A supramolecular-hydrogel-encapsulated hemin as an artificial enzyme to mimic peroxidase

Article abstract of DOI:10.1002/anie.200700404

(Figure Presented) Faking it: The use of a supramolecular hydrogel as the structural component of artificial enzymes provides a new and useful approach to the development of biomimetic catalysts. In toluene, hemin chloride encapsulated in such a hydrogel achieves about 60% nascent catalytic activity of horseradish peroxidase. Additionally, the activity of hemin in the hydrogel is 387.1 times greater than that of free hemin.

Full text of DOI:10.1002/anie.200700404

Angewandte
Chemie
DOI: 10.1002/anie.200700404
Enzyme Mimetics
A Supramolecular-Hydrogel-Encapsulated Hemin as an Artificial
Enzyme to Mimic Peroxidase**
Qigang Wang, Zhimou Yang, Xieqiu Zhang, Xudong Xiao, Chi K. Chang,* and Bing Xu*
A challenge in chemistry is an artificial enzyme[1] that mimics
the functions of the natural system but is simpler than
proteins. The intensive development of artificial enzymes that
use a variety of matrices,[2] the rapid progress in supramolec-
ular gels,[3] and the apparent “superactivity” exhibited by
supramolecular hydrogel-immobilized enzymes,[4] prompted
us to evaluate whether supramolecular hydrogels will
improve the activity of artificial enzymes for catalyzing
reactions in water or in organic media. To demonstrate the
concept, we used hemin as the prosthetic group to mimic
peroxidase, a ubiquitous enzyme that catalyzes the oxidation
of a broad range of organic and inorganic substrates by
hydrogen peroxide or organic peroxides. The structures of the
active site as well as the reaction mechanism of peroxidases
are well studied.[5] Much effort has been focused on incorpo-
rating metalloporphyrins[6] into protein-like scaffolds[7] in the
quest towards peroxidase mimetics, which do not show
satisfactory activity and selectivity mainly owing to the lack
of the peptidic microenvironment that exists in the native
peroxidase. The shape of the protein pocket and the amino
acid residues or functional groups that surround the active site
bring about the special inclusion behavior between the
enzyme and the substrate. As a result, b-cyclodextrins (b-
CDs), which are a frequently used model system, act as an
excellent enzyme model owing to their appropriate size and
their fairly rigid and hydrophobic cavities that provide
favorable binding of the prosthetic group as well as substrates.
Experimentally, b-CD-modified hemins have showed higher
activity relative to free hemin,[8] suggesting that supramolec-
ular hydrogels may act as an alternative matrix to encapsulate
hemin for the mimetic of peroxidase.
biomineralization,[15] and as biomaterials for wound heal-
ing.[13] The application of supramolecular hydrogels as the
skeletons of artificial enzymes has yet to be explored. Similar
to peptide chains that form active sites in enzymes, the self-
assembled nanofibers of amino acids in the supramolecular
hydrogels could act as the matrices of artificial enzymes. Thus,
the supramolecular-hydrogel systems serve two functions:
1) as the skeletons of the artificial enzyme to aid the function
of the active site (e.g., hemin) and, 2) as the immobilization
carriers to facilitate the recovery of the catalysts in practical
applications.
Herein, we mixed hemin chloride (3) into the hydrogel
formed by the self-assembly of two simple derivatives of
amino acids (1 and 2). The activity of this new type of artificial
enzyme is higher than the activity of free hemin, hemin in b-
CD, or hemin in polymeric hydrogels. This artificial enzyme
shows the highest activity in toluene for an oxidation reaction,
reaching about 60% of the nascent activity of horseradish
peroxidase (HRP). This result is particularly interesting
because it implies that the control of the structure of
hydrogelators could tailor the nanofibers as an adjustable
microenvironment around active centers and thereby affect
the performance of artificial enzymes. Moreover, the supra-
molecular hydrogel acts as an effective carrier to minimize the
dimerization and oxidative degradation of free hemin in the
peroxidization reaction. Overall, the supramolecular-hydro-
gel-based artificial enzyme offers a new opportunity to
achieve catalysis with high operational stability and reusabil-
ity, which ultimately would benefit industrial biotransforma-
tion.
Scheme 1 illustrates the simple procedure of using supra-
molecular hydrogels to encapsulate hemin. Equal molar
equivalents of 1 and 2 and two equivalents of Na2CO3 in
water formed a suspension, which turned into a clear solution
at about 608C. Then, 3 was added and dissolved immediately
in the solution. The subsequent cooling of the solution to
room temperature afforded a supramolecular hydrogel con-
taining hemin molecules (Gel II). Without the addition of 3,
the same procedure gave the control (Gel I). As a point of
reference, we made the artificial peroxidases by using b-CD
or a polyacrylamide hydrogel to encapsulate hemin according
to the literatures.[16]
Supramolecular hydrogels, formed by the self-assembly of
nanofibers of amphiphilic oligopeptides[9,10] or small mole-
cules,[11–13] have served as scaffolds for tissue engineering,[9]
a
medium for screening inhibitors of enzymes,[12,14] a matrix for
[*] Dr. Q. Wang, Dr. Z. Yang, Prof. C. K. Chang, Prof. B. Xu
Department of Chemistry
The Hong Kong University of Science and Technology
Clear Water Bay, Hong Kong (P.R. China)
Fax: (+852)2358-1594
E-mail: chang@ust.hk
chbingxu@ust.hk
As shown in the TEM and AFM images in Figure 1, Gel I
and Gel II have different morphologies. The networks of
Gel I have 50–500-nm pores formed by the nanofibers
(roughly 20 nm in diameter) of the self-assembled 1 and 2.
Besides the relatively large pores, the TEM image of the
nanofibers in Gel II, however, shows two distinct regions: the
dark part (fibers of approximately 20 nm in diameter) and the
gray part (surface layer of approximately 6 nm thickness).
X. Zhang, Prof. X. Xiao
Department of Physics
The Hong Kong University of Science and Technology
Clear Water Bay, Hong Kong (P.R. China)
[**] This work was partially supported by RGC of Hong Kong (HKU2/
05C, 604905, 600504) and EHIA (HKUST).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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4285

Communications
400 nm with a shoulder at
365 nm and a weak band
at 585 nm. The Soret
bands of the hemin
inside the hydrogel (at
400 nm, the bands are
similar to hemin in aque-
ous micelle solutions and
artificial
agree with the spectra of
hemin chloride in
proteins[19,20]
)
dimethyl sulfoxide and
methanol,[21] suggesting
monomeric hemin chlo-
ride. As reported,[19,20]
the weak absorbance
band at 585 nm should
be ascribed to charge-
transfer transition. The
weak shoulder at 365 nm
in the spectra of Gel II
indicates the presence of
a small amount of mono-
meric haematin. Overall,
the nanofibers in the
supramolecular hydrogel
effectively reduce the
dimerization of hemin
through supramolecular
interactions to localize
Scheme 1. A) Structures of the molecules and the procedure for making the supramolecular hydrogels containing
hemin chloride. B) Illustration of the artificial enzyme-catalyzed peroxidation of pyrogallol to purpurogallin
(S=substrate, P=product, and Solvent=aqueous buffer solution (0.01m, pH 7.4, phosphate) or toluene).
The similar size of the dark part in Gel II compared with the
diameter of the nanofiber in Gel I suggests that the dark part
mainly consists of the nanofibers of 1 and 2. The gray part
likely consists of less-ordered aggregates of 1 and 2. The AFM
spectrum of Gel II also shows that a loose layer surrounds the
dense nanofibers (Figure 1C, the bright region is about 30 nm
in height and agrees with the TEM result). The TEM image of
Gel II clearly reveals that the dark nanofibers are surrounded
by the gray part (Figure 1D), which agrees with the AFM
result. The energy-dispersive X-ray spectroscopic (EDX)
analysis of different areas in the high-resolution (HR)TEM
image of Gel II (areas E and F in Figure 1D) indicates that
the blank area (Figure 1E) has no Fe signal, whereas the area
around the nanofibers (Figure 1F) has about 2.23 wt% Fe,
confirming that the hemin molecules localize on the nano-
fibers. Moreover, the presence of hemin in the outer layer of
the nanofibers would allow the substrate to approach hemin
easily.
Figure 2 shows the UV/Vis spectra of hemin in a buffer
solution and in the hydrogel. The free hemin chloride in
pH 7.4 buffer solution displays a Soret peak at 385 nm along
with a shoulder at 365 nm, indicating the presence of a
mixture of the m-oxo bihemin (the dimer of hemin) along with
the monomeric hemin hydroxide (haematin).[17] Additionally,
a low-intensity band around 610 nm also agrees with the
Q band value of m-oxo bihemin.[17,18] Therefore, the predom-
inant structures of hemin in pH 7.4 phosphate buffer solution
Figure 1. The TEM images of A) Gel I and B) Gel II. C) The AFM image
of the nanofiber in Gel II. D) The high-resolution TEM image of Gel II.
E) The EDX analysis of the selected areas (E and F) shown in (D).
are the hemin dimers connected by m-oxo bridges and a small
amount of haematins. Gel II displays a broad Soret band at
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Table 1: Comparison of the activity of the artificial enzymes in water and
toluene.[a]
kcat in water [minÀ1
]
kcat in toluene [minÀ1
]
hemin(Phe)
19.9
49.7
2.4
370.7
1045.3
2.7
hemin(Phe+His)
hemin(Free)
hemin(Mix)
3.1
3.9
hemin(b-CD)
hemin(b-CD+His)
hemin(polymer)
hemin(polymer+His)
Met-Hb
4.8
5.6
7.6
9.2
6.1
7.8
2.4
2.8
1.0
1.8
37.4
1740.0
HRP
[a] The background of all carriers without hemin has been subtracted.
Hb=hemoglobin.
Figure 2. UV/Vis spectra of free hemin in pH 7.4 phosphate buffer
solution (hemin(Free); d), hemin in the hydrogel (hemin(Phe); c),
and hemin in the hydrogel with histidine (hemin(Phe+His); a). The
spectra at the weak absorption region (525–675 nm) are enlarged four
times.
high activity of hemin(Phe) in pH 7.4 buffer solution: 1) local-
ization of hemin on the nanofibers preserves the catalytic
species, monomeric hemin, and 2) the nanoporous channels in
the hydrogel facilitate the substrate across the hydrogel
network to access the hemin in the hydrogel.
monomeric hemin chloride within/around the nanofibers.
With the addition of histidine, the spectrum of Gel II shows a
red shift in the Soret band from 400 nm to 406 nm, which is
consistent with the hemin titrated by imidazole at 1:1 ratio in
methanol,[20,21] indicating the formation of a hemin–histidine
complex.
By using the oxidation of pyrogallol as a model reaction,
we obtained the catalytic rate of hemin in various environ-
ments. As shown in Figure 3, Gel I (i.e., the control) exhibits
A notable feature of the supramolecular-hydrogel-based
artificial enzyme is its high activity in an organic solvent. The
kinetic data in Table 1 indicate that the kcat of hemin(Phe) in
toluene (370.7 minÀ1) is 136 times that of hemin(Free) in toluene
(2.7 minÀ1). Relative to hemin(Phe) in aqueous solution, the kcat
of hemin(Phe) in toluene increases about 17.6 times. The
increase in the activity could be ascribed to the phase
equilibrium of reactants and products between hydrophobic
toluene solution and the aqueous environment of artificial
enzymes and can specifically be attributed to two causes:
1) the enrichment of hydrophilic substrate (pyrogallol logP
0.294) in the aqueous environment of the artificial enzyme
directly enhances the activity, and 2) the easy diffusion of the
hydrophobic product (purpurogallin logP 2.416) to the
similarly hydrophobic toluene (logP 2.678) also promotes
the catalytic conversion.
The supramolecular hydrogel greatly outperforms other
hemin carriers in toluene (Table 1). Therefore, the unique
molecular arrangement of the amino acids in the supramolec-
ular hydrogels/nanofibers likely contributes to the high
overall activity of the artificial enzyme in toluene. Specifically,
the nanoporous channel and amphiphilic structure of the
supramolecular hydrogel should assist the mass transfer
between the hydrogel phase and the toluene solution. To
further verify the effect of the supramolecular hydrogels, we
tested the activity of hemin in the solution of N-acylated
phenylalanine and lysine (see the Supporting Information).
The kcat values of this control (hemin(Mix), Table 1) are
essentially the same as those of free hemin, indicating that
the formation of the hydrogels is the major factor contributing
to the activity of hemin in the artificial enzymes.
Figure 4 also shows the catalytic courses of the oxidation
of pyrogallol catalyzed by hemin(Phe+His) in water and toluene,
verifying that histidine significantly increases the perfor-
mance of the artificial enzyme. As indicated by the kinetic
parameters listed in Table 1, the addition of histidine dra-
matically enhances the activity of hemin in the artificial
enzyme by about 2.5 times in water and 2.8 times in toluene,
Figure 3. The initial reaction courses of pyrogallol (10.0 mm) and H2O2
(40.0 mm) in 0.01m pH 7.4 phosphate buffer solution catalyzed by
~
&
*
5 mm hemin(Phe+His) ( ), hemin(Phe) ( ), hemin(Free) ( ), and Gel I control
with 0.5 mLLÀ1 concentration ( ). The reactions in the first minute
!
display zero-order kinetics, thus being defined as the initial activity.
no catalytic activity and Gel II (i.e., hemin(Phe)) exhibits a
higher activity than that of free hemin (i.e., hemin(Free)) in the
buffer solution (10 mm, pH 7.4, phosphate buffer solution),
confirming the effectiveness of the artificial enzyme. The
Lineweaver–Burk plots constructed by the initial rates of
reactions give the activity of the artificial enzyme. As shown
in Table 1, the artificial enzyme has a higher turnover number
(kcat) than that of free hemin (hemin(Free)), b-CD-bound hemin
(hemin(b-CD)),
or
polyacrylamide-hydrogel-encapsulated
hemin (hemin(polymer)). Two factors likely contribute to the
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Communications
In summary, we have demonstrated that nanofibers in
supramolecular hydrogels function as the skeleton of the
artificial enzyme and serve as the immobilizing carrier to
enhance the catalytic activity of hemin chloride for perox-
idation in water or in organic media. The supramolecular
hydrogels protect the hemin monomer by preventing dimmer-
ization and degradation and facilitate the catalytic reaction by
providing nanoporous diffusion channels, which possess
unique flexibility to allow the transport of substrates.
Although it is still difficult to obtain the complete molecular
details in such artificial enzymes owing to current limitations
on the characterization of supramolecular hydrogels, the
noncovalent interactions among the hydrogelators and
between the hydrogelators and water molecules would
permit sufficient flexibility to assist the transport of the
substrates and the products, which may be essential for this
type of artificial enzymes.
Figure 4. The conversion courses of pyrogallol (10.0 mm) and H2O2
*
(40.0 mm) catalyzed by hemin(Phe+His) in toluene ( ), hemin(Phe) in
~
&
*
toluene ( ), hemin(Free) in toluene ( ), hemin(Phe+His) in water ( ),
~
hemin(Phe) in water ( ), hemin(Free) in water (&).
indicating that the coordination of histidine to the FeIII center
in the hemin increases the activity in the artificial enzymes. As
shown in Figure 3, the Soret band of hemin(Phe+His) is similar to
that of methemoglobin (408 nm) and peroxidase (405 nm),
both of which have a proximal histidine ligand coordinating
the heme iron.[22] It is well known that the proximal histidine
in preoxidase enhances the activity.[23] Therefore, the perox-
idase-like hemin–histidine complex in the artificial enzymes
should be the major cause of the high activity. This is
evidenced in that the histidine-derived enhancements of
activity are the same magnitude in different solvents and
different carriers (Table 1).
The supramolecular-hydrogel-immobilized hemin also
exhibits high stability and excellent reusability, which is
particularly useful for industrial applications. Figure 4 shows
the 360-min courses of the oxidation of pyrogallol catalyzed
by various hemins in water and toluene. The hemins in the
hydrogels all maintained high catalytic activity during the first
120-min course of the reaction in toluene or in water, whereas
the free hemin lost most of its catalytic activity after about 5–
10 min of reaction. The highest conversion of hemin(Phe+His) is
about 96% after reaction for 360 min. One plausible explan-
ation for the high stability of the artificial enzyme is that the
hemin molecules are immobilized and isolated from each
other within/around the nanofiber, and the hydrogels protect
hemin from oxidative inactivation. To test its reusability, we
compared the fresh and recovered artificial enzyme (hem-
in(Phe)) during the 15-min peroxidization of pyrogallol in
toluene. The result shows that the amount of product in the
third run reaches 82% of that in the first run. Therefore, the
artificial enzyme in toluene exhibits excellent reusability.
To verify the generality of this approach, we evaluated the
activity of hemin in the hydrogels formed by other 9-
fluorenylmethoxycarbonyl (Fmoc) amino acids. In either
water or toluene, hemin(Ala), hemin(Val), and hemin(Leu) have
activities similar to that of hemin(Phe). Various substrates also
were employed to evaluate the catalytic ability of artificial
enzyme in toluene. The result shows that the activity of
hemin(Phe+His) in toluene can reach up to 20% and 4.2% of
that of HRP in water with o-phenyldiamine and o-amino-
phenol as the substrate, respectively.
Received: January 30, 2007
Revised: March 9, 2007
Published online: April 19, 2007
Keywords: biotransformations · enzyme mimetics · hemin ·
.
heterogeneous catalysis · hydrogels
[1] R. Breslow, Acc. Chem. Res. 1995, 28, 146; R. Breslow, Artificial
Enzymes, Wiley-VCH, Weinheim, 2005.
[2] A. J. Kirby, Angew. Chem. 1996, 108, 770; Angew. Chem. Int. Ed.
Engl. 1996, 35, 706; R. Breslow, S. D. Dong, Chem. Rev. 1998, 98,
1997; W. B. Motherwell, M. J. Bingham, Y. Six, Tetrahedron
2001, 57, 4663; G. Wulff, Chem. Rev. 2002, 102, 1; G. Wulff, B. O.
Chong, U. Kolb, Angew. Chem. 2006, 118, 3021; Angew. Chem.
Int. Ed. 2006, 45, 2955; S. C. Zimmerman, M. S. Wendland, N. A.
Rakow, I. Zharov, K. S. Suslick, Nature 2002, 418, 399; L. G.
Marinescu, M. Bols, Angew. Chem. 2006, 118, 4706; Angew.
Chem. Int. Ed. 2006, 45, 4590; D. M. Vriezema, M. C. Aragones,
J. A. A. W. Elemans, J. J. L. M. Cornelissen, A. E. Rowan,
R. J. M. Nolte, Chem. Rev. 2005, 105, 1445.
[3] P. Terech, R. G. Weiss, Chem. Rev. 1997, 97, 3133; L. A. Estroff,
A. D. Hamilton, Chem. Rev. 2004, 104, 1201; M. George, R. G.
Weiss, Acc. Chem. Res. 2006, 39, 489; R. G. Weiss, P. Terech,
Molecular Gels: Materials with Self-Assembled Fibrillar Net-
works, Springer, Dordrecht, 2006.
[4] Q. Wang, Z. Yang, L. Wang, M. Ma, B. Xu, Chem. Commun.
2007, 1032.
[5] H. Pelletier, J. Kraut, Science 1992, 258, 1748.
[6] C. K. Chang, T. G. Tralor, Proc. Natl. Acad. Sci. USA 1973, 70,
2647; R. K. Z. Pandey, G. , The Porphyrin Handbook, Academic
Press, Boston, 2000; W. H. Cui, B. B. Wayland, J. Am. Chem. Soc.
2004, 126, 8266; J. Mosinger, M. Janoskova, K. Lang, P. Kubat, J.
Photochem. Photobiol. A 2006, 181, 283.
[7] Y. Lu, S. M. Berry, T. D. Pfister, Chem. Rev. 2001, 101, 3047; L.
Fruk, C. M. Niemeyer, Angew. Chem. 2005, 117, 2659; Angew.
Chem. Int. Ed. 2005, 44, 2603; J. Yang, R. Breslow, Angew. Chem.
2000, 112, 2804; Angew. Chem. Int. Ed. 2000, 39, 2692.
[8] Y. P. Huang, W. H. Ma, J. Li, M. M. Cheng, J. C. Zhao, L. J.
Wang, J. C. Yu, J. Phys. Chem. B 2003, 107, 9409.
[9] G. A. Silva, C. Czeisler, K. L. Niece, E. Beniash, D. Harrington,
J. A. Kessler, S. I. Stupp, Science 2004, 303, 1352; H. A. Behanna,
J. J. J. M. Donners, A. C. Gordon, S. I. Stupp, J. Am. Chem. Soc.
2005, 127, 1193; S. G. Zhang, Nat. Biotechnol. 2003, 21, 1171; S.
Vauthey, S. Santoso, H. Y. Gong, N. Watson, S. G. Zhang, Proc.
Natl. Acad. Sci. USA 2002, 99, 5355.
4288 www.angewandte.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 4285 –4289

Angewandte
Chemie
[10] A. Aggeli, M. Bell, N. Boden, J. N. Keen, P. F. Knowles, T. C. B.
McLeish, M. Pitkeathly, S. E. Radford, Nature 1997, 386, 259.
[11] H. Kobayashi, M. Amaike, J. H. Jung, A. Friggeri, S. Shinkai,
D. N. Reinhoudt, Chem. Commun. 2001, 1038; M. Suzuki, M.
Yumoto, M. Kimura, H. Shirai, K. Hanabusa, Chem. Commun.
2002, 884; B. G. Xing, C. W. Yu, K. H. Chow, P. L. Ho, D. G. Fu,
B. Xu, J. Am. Chem. Soc. 2002, 124, 14846; S. M. Park, Y. S. Lee,
B. H. Kim, Chem. Commun. 2003, 2912; M. Ikeda, M. Takeuchi,
S. Shinkai, Chem. Commun. 2003, 1354; Z. M. Yang, H. W. Gu,
D. G. Fu, P. Gao, K. J. K. Lam, B. Xu, Adv. Mater. 2004, 16, 1440;
Z. M. Yang, H. W. Gu, Y. Zhang, L. Wang, B. Xu, Chem.
Commun. 2004, 208; Y. Zhang, Z. M. Yang, F. Yuan, H. W. Gu, P.
Gao, B. Xu, J. Am. Chem. Soc. 2004, 126, 15028; S. Tamaru, S.
Kiyonaka, I. Hamachi, Chem. Eur. J. 2005, 11, 7294; N.
Sreenivasachary, J. M. Lehn, Proc. Natl. Acad. Sci. USA 2005,
102, 5938; V. Jayawarna, M. Ali, T. A. Jowitt, A. E. Miller, A.
Saiani, J. E. Gough, R. V. Ulijn, Adv. Mater. 2006, 18, 611; Z. M.
Yang, G. L. Liang, L. Wang, B. Xu, J. Am. Chem. Soc. 2006, 128,
3038.
[13] Z. M. Yang, K. M. Xu, L. Wang, H. W. Gu, H. Wei, M. J. Zhang,
B. Xu, Chem. Commun. 2005, 4414.
[14] Z. M. Yang, B. Xu, Chem. Commun. 2004, 2424.
[15] Z. A. C. Schnepp, R. Gonzalez-McQuire, S. Mann, Adv. Mater.
2006, 18, 1869.
[16] Y. Z. Li, N. He, X. Q. Wang, W. B. Chang, Y. X. Ci, Analyst 1998,
123, 359; Y. P. Huang, R. X. Cai, L. Y. Mao, H. P. Huang, Anal.
Lett. 2000, 33, 2883.
[17] J. Silver, B. Lukas, Inorg. Chim. Acta 1983, 78, 219.
[18] J. R. Miller, J. A. Taies, J. Silver, Inorg. Chim. Acta 1987, 138,
205; J. W. Smalley, A. J. Birss, R. Withnall, J. Silver, Biochem. J.
2002, 362, 239.
[19] P. K. Shantha, G. S. S. Saini, H. H. Thanga, A. L. Verma, J.
Raman Spectrosc. 2001, 32, 159.
[20] E. Monzani, B. Bonafe, A. Fallarini, C. Redaelli, L. Casella, L.
Minchiotti, M. Galliano, Biochim. Biophys. Acta Protein Struct.
Mol. Enzymol. 2001, 1547, 302.
[21] E. S. Ryabova, A. Dikiy, A. E. Hesslein, M. J. Bjerrum, S. Ciurli,
E. Nordlander, J. Biol. Inorg. Chem. 2004, 9, 385.
[22] G. Smulevich, M. Paoli, G. DeSanctis, A. R. Mantini, F. Ascoli,
M. Coletta, Biochemistry 1997, 36, 640.
[12] S. Kiyonaka, K. Sada, I. Yoshimura, S. Shinkai, N. Kato, I.
Hamachi, Nat. Mater. 2004, 3, 58; I. Yoshimura, Y. Miyahara, N.
Kasagi, H. Yamane, A. Ojida, I. Hamachi, J. Am. Chem. Soc.
2004, 126, 12204.
[23] S. Nagano, M. Tanaka, K. Ishimori, Y. Watanabe, I. Morishima,
Biochemistry 1996, 35, 14251.
Angew. Chem. Int. Ed. 2007, 46, 4285 –4289
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 4289