H2O2-dependent substrate oxidation by an engineered diiron site in a bacterial hemerythrin

Article abstract of DOI:10.1039/c3cc48108e

The O₂-binding carboxylate-bridged diiron site in DcrH-Hr was engineered in an effort to perform the H₂O₂-dependent oxidation of external substrates. A His residue was introduced near the diiron site in place of a conserved residue, Ile119. The I119H variant promotes the oxidation of guaiacol and 1,4-cyclohexadiene upon addition of H ₂O₂.

Full text of DOI:10.1039/c3cc48108e

Volume 50 Number 26 4 April 2014 Pages 3379–3520
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Akira Onoda, Takashi Hayashi et al.
H O -dependent substrate oxidation by an engineered diiron site in a
2
2
bacterial hemerythrin

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H O -dependent substrate oxidation by an
2
2
engineered diiron site in a bacterial hemerythrin†
Cite this: Chem. Commun., 2014,
0, 3421
5
a
a
b
c
d
Yasunori Okamoto, Akira Onoda,* Hiroshi Sugimoto, Yu Takano, Shun Hirota,
e
b
a
Received 22nd October 2013,
Accepted 9th December 2013
Donald M. Kurtz Jr., Yoshitsugu Shiro and Takashi Hayashi*
DOI: 10.1039/c3cc48108e
www.rsc.org/chemcomm
9,10
The O
2
-binding carboxylate-bridged diiron site in DcrH-Hr was tunnel within the four-helix bundle compared to classical Hrs.
-dependent oxidation of As for classical Hrs, the diiron site of DcrH-Hr reversibly binds O2
engineered in an effort to perform the H
2 2
O
external substrates. A His residue was introduced near the diiron site in but shows more rapid autoxidation. Recently, we succeeded in
place of a conserved residue, Ile119. The I119H variant promotes the altering the first coordination sphere of the diiron site in DcrH-Hr
oxidation of guaiacol and 1,4-cyclohexadiene upon addition of H
2
O
2
.
by replacing residue Ile119 located close to the exogenous ligand
binding site with a Glu residue. The I119E variant showed
11
2
O is utilized in several non-heme diiron-carboxylate proteins per- altered properties of exogenous ligand binding and redox beha-
forming essential biological processes ranging from transport of O
2
vior in the wild type DcrH-Hr (WT). Herein, we report another
1,2
(
hemerythrin, Hr), hydroxylation of C–H bonds (methane mono- engineered variant DcrH-Hr, I119H, of the same residue, in which
3
oxygenase hydroxylase), generation of a tyrosyl radical for DNA alteration of the coordination sphere generates the capability of
synthesis (ribonucleotide reductase), and reduction of unsaturated H O -dependent oxidation of exogenous substrates.
4
2
2
5
fatty acids (D9 desaturase). Despite these functional differences, all of
I119H was expressed from Escherichia coli and purified as a holo-
these proteins have a carboxylate-bridged diiron active site within a form containing approximately 1.6 iron atoms per protein, as
2
a,6
12
four-helix bundle protein folding motif.
and the environment surrounding the diiron site in these proteins exhibited two absorption maxima at 325 and 375 nm, which are well-
have apparently evolved to optimize a variety of reactions with O established to arise from oxo to ferric LMCT transitions in the oxo–
Engineering of the first or the second coordination sphere of the dicarboxylate-bridged met-Hrs.
The coordination sphere determined by ferrozine analysis. The diferric form (met-I119H)
2
.
1
b,c,8
Resonance Raman (rR) spectra of
7
À1
diiron site constitutes one approach to understanding the effects of I119H gave a characteristic peak at 499 cm for the symmetric
the protein environment, which distinguish reversible O binding stretching Fe–Ooxo–Fe vibrational mode. The met-I119H protein was
from O activation. As a target protein for engineering the coordina- crystallized, and the structure of the oxo-bridged diiron site was
2
tion sphere, we have selected DcrH-Hr, which contains the histidine- determined by X-ray crystallographic analysis at a resolution of 1.9 Å.
rich coordination sphere and exhibits spectroscopic properties The overall structure of the met-I119H is similar to that of the met
8
2
13
8
10
characteristic of all Hrs, but contains a larger substrate-accessible form of WT (met-WT) (RSMD value, 0.324). Surprisingly, H119
coordinates to Fe1 in the crystal structure in place of the conserved
H118 ligand residue in WT (Fig. 1). The coordination geometry of
other His residues and carboxylate ligands is almost identical to that
a
Department of Applied Chemistry, Graduate School of Engineering,
Osaka University, Suita, Osaka 565-0871, Japan.
of met-WT (Fig. S1, ESI†). A computational study of the I119H diiron
E-mail: onoda@chem.eng.osaka-u.ac.jp, thayashi@chem.eng.osaka-u.ac.jp
Biometal Science Laboratory, RIKEN SPring-8 Center, Sayo, Hyogo 679-5148, Japan
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan
Graduate School of Materials Science, Nara Institute of Science and Technology,
Ikoma 630-0192, Japan
b
c
site yielded alternative structures in which either H118 or H119
14
coordinates to the Fe1 (Fig. 2). It is, thus, expected that either H118
d
or H119 coordination would be stable in I119H in solution.
The exogenous ligand binding properties of the met-I119H diiron
e
Department of Chemistry, University of Texas at San Antonio, San Antonio,
Texas 78249, USA
site were investigated using azide, which is known to coordinate to
9
†
Electronic supplementary information (ESI) available: Information on materials, Fe2 in place of chloride in met-WT. The azide adduct of met-WT
8
instrumentation, experimental details and additional data on preparation of
proteins, crystal structure analysis, resonance Raman and FTIR spectroscopy,
exhibits a UV-vis absorption at 443 nm, while that of I119H showed
a shifted absorption maximum at 425 nm, indicating a change in
conformation or orientation of the bound azide ligand (Fig. S2,
reaction of reduced I119H with O
of guaiacol and 1,4-cyclohexadiene. The atomic coordinates and structure factors
PDB code 3WHN) have been deposited into the Protein Data Bank, http://www.
2 2 2
, consumption of H O , and oxidation reactions
ESI†). The rR spectrum of I119H showed the Fe–N
3
stretching
(
À1
14 À 15
À
rcsb.org/. See DOI: 10.1039/c3cc48108e
vibration at 361 cm , which was confirmed by
N
3
/ N
3
isotope
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labeling experiments (Fig. 3a). The Fe–N
I119H is downshifted compared to that of the WT at 372 cm
3
stretching vibration of
À1 8
.
This finding suggests weaker Fe–N bonding in I119H than that
3
observed in WT.
Additional structural details of the bound azide ligand were
obtained from FTIR spectroscopy in the region of the asymmetric
stretch of the coordinated azide nas(NNN). The azide adduct of
À1
met-WT showed a nas(NNN) band at 2050 cm (Fig. 3b). The
1
5
isotope labeling experiment using NN
2
indicates that the term-
1
inal nitrogen of the azide ligand binds in a Z fashion, as expected
from the X-ray crystal structure of the met-WT azide adduct.
1,9,16
The azide adduct of met-I119H exhibits two n (NNN) bands at
as
Fig. 1 Crystal structures of (a) met-WT (PDB code: 3AGT) and (b) met-
À1
I119H (iron in brown, oxygen in red, nitrogen in blue, carbon in white, sulfur 2050 and 2060 cm at 5 K (Fig. 3b). The newly observed band,
À1
in yellow, and chloride in green). The 2Fobs À Fcalc electron density in light 10 cm -higher than that of met-WT, suggests a resonance form
blue grid (contoured at 1.5s) around the diiron site. The structure drawings
3+
À
+
À
shifted farther from the symmetric [Fe –N QN QN ] towards
15
were generated in PyMOL.
3+
2À
+
the asymmetric [Fe –N –N RN]. The increased asymmetry of
the two N–N bonds could be stabilized by a hydrogen bond
interaction with the 1N atom of the coordinated azide, possibly
17
with the displaced His119 ligand. No such hydrogen bonding
interaction is observed in the structure of the met-WT azide
9
adduct. The two n (NNN) bands observed at 5 K coalesced at
as
25 1C (Fig. 3b). Based on this observation together with the crystal
structure and calculated optimized structures, we propose two
conformations for I119H: H118off/H119on and H118on/H119off.
In the latter case, the H119 residue is capable of forming a
hydrogen bonding interaction with an exogenous ligand.
Fig. 2 Depiction of computationally optimized structures of I119H diiron
site models (a) where H118 coordinates to Fe1 and (b) where H119
coordinates to Fe1 (iron in brown, carbon in white, oxygen in red, nitrogen
in blue, chloride in green, and sulfur in yellow).
We evaluated the reactivity of I119H toward H O as a potential
2
2
exogenous ligand, which, like azide, could form a hydrogen bond
with the pendant H119 residue. The met-WT and met-I119H in
5
0 mM HEPES (pH 7.0) were individually treated with 50 eq. of
from an aqueous solution, and the amount of H con-
sumed was determined by the iodometry method. Disproportio-
nation of H into O and H O has been reported to occur upon
reaction of H with a synthetic Hr model complex. We found
that the met-WT reacted with H O within 10 min with a con-
H O
2 2
2 2
O
2
O
O
2 2
2
2
2
18
2
2
comitant evolution of O , which was confirmed by gas chromato-
2
graphy (Fig. S4, ESI†). In addition, the deoxy-WT was oxidized to
the met form by H
the disproportionation reaction of H
mechanism analogous to that proposed for the Hr model complex
Scheme S1, ESI†). The consumption rate of H by I119H was
significantly slower than that of WT (Fig. 4a).
O
2 2
(Fig. S5, ESI†). These results indicate that
2 2
O
could proceed via a
(
2 2
O
Motivated by the differing reactivity of WT and I119H toward
H O , we tested the promotion of oxidation of external substrates
2
2
by these proteins. Fig. 4b demonstrates that I119H promotes
oxidation of guaiacol by H , as shown by the clear increase in
2 2
O
absorption at 470 nm assigned to the oxidized guaiacol product,
whereas, under the same conditions, WT does not. Similarly,
I119H promotes the oxidation of 1,4-cyclohexadiene by H O ,
2 2
producing benzene as a product determined by GC-MS analysis,
whereas WT showed no such reactivity (Fig. S6, ESI†). These
findings support the generation of an oxidizing intermediate from
the altered diiron site in I119H which cannot be generated in WT.
According to the reaction analysis, the oxidized product yields for
both the guaiacol and cyclohexadiene reactions are approximately
0.26 mol product per mol of a diiron site under the conditions as
Fig. 3 (a) Resonance Raman spectra of the azide adduct of (i) met-WT
14
14
15
with Na
N
3
, (ii) met-I119H with Na
N
3
14
, (iii) met-I119H with Na
N
3
, and
15
(iv) the difference spectrum
N
3
minus
3
N adducts of met-I119H. Peaks
are labeled according to their vibrational assignments, as described in the
text. (b) FTIR spectra of the azide adduct of (i) met-WT at 5 K, (ii) met-I119H
at 5 K, and (iii) met-I119H at 25 1C.
3
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The computations were performed at the Research Center for
Computational Science, Okazaki, Japan.
Notes and references
1
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(a) D. M. Kurtz Jr., JBIC, J. Biol. Inorg. Chem., 1997, 2, 159; (b) D. M.
Kurtz Jr., Dioxygen-binding Proteins, in Comprehensive Coordination
Chemistry II, ed. J. A. McCleverty and T. J. Meyer, Elsevier, Oxford,
U.K., 2004, vol. 8, p. 229.
3
4
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(
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Fig. 4 (a) Consumption of H
2
O
2
in the presence of met-I119H (solid line)
] = 5 mM. All experi-
2 2
and met-WT (dashed line); [protein] = 100 mM, [H O
ments were carried out in 50 mM HEPES (pH 7.0) at 25 1C. (b) Oxidation
reaction of guaiacol by met-I119H (solid line) and met-WT (dashed line) at
5
6
2
5 1C. The absorption of the guaiacol product at 470 nm was monitored;
[protein] = 100 mM, [guaiacol] = 5 mM, [H ] = 5 mM.
2 2
O
(
b) M. H. Sazinsky and S. J. Lippard, Acc. Chem. Res., 2006, 39, 558.
7
(a) J. Xiong, R. S. Phillips, D. M. Kurtz Jr., S. Jin, J. Ai and J. Sanders-
Loehr, Biochemistry, 2000, 39, 8526; (b) C. S. C. Farmer, D. M. Kurtz
Jr., R. S. Phillips, J. Ai and J. Sanders-Loehr, J. Biol. Chem., 2000,
described in Fig. 4b. During the substrate oxidation reaction,
is also expected to be consumed by its concurrent dispro-
portionation. We confirmed that the oxidation of guaiacol could
be continued upon the addition of H to the initial reaction
2
75, 17043; (c) C. S. Farmer, D. M. Kurtz Jr., Z. J. Liu, B. C. Wang,
J. Rose, J. Ai and J. Sanders-Loehr, JBIC, J. Biol. Inorg. Chem., 2001,
, 418; (d) M. Faiella, C. Andreozzi, R. T. M. de Rosales, V. Pavone,
2 2
H O
6
2 2
O
O. Maglio, F. Nastri, W. F. DeGrado and A. Lombardi, Nat. Chem.
Biol., 2009, 5, 882; (e) A. J. Reig, M. M. Pires, R. A. Snyder, Y. Wu,
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solution (Fig. S7, ESI†). The key for the generation of the oxidizing
intermediate could be a hydrogen bonding interaction between a
hydroperoxide coordinating in a fashion similar to that in oxyHr
and the displaced H119 ligand. Previous studies have invoked
hydrogen bonding to a hydroperoxide bound to Fe(III) as
8 J. Xiong, D. M. Kurtz Jr., J. Ai and J. Sanders-Loehr, Biochemistry,
000, 39, 5117.
2
9
C. E. Isaza, R. Silaghi-Dumitrescu, R. B. Iyer, D. M. Kurtz Jr. and
M. K. Chan, Biochemistry, 2006, 45, 9023.
1
9
promoting formation of a high-valent iron-oxo species. Sup- 10 A. Onoda, Y. Okamoto, H. Sugimoto, Y. Shiro and T. Hayashi, Inorg.
Chem., 2011, 50, 4892.
1 Y. Okamoto, A. Onoda, H. Sugimoto, Y. Takano, S. Hirota, D. M. Kurtz
Jr., Y. Shiro and T. Hayashi, Inorg. Chem., 2013, 52, 13014.
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azide ligation experiment monitored by FTIR spectroscopy.
1
An additional contribution could involve dynamic coordination 12 L. L. Stookey, Anal. Chem., 1970, 42, 779.
1
3 The diferrous form of the I119H variant is oxidized by O
diferric form (Fig. S3, ESI†). Although this fact suggests the binding
of O , the oxy form was not observed due to more rapid autoxidation
and slower O binding relative to those of WT DcrH-Hr.
4 The total energy of the first coordination sphere in the H118on/
H119off and H118off/H119on models differs only 0.67 kcal mol
In the H118on/H119off model, the hydrogen bonding interaction
between H119 and chloride was formed. The interaction energy
between H118 and H119 in the H118on/H119off model and that
2
to the
of H118 and H119, which transiently forms a five-coordinated
Fe1 site via a conformational transition during the reaction.
This transient coordination site may allow formation of a
m-1,2-peroxo-diferric species, which is the most commonly
proposed precursor to high-valent iron–oxo species in non-heme
diiron-carboxylate enzymes.
In conclusion, we have engineered the diiron site of DcrH-Hr
to promote the H -dependent oxidation of exogenous sub-
2
2
1
À1
.
20
between H118 and M120 in the H118off/H119on model is 5.88 and
2
O
2
À1
1.84 kcal mol , respectively.
strates. A His residue introduced proximal to the diiron site will 15 W. L. DeLano, The PyMOL Molecular Graphics System, DeLano
Scientific, San Carlos, CA, 2008.
participate in a hydrogen bonding interaction with the exogenous
H O ligand, thereby, activating the peroxide. To the best of our
À1
1
6 The peak at 2043 cm was assigned as nas(NNN) of a free azide. The
15
2
2
azide ( NN ) adduct of met-WT gave two n (NNN) peaks at 2033
2
as
À1
knowledge, this is the first example that successfully converts an
-binding non-heme diiron-carboxylate protein to an oxidatively
and 2045 cm .
1
1
1
2
7 S. Lu, M. H. Sazinsky, J. W. Whittaker, S. J. Lippard and P. Mo ¨e nne-
Loccoz, J. Am. Chem. Soc., 2005, 127, 4148.
8 B. Mauerer, J. Crane, J. Schuler, K. Wieghardt and B. Nuber, Angew.
Chem., Int. Ed., 1993, 32, 289.
O
2
active one. Efforts to characterize the oxidatively active species of
the I119H variant and the further engineering of the diiron site
are in progress.
This work was financially supported by Grants-in-Aid for
Scientific Research ((C), JSPS KAKENHI Grant Number 205590020,
and Innovative Areas ‘‘Molecular Activation’’, area 2204, MEXT
KAKENHI Grant Number 221050130). Y.O. appreciates support
from the Research Fellowship of JSPS and Global COE Program
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‘‘Global Education and Research Center for Bio-environmental
Chemistry’’ of Osaka University. D. M. K., Jr. acknowledges support
from the National Institutes of Health (grant R01 GM040388).
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 3421--3423 | 3423