2263–2268.
[38] N. Ueyama, M. Nakata, A. Nakamura, J. Mol. Catal. 14 (1982) 341–350.
[39] C.J. Casewit, D.E. Coons, L.L. Wright, W.K. Miller, M.R. DuBois, Organometallics 5
(1986) 951–955.
[24] M. Good, M. Vašák, Biochemistry 25 (1986) 3328–3334.
[25] M. Vašák, J.H.R. Kägi, Proc. Natl Acad. Sci. USA 78 (1981) 6709–6713.
[26] M. Vašák, J.H.R. Kägi, B. Holmquist, B.L. Vallee, Biochemistry 20 (1981) 6659–6664.
[27] M. Vašák, J. Am. Chem. Soc. 102 (1980) 3953–3955.
[40] L.H. Andrews, A. Lowy, J. Am. Chem. Soc. 56 (1934) 1411–1412.
[41] S. Zbaida, W.G. Levine, Chem. Res. Toxicol. 4 (1991) 82–88.