A Structural and Functional Model for the Tris-Histidine Motif in Cysteine Dioxygenase

Article abstract of DOI:10.1002/chem.201901005

The iron(II) complexes [Fe(L)(MeCN)₃](SO₃CF₃)₂ (L are two derivatives of tris(2-pyridyl)-based ligands) have been synthesized as models for cysteine dioxygenase (CDO). The molecular structure of one of the complexes exhibits octahedral coordination geometry and the Fe?N_py bond lengths [1.953(4)–1.972(4) ?] are similar to those in the Cys-bound Fe^II-CDO; Fe?N_His: 1.893–2.199 ?. The iron(II) centers of the model complexes exhibit relatively high Fe^III/II redox potentials (E_1/2=0.988–1.380 V vs. ferrocene/ferrocenium electrode, Fc/Fc⁺), within the range for O₂ activation and typical for the corresponding nonheme iron enzymes. The reaction of in situ generated [Fe(L)(MeCN)(SPh)]⁺ with excess O₂ in acetonitrile (MeCN) yields selectively the doubly oxygenated phenylsulfinic acid product. Isotopic labeling studies using ¹⁸O₂ confirm the incorporation of both oxygen atoms of O₂ into the product. Kinetic and preliminary DFT studies reveal the involvement of an Fe^III peroxido intermediate with a rhombic S= (Formula presented.) Fe^III center (687–696 nm; g≈2.46–2.48, 2.13–2.15, 1.92–1.94), similar to the spectroscopic signature of the low-spin Cys-bound Fe^IIICDO (650 nm, g≈2.47, 2.29, 1.90). The proposed Fe^III peroxido intermediates have been trapped, and the O?O stretching frequencies are in the expected range (approximately 920 and 820 cm^?1 for the alkyl- and hydroperoxido species, respectively). The model complexes have a structure similar to that of the enzyme and structural aspects as well as the reactivity are discussed.

Full text of DOI:10.1002/chem.201901005

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Title: A Structural and Functional Model for the Tris-Histidine Motif in
Cysteine Dioxygenase
Authors: Karunanithi Anandbabu, Ramamoorthy Ramasubramanian,
Hubert Wadepohl, Peter Comba, Neethinathan Johnee Britto,
Madhavan Jaccob, and Ramasamy Mayilmurugan
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A Structural and Functional Model for the Tris-Histidine Motif in
Cysteine Dioxygenase
Karunanithi Anandababu,[a]+ Ramamoorthy Ramasubramanian,[a]+ Hubert Wadepohl,[b] Peter Comba, [b]*
Neethinathan Johnee Britto,[c] Madhavan Jaccob,[c] and Ramasamy Mayilmurugan[a]*
Dedicated to Professor Gabor Laurenczy on occasion of his 65th birthday
Abstract: The iron(II) complexes [Fe(L)(MeCN)3](SO3CF3)2 (L are two
derivatives of tris(2-pyridyl)-based ligands) have been synthesized as
models for cysteine dioxygenase (CDO). The molecular structure of
one of the complexes exhibits octahedral coordination geometry and
the Fe-Npy bond lengths [1.953(4) - 1.972(4) Å] are similar to those in
the Cys-bound FeII-CDO; Fe-NHis: 1.893 - 2.199 Å. The iron(II)
centers of the model complexes exhibit relatively high FeIII/II redox
potentials (E1/2 = 0.988 - 1.380 V vs. Fc/Fc+), within the range for O2
activation and typical for the corresponding nonheme iron enzymes.
The reaction of in situ generated [Fe(L)(MeCN)(SPh)]+ with excess O2
in acetonitrile (MeCN) yields selectively the doubly oxygenated
phenylsulfinic acid product. Isotopic labeling studies using 18O2
confirm the incorporation of both oxygen atoms of O2 into the
product. Kinetic and preliminary DFT studies reveal the involvement
of an FeIII peroxido intermediate with a rhombic S = ½ FeIII center (687-
696 nm; g  2.46 - 2.48, 2.13 - 2.15, 1.92 - 1.94), similar to the
spectroscopic signature of the low-spin Cys-bound FeIIICDO (650 nm,
g  2.47, 2.29, 1.90). The proposed FeIII peroxido intermediates have
been trapped, and the O-O stretching frequencies are in the expected
range (approx. 920 and 820 cm-1 for the alkyl- and hydroperoxido
species, respectively). The model complexes have a structure similar
to that of the enzyme and structural aspects as well as the reactivity
are discussed.
The tris-histidine motif (3-His) in the active site of CDO is
different from the typical 2-His-1-carboxylate facial triad
adopted in the active site of many non-heme FeII
dioxygenases and, in addition to CDO, it is found in the -
diketone dioxygenase (Dke1), gentisate dioxygenase, salicylate
5
dioxygenase, and other oxygenases.[ ]
Scheme 1. Chemical structures of the CDO active site and the CDO-
Cys adduct.
The biomimetic chemistry of the 2-His-1-carboxylate
motif in FeII-dependent oxygenases has been studied
5,6
]
extensively[
but there is less knowledge about the
and only a few model systems
properties of tris-His residues
have been reported so far:[
,
7-9
Introduction
]
Goldberg and co-workers
Oxygen activating enzymes are involved in several
important biochemical reactions, ranging from drug
metabolism and biosynthesis of hormones to detoxification
processes.[1] Cysteine dioxygenase (CDO) converts
selectively L-cysteine (Cys) to L-cysteine sulfinic acid, using
reported the FeII-thiolate complexes [(iPrBIP)FeII(SPh)X] (X =
Cl, SO3CF3) of the bis(imino)pyridine BIP with three nitrogen
donors, and their reaction with O2, leading to S-oxygenation
7
of the coordinated thiolate ligand.[ ] The enzyme-substrate
model complexes [FeII(N3S)(SO3CF3)] (N3 = BIP) and
[FeII(N3PyS)(CH3CN)]BF4 (N3Py=N-[di(2-pyridinyl)-methyl]-
N-(2-pyridinylmethyl)amine), where the thiolate substrate is
linked to the ligand, have also been reported. Oxygenation
of these complexes yield selectively the triply S-oxygenated
2
O2 in a key step of the biosynthesis of pyruvate and taurine.[ ]
In mammalian systems, the sulfur metabolism is of
significant medicinal interest because higher levels of
cysteine have been associated with a variety of neurological
disorders, including motor neuron, Alzheimer’s and
8
sulfonato product.[ ] Limberg and co-workers reported
3
Parkinson’s disease.[ ] CDO has been isolated from mouse,
cysteine and cystamine adducts of the well-known tris-
pyrazolylborate ligand (Tp), serving as biomimetic models of
the 3-His-FeII system, and these produce doubly oxygenated
rat and human sources and four crystal structures have been
4
published.[ ] These reveal that a high-spin FeII center is
coordinated to three histidine residues (His-86, His-88, His-
140), one to three solvent (H2O) molecules and a cysteine-
tyrosine crosslink (C93-Y157) in the second coordination
sphere. The substrate Cys is coordinated to the FeII center
9
products with O2.[ ] Brunold and Fiedler studied FeII
complexes of the tris(4,5-diphenyl-1-methyl-imidazol-2-
yl)phosphine ligand Ph2TIP as a model for the 3-His triad of
10
]
the thiol dioxygenase (TDO).[
4
in a bidentate mode via RS- and RNH2 donors (Scheme 1).[ ]
We use a tris(pyridyl) based ligand system (Scheme 2),
which enforces an environment to the metal center
(geometry and electronics) that is very similar to the tris-His
active site of CDO (vide infra for the observed structural
properties; pKa (protonated heterocycles): 7.0 vs. 5.2 vs. 2.5
for imidazole, pyridine and pyrazole, respectively). Here, we
present results that show that the FeII complex of our ligand
is not only a structural model but, upon coordination of a
thiolate substrate and reaction with O2, produces a low-spin
[a]
[b]
[c]
Bioinorganic Chemistry Laboratory/Physical Chemistry, School of
Chemistry, Madurai Kamaraj University, Madurai 625021, India.
E-mail: mayilmurugan.chem@mkunuversity.org;
ramamayil@gmail.com
Universität Heidelberg, Anorganisch-Chemisches Institut and
Interdisciplinary Center for Scientific Computing, Im Neuenheimer
Feld 270, D-69120 Heidelberg, Germany.E-mail:
peter.comba@aci.uni-heidelberg.de
Department of Chemistry, Loyola College, Chennai 600034, India.
Supporting information for this article is given via a link at the end of
the document.
+These authors contributed equally to this work.
1
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FeIII-peroxido intermediate and a doubly oxygenated sulfinic
electronic effects (e.g., the pKa values, see above) and the
7-10
acid as a product
.
geometries (5- vs. 6-ring heterocycles).[ ] The similarity of
the structurally well-defined FeII model
2
with that of the
2 with the active
enzyme is visualized by the overlay plot of
site structure of CDO (Figure 1): the main difference between
the two geometries is the torsion around the Fe-Nheterocycle
axes, well visualized in Figure 1. We propose that this is due
to the rigidity of our model ligands L, where the only flexibility
Results and Discussion
Syntheses and Characterization.
The ligands tris(2-pyridyl)ethane and tris(2-pyridyl)-carbinol
(L1 and L2, respectively, see Scheme 2) were prepared with a
procedure slightly modified to that published before,[ ] and their
11
in the tripodal structure is the torsion around the Calkyl
-
FeII complexes were obtained by reaction with Fe(SO3CF3)2 in
MeCN under argon. Complexes [Fe(L1)(MeCN)3](SO3CF3)2 1 and
[Fe(L2)(MeCN)3]-(SO3CF3)2 2 were obtained as red-orange solids
in good yields (74-82 %). Recrystallization of 1 and 2 in CH3CN
yielded pure complexes for further studies. The elemental and
mass spectrometric analyses (MS) confirm their formation: the
HR-ESI-MS shows prominent molecular ion peaks for 1:
[FeN3O3SC18H15F3]+ m/z, 466.01309 and 2: [FeN3O4SC17H13F3]+
m/z, 467.99263 (Figure S1; coordinated solvents may dissociate
during the measurement). Attempts to synthesize 1 and 2 with
non-coordinating solvents such as CH2Cl2 and CHCl3 yielded bis-
complexes of the type [(L)2Fe](SO3CF3)2. This is not surprising
since the formation of bis-complexes by structurally similar
ligands has been reported for CuII, FeII, RuII, CoII, NiII, ZnII and MnII
complexes,[12] and the formation of bis-complexes was only
avoided in a few cases by the substitution at the ortho-position of
the pyridyl arms of the ligand.[13] The solution magnetic
susceptibilities of 1 and 2 (Evans method) are µeff = 5.1 - 5.3 µB,
i.e. close to the spin-only value for a high-spin FeII center. That is,
in solution the FeII centers have high-spin electronic configuration
as in the enzyme.
Cheterocycle bond, and the lowest energy conformation
obviously is that with the three pyridine rings parallel to the
molecular C3 axis. However, we anticipate only a minor
electronic perturbation due to this major structural difference,
since π-bonding involving pyridine and imidazole groups
coordinated to an iron center is known to be of only minor
importance at most.[15] It therefore is not obvious how this
structural “mismatch” relates to differences in the reactivities.
O1
His86
His140
N3
N1
N2
His88
Fe1
Fe
N5
N6
N4
WO
OW
OW
-
Figure 1. Left: molecular structure of 2; H-atoms and SO3CF3 ions
are omitted for clarity. Right: overlay plot of the structures of the CDO
active site and 2.
Due to the inductive effect of the methyl group in L1, the iron
complex 1 exhibits a lower FeIII/II redox potential (E1/2, 0.988 V vs.
Fc/Fc+) than 2 (E1/2, 1.38 V vs. Fc/Fc+, Supporting Information,
Figure S2, Table S3). However, the FeIII/II potential of 1 is higher
than the reported redox barrier for O2 activation by non-heme iron
7,15
centers of less than -0.10 V vs. Fc/Fc+.[
Complex 1 shows
]
electronic absorption bands at 449 nm (, 704 M-1cm-1) and 383
nm (, 3172 M-1cm-1), assigned to arise from FeII-to-ligand charge
transfer transitions (MLCT),[ ] and the more intense bands below
16
342 - 247 nm are due to π
→π* transitions within the ligand. Very
similar transitions are observed for 2, (, : 438 nm, 1304 M-1cm-
1; 379 nm, 3790 M-1cm-1; Supporting Information, Figure S3, Table
S3). Thiophenol was used as a model substrate for studying
CDO-type reactivity in presence of dioxygen. On addition of
Scheme 2. Synthesis of the FeII complexes and their adducts.
Structure, Spectroscopic and Redox Properties.
The unit cell of
2 shows two crystallographically
sodium thiophenolate (PhSNa) to
1 and 2, there is formation
independent molecules with identical coordination geometry
and almost identical structural parameters (Supporting
Information, Tables S1, S2). In analogy to the three histidine
residues (His-86, His-88, His-140) and three water
molecules in the active site of CDO, the tris-pyridine ligand
and three MeCN molecules complete the octahedral
of the thiophenolate adducts [Fe(L1)(MeCN)(SPh)]+,
3
4
(HR-
(HR-
ESI-MS, m/z, 467.0955), and [Fe(L2)(MeCN)(SPh)]+,
ESI-MS, m/z, 469.3512), respectively (Supporting
Information, Figure S4). The FeIII/II redox potentials of 3 (E1/2
,
1.06 V) and 4 (E1/2, 1.26 V, Supporting Information, Figure S5,
Table S3) are slightly different from those of 1 and 2. The
4
geometry around the FeII center.[ ] Interestingly, the Fe-N1py
formation of the thiolate adducts is supported by PhS
→
FeII
bond (1.953 (4), 1.958 (4) Å) is slightly shorter than the other
two Fe-Npy bonds (1.969 (4), 1.972 (4) Å, 1.965 (4), 1.965 (4)
Å, respectively). A similar but more pronounced difference of
the Fe-NHis bond distances was observed in the active site of
a Cys-bound FeII-CDO (Fe-NHis86,1.893; Fe-NHis88, 2.199; Fe-
charge transfer (CT) transitions around 550 nm (L1) and 560
nm (L2) (Supporting Information, Figure S3, S8). Similar
spectral features were observed for the Cys-bound FeII-
4,6
7-10
]
CDO[ ] as well as in other model complexes.[
All possible electronic spin states of
optimized using DFT methods. The optimized geometries of
and along with computed bond parameters, spin density
4,14
3
and 4 were
NHis140, 2.110 Å).[ ] Interestingly, the Fe-NHis distances are
almost identical in the resting state CDO structure (2.0 - 2.1
4
3
4
Å).[ ] The Fe-Npy distances in
2 are shorter than the Fe-NHis
bonds in CDO, while those of the FeII-Tp complexes and their
derivatives (2.11 - 2.28 Å) are longer, and this is due to
plots and relative energies between different spin states are
given in the Supporting information, Table S4. Calculations
reveal that the high-spin (S = 2) surface is the ground state
2
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for both
3
and
4
. Spin density plots show that the four
Further, the stoichiometric reaction of 1 with thiophenol
unpaired electrons are predominantly localized on the metal
center (ρFe = 3.66). TD-DFT-simulated absorption spectra of
and thioanisole in presence of O2 yielded phenylsulfinic acid
and methyl phenyl sulfone (GC yield of the latter: 84%),
respectively. With 18O2 the mass spectra exhibited shifts of
four mass units for phenylsulfinic (m/z, 142 vs. 146) and
methyl phenyl sulfone (m/z, 160 vs. 156; Supporting
Information, Figure S10) over 12 h, indicating that both atoms
of 18O2 are incorporated into the products. However, mixtures
of methyl phenyl sulfoxide and methyl phenyl sulfone were
observed in the reaction with thioanisole as substrate, when
the reaction was stopped after 5 h instead of 12 h reaction
time. Mixed labeling experiments using equal amounts of 16O2
and 18O2 show some selectivity for the pure isotopomers but also
some mixed-labeled sulfone product (m/z, 156 : 158 : 160 = 2.6 :
1 : 1.2 (Supporting Information, Figure S10a).
high-spin
transfer transition from PhS
3
show a band at 427 nm, which is due to a charge
FeII. This supports the
→
experimentally observed band at around 550 nm
corroborating the formation of thiolate adduct (see
Supporting Information, Figure S6).
Dioxygenation Kinetics and Product Analysis.
On treatment of
transition at 550 nm, assigned to a PhS
decreases with a second order reaction rate kO2 = (5.14
0.09) × 10-2 M-1s-1 (t1/2, 0.46 h; Figure 2, S7, Table S3).
Complex shows a decrease in intensity of the PhS
Fe2+
3
with O2 at -40˚C, the electronic
→
FeII CT of
3

4
→
CT band around 514 nm together with the formation of a
transition around 455 nm. The corresponding rate of
Dioxygenation Mechanism.
oxygenation is kO2 = (11.46
h; Supporting Information, Figure S8, S9; Table 1, S3), i.e.
two times faster than for . The reaction mixtures from kinetic
studies show complete regeneration of and , supported
by HR-ESI-MS with prominent ions at m/z, 466.01312 and
m/z, 467.99215, respectively, for and . The GC-MS

0.01) × 10-2 M-1s-1 (t1/2 = 0.21
Adducts 3 and 4 are spectroscopically distinct, allowing
to mechanistically interrogate the O2 activation and study the
kinetics of the formation of intermediates. EPR spectra of
adducts 3 and 4 in MeOH at ambient atmosphere (Figure 3;
Supporting Information, Figure S11, Table1, S3) reveal
signals for at least three different species, a high-spin FeIII (S
3
1
2
1
2
analyses of the reaction solutions confirm the formation of
the doubly oxygenated phenylsulfinic acid as product. In
= 5/2) (
3
: g = 8.62, 6.49, 4.30;
4
: g = 7.89, 5.61, 4.30), a
: g = 2.17, 2.07, 2.04; : g =
2.47, 2.13, 1.94),[
and minor components at g =
12.17 and 11.50 for 3 as well as a signal at g = 2.32, 2.09,
rhombic low-spin FeIII (S = ½) (
3
4
15,17,18,21
]
separate experiments,
a
10-fold excess of sodium
and in
thiophenolate was treated with the iron complexes
1
2
presence of O2 at 25˚C. After 12 hours, the GC-MS analysis
showed 100% conversion of the substrate to phenylsulfinic
1.98 for
3/2) or low-spin (S = ½) species.[ ] The high-spin FeIII signals
of and are similar to previously reported high-spin Cys-
bound FeIII−CDO species.[
4, which might correspond to intermediate-spin (S =
22
acid (GC yield:
1
, 98% and
2
, 93%). In contrast to previously
3
4
9,10
16,18
]
reported systems,[ ] no disulfide-bridged dimer product of
the substrate or triple oxygenation were observed.
1.6
1.4
0.9
0.8
Table 1. EPR parameters, kinetic data and product analysis
of and
0.7
1.2
3
4
0.6
1.0
0.5
0.8
0.4
0
500 1000 1500 2000 2500
Time(s)
0.6
0.4
0.2
0.0
500
600
700
800
900
1000
Wavelength (nm)
0.5
0.4
0.3
0.2
0.1
0.0
0.30
0.25
0.20
0.15
0.10
0
100
200
300
400
500
Time(s)
400
500
600
700
800
900
1000
Wavelength (nm)
Figure 2. (A) UV-vis-NIR spectral changes for the reaction of 3 (6
×10-4 M) with saturated O2 at -40˚C and (B) decay of its
alkylperoxido-FeIII species at 25˚C in MeCN. Insets: plot of the
change in absorbance vs. time.
3
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