Preparation and characterization of myoglobin reconstituted with Fe(II) oxaporphyrin: The monoanionic macrocycle provides unique cyanide binding behavior for the ferrous species

Article abstract of DOI:10.1016/j.ica.2017.06.054

Iron-oxaporphyrin possessing two propionate side chains (FeOP) was synthesized as an artificial cofactor for myoglobin. The autoxidation of FeOP provides a ferric μ-oxo bridged diiron structure. The Fe^II/Fe^III redox potential of FeOP dimethyl ester in acetonitrile is +310 mV vs an Ag|AgCl electrode as determined by cyclic voltammetry. The value is positively shifted by 710 mV from that of the native heme cofactor, indicating that the ferrous species is stabilized in the oxaporphyrin framework. Myoglobin reconstituted with Fe^IIOP was prepared in the presence of dithionite and characterized by UV–vis spectroscopy, ESI-TOF MS, and size exclusion chromatography. Interestingly, autoxidation of the reconstituted protein is found to release the cofactor from the heme pocket, suggesting that the affinity of Fe^IIIOP for the apoprotein is dramatically reduced. Furthermore, cyanide binds to Fe^IIOP in the heme pocket of myoglobin with a binding constant of 1.2 × 10⁴ M^?1, although native deoxymyoglobin has no affinity for cyanide. These findings demonstrate that FeOP is a new type of artificial cofactor for myoglobin which provides a ferrous species with unique characteristics.

Full text of DOI:10.1016/j.ica.2017.06.054

Inorganica Chimica Acta xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Preparation and characterization of myoglobin reconstituted with Fe(II)
oxaporphyrin: The monoanionic macrocycle provides unique cyanide
binding behavior for the ferrous species
Hiroyuki Meichin a, Koji Oohora a,b,c, Takashi Hayashi a,
⇑
a Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan
b Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan
c PRESTO, Japan Science and Technology Agency (JST), Kawaguchi 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Iron-oxaporphyrin possessing two propionate side chains (FeOP) was synthesized as an artificial cofactor
for myoglobin. The autoxidation of FeOP provides a ferric
l
-oxo bridged diiron structure. The FeII/FeIII
Received 27 April 2017
Received in revised form 19 June 2017
Accepted 21 June 2017
Available online xxxx
redox potential of FeOP dimethyl ester in acetonitrile is +310 mV vs an Ag|AgCl electrode as determined
by cyclic voltammetry. The value is positively shifted by 710 mV from that of the native heme cofactor,
indicating that the ferrous species is stabilized in the oxaporphyrin framework. Myoglobin reconstituted
with FeIIOP was prepared in the presence of dithionite and characterized by UV–vis spectroscopy, ESI-
TOF MS, and size exclusion chromatography. Interestingly, autoxidation of the reconstituted protein is
found to release the cofactor from the heme pocket, suggesting that the affinity of FeIIIOP for the apopro-
tein is dramatically reduced. Furthermore, cyanide binds to FeIIOP in the heme pocket of myoglobin with
a binding constant of 1.2 ꢀ 104 Mꢁ1, although native deoxymyoglobin has no affinity for cyanide. These
findings demonstrate that FeOP is a new type of artificial cofactor for myoglobin which provides a ferrous
species with unique characteristics.
This article is dedicated to Prof. Sóvágó
Keywords:
Reconstituted myoglobin
Monoanionic macrocycle
Iron oxaporphyrin
Cyanide binding
Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction
involved in the catalytic cycle. The corrin framework of cobalamin
also has monoanionic character.
Porphyrin, a macrocycle with 18
p
-electrons, includes four pyr-
Myoglobin (Mb), a ubiquitous mammalian oxygen storage
hemoprotein, uses the heme cofactor which is also used in various
oxidative heme enzymes such as cytochrome P450 and heme-con-
taining peroxidases such as horseradish peroxidase. Over several
decades, various efforts in the field of protein engineering have
demonstrated enhancement of enzymatic activity of Mb as well
as ligand binding affinities [12,13]. One of the methods used in
these engineering efforts is reconstitution of Mb with its native
heme molecule being substituted for an artificially-synthesized
heme analogue [14–27]. Recently, our group has been focusing
on reconstitution in efforts to modify the function of Mb. For
instance, Mb reconstituted with iron porphycene, a constitutional
isomer of iron porphyrin, dramatically enhances O2 binding affin-
ity. In addition, O2/CO discrimination is reversed compared to
native Mb [20]. Furthermore, manganese porphycene in the heme
pocket of Mb has catalytic activity toward hydroxylation of inert
alkanes [21]. Porphyrin analogues such as corrole and corrin
derivative have also been found to have interesting influences as
artificial cofactors for Mb [22–24]. For instance, an iron complex
role rings. Iron protoporphyrin IX complex, heme b, plays impor-
tant roles in binding of small molecules such as O2, NO and CO
[1] as well as in enzymatic activities including biosynthesis of hor-
mones [2], drug metabolism [3], and hypochlorite production [4].
These functions harness various redox states of the iron center.
Other enzymes utilize different metal porphyrinoid compounds
such as a nickel corphin complex (F430) and a cobalt corrinoid
complex (cobalamin) (Fig. 1). F430 is used in the active site of
methyl coenzyme M reductase, a methane-evolving enzyme of
methanogenic archaea [5,6]. The corphin framework has monoan-
ionic character which stabilizes a Ni(I) species as an active inter-
mediate. Cobalamin is found in cobalamin-dependent enzymes
such as diol dehydratase [7–9] and methionine synthase [10,11].
In methionine synthase, a specific Co(I) species is known to be
⇑
Corresponding author.
E-mail address: thayashi@chem.eng.osaka-u.ac.jp (T. Hayashi).
http://dx.doi.org/10.1016/j.ica.2017.06.054
0020-1693/Ó 2017 Elsevier B.V. All rights reserved.
Please cite this article in press as: H. Meichin et al., Inorg. Chim. Acta (2017), http://dx.doi.org/10.1016/j.ica.2017.06.054

2
H. Meichin et al. / Inorganica Chimica Acta xxx (2017) xxx–xxx
2. Experimental
2.1. Instruments
UV–vis spectral measurements were carried out with a UV-
2550 or UV-3150 double-beam spectrophotometer (Shimadzu) or
V-670 UV–vis-NIR Spectrophotometer (JASCO). pH values were
monitored using an F-52 pH meter (HORIBA). ESI-TOF MS analyses
were performed on a micrOTOF-II mass spectrometer (Bruker). 1H
and 13C NMR spectra were recorded on a DPX 400 or Avance III HD
spectrometer (Bruker). Chemical shifts are reported in ppm relative
to the internal TMS signal (0.00 ppm). Electrochemical studies
were carried out using a potentiostat (CompactStat, Ivium Tech-
nologies) using a platinum wire as a counter electrode, an Ag|AgCl
(saturated NaClaq: BAS) electrode as a reference and a polished
platinum disk as a working electrode under anaerobic conditions.
The detection of transiently formed species and kinetic measure-
ments were conducted with a rapid scan stopped-flow system
(Unisoku) constructed with a Xe source probe light. CD spectra
were recorded at 25 °C on a J-820AC spectropolarimeter (JASCO).
Air sensitive manipulations were performed in a UNILab glove
box (MBRAUN).
Fig. 1. Molecular structures of representative metal porphyrinoid complexes.
consisting of a corrole ring, which is a trianionic macrocycle, has
been found to accelerate one-electron oxidation of a peroxidase
substrate, relative to native Mb [22]. Moreover, a corrinoid frame-
work as a monoanionic macrocycle forms a cobalt complex in Mb
which promotes a reaction similar to that of cobalamin-dependent
methionine synthase [23,24]. Recently, progress has also been
made in incorporating porphyrins containing noble metals such
as iridium and ruthenium into apoMb, and their activities have
been tested [25–27]. We have conducted a series of studies of
metal complexes of tetrapyrrole macrocycles to modulate the
physicochemical properties and reactivities of the metal center.
Another promising strategy toward development of unique artifi-
cial hemoproteins is represented by reconstitution with an artifi-
cial cofactor where one of the four pyrrolic nitrogen atoms is
substituted with another heteroatom such as oxygen or sulfur.
The O- and S-substituted porphyrins, which are known as oxapor-
phyrin and thiaporphyrin, respectively, provide the porphyrin with
monoanionic character at the macrocycle core [28]. It is particu-
larly interesting that these porphyrinoids stabilize a low-valent
species of a coordinated metal in the framework [29,30]. Therefore,
we have recently prepared an iron complex of oxaporphyrin with a
furan ring instead of a pyrrole ring (Fig. 2). The present work
describes preparation and physicochemical properties of a new
water-soluble iron oxaporphyrin (FeOP) with two propionate side
chains and Mb reconstituted with FeOP as an artificial cofactor.
2.2. Synthesis of FeOP
All chemicals were purchased from Wako, TCI, Nacalai, and
Sigma-Aldrich and used as received unless otherwise noted. Pyr-
role derivatives 1, 2 [22], 4 [31] and 1,4-diformylfuran 6 [32] were
prepared according to reported procedures. FeOP was prepared as
described below (Scheme 1).
2.2.1. Preparation of dipyrromethane 3
Pyrrole 1 (1.00 g, 2.68 mmol) and pyrrole 2 (0.91 g, 2.68 mmol)
were dissolved in a mixture of glacial acetic acid (65 mL) and water
(13 mL). To the solution, p-toluenesulfonic acid monohydrate
(90 mg, 0.47 mmol) was added and the mixture was stirred for
24 h at ambient temperature. The reaction mixture was then
poured into ice-water (300 mL) and carefully neutralized with
sodium bicarbonate, followed by extraction with dichloromethane.
Fig. 2. (a) Molecular structures of heme and iron oxaporphyrin (FeOP) and (b) schematic representation of the reconstitution of myoglobin.
Please cite this article in press as: H. Meichin et al., Inorg. Chim. Acta (2017), http://dx.doi.org/10.1016/j.ica.2017.06.054

H. Meichin et al. / Inorganica Chimica Acta xxx (2017) xxx–xxx
3
Scheme 1. Synthesis of FeOP.
After washing with saturated NaHCO3aq and brine, the organic
phase was dried over Na2SO4 and dried using a rotary evaporator.
The crude product was then purified by silica gel column chro-
matography (Hexane/AcOEt = 47/3–1/1) to afford pure product 3
(544 mg, 0.94 mmol, 35%). 1H NMR (400 MHz, CDCl3) d = 10.0
(1H, s), 9.31 (1H, s), 7.17–7.09 (5H, m), 5.07 (2H, s), 3.84 (2H, s),
3.49 (3H, s), 3.43 (3H, s) 2.60 (4H, m), 2.25 (4H, m), 2.14 (3H, s),
2.10 (3H, s), 1.36 (9H, s) (Fig. S1). 13C NMR (100 MHz, CDCl3)
d = 173.49, 173.36, 161.53, 161.18, 136.22, 131.15, 129.59,
128.19, 128.19, 127.67, 127.55, 125.50, 120.05, 119.61, 119.19,
117.47, 79.94, 65.33, 60.13, 51.35, 34.43, 28.19, 22.43, 20.65,
19.15, 13.91, 10.54, 10.45 (Fig. S2). ESI-TOF MS (positive mode) cal-
culated for [M + Na]+, 603.268; found 603.272.
10.82 (1H, s), 10.61 (1H, s), 10.45 (1H, s), 10.36 (1H, s), 10.08
(1H, s), 4.59–4.56 (2H, t, J = 6.4 Hz) 4.40–4.37 (2H, t, J = 7.2 Hz),
3.89 (3H, s), 3.63 (3H, s), 3.62 (3H, s), 3.59 (3H, s), 3.34–3.27 (4H,
m), 1.25 (6H, s). 13C NMR (100 MHz, CDCl3) d = 173.41, 52.06,
52.00, 36.52, 36.11, 29.85, 21.77, 21.70, 12.50, 12.30, 12.22,
12.04, 1.20, 0.14. ESI-TOF MS (positive mode) calculated for [M
+ H]+, 540.249; found 540.250.
2.2.4. Preparation of FeOP dimethyl ester (FeOPDME)
To oxaporphyrin
(200 mg), degassed acetonitrile (20 mL), dichloromethane
(20 mL), and FeCl3 (23 mg, 142 mol) were added under a nitrogen
7 (6.4 mg, 11.8 mmol) and iron powder
l
atmosphere. The solution was refluxed for 2 h and residual solid
iron was filtered and followed by evaporation of solvent. The crude
product was purified by gel filtration column chromatography in
2.2.2. Preparation of tripyrrin 5
Dipyrromethane 3 (200 mg, 0.35 mmol) was dissolved in 4 mL
of trifluoroacetic acid (TFA) under a nitrogen atmosphere and stir-
red for 5 min at ambient temperature. To this solution, formylpyr-
role 4 (116 mg, 0.45 mmol) in 10 mL of methanol was added and
the solution was further stirred for 90 min at ambient temperature.
methanol to give FeOPDME (5.5 mg, 8.7 lmol, 74%). HR-ESI-TOF
MS (positive mode) calculated for [M–Cl]+, 594.1686; found
594.1690.
Then 30% HBr/AcOH (70 lL, 0.36 mmol) was added. The solution
2.2.5. Preparation of FeOP
was then stirred for an additional 30 min before removal of the sol-
vent on a rotary evaporator to provide a red crude oil 5 (381 mg).
The obtained oil was then used in the next reaction without
purification.
FeOPDME (5.5 mg, 8.7 lmol) was dissolved in 1 mL of THF
under a nitrogen atmosphere, and 1 mL of 0.05 M KOH solution
was added. This mixture was stirred for 2 h at ambient tempera-
ture. The reaction mixture was carefully neutralized with 0.05 M
HCl solution. After evaporation, the crude product was purified
by gel filtration column chromatography in methanol to afford pro-
2.2.3. Preparation of oxaporphyrin 7
Crude tripyrrin 5 (330 mg) was stirred in a mixture of 30% HBr/
AcOH (1.4 mL) and TFA (7 mL) for 6 h under a nitrogen atmo-
sphere. The solution was diluted with 500 mL of methanol and
then diformylfuran 6 (54 mg, 0.44 mmol) in 10 mL of methanol
was added to this solution. Then the mixture was stirred for 12 h
before removal of the solvent on a rotary evaporator. The obtained
red oil was dissolved in dichloromethane and washed with brine
three times. The organic phase was dried over Na2SO4, evaporated
and purified by silica gel column chromatography (CHCl3/CH3-
OH = 49/1–3/2) to afford oxaporphyrin 7 (32 mg, 0.059 mmol,
17% in two steps). 1H NMR (400 MHz, CDCl3) d = 11.16 (1H, s),
duct FeOP (1.2 mg, 2.0 lmol, 23%). HR-ESI-TOF MS (positive mode)
calculated for [M–Cl]+, 566.1373; found 566.1361.
2.3. Electrochemical measurement of FeOPDME
Cyclic voltammograms (CVs) were obtained in the potential
range of ꢁ1.5 to 0.7 V (vs saturated Ag|AgCl reference electrode)
at scan rate of 10 to 300 mV/s in degassed acetonitrile containing
5% DMF and 54 mM of tetrabutylammonium hexafluorophosphate
as an electrolyte at 25 °C.
Please cite this article in press as: H. Meichin et al., Inorg. Chim. Acta (2017), http://dx.doi.org/10.1016/j.ica.2017.06.054

4
H. Meichin et al. / Inorganica Chimica Acta xxx (2017) xxx–xxx
ꢀ
ꢁ
ꢀ
ꢁ
½HCNꢂ
C0 ꢁ ½CNꢁꢂ
2.4. 1H NMR analysis of FeOPDME using Evans method
pH ¼ pKa ꢁ log
¼ pKa ꢁ log
ð4Þ
½CNꢁꢂ
½CNꢁꢂ
The molar susceptibility of FeOPDME was determined using the
Evans technique. 1H NMR spectra of FeOPDME (2.5 mM) were
measured in chloroform-d1 solution containing dichloromethane
as a reference. The equations below were used in the analysis
(Eqs. (1)(3)).
where pKa is 9.2 [34] and C0 is the concentration of added KCN.
3. Results and discussion
Despite the large number of known porphyrinoid compounds,
reports on oxaporphyrin are quite limited [29,30,35–38]. In 1971,
A. W. Johnson and coworkers reported the synthesis and properties
of alkyl group-substituted oxaporphyrin as a monohydrobromide
adduct. In this work, oxaporphyrin 7 was isolated in its protonated
form. The protonation and deprotonation of the inner nitrogen
atom are reversible upon addition of HClaq or NaOHaq to oxapor-
phyrin dissolved in methanol, as confirmed by UV–vis spectral
changes in Fig. 3. This behavior is consistent with the report of
Johnson et al. [35].
Insertion of iron into oxaporphyrin 7 was carried out in a sim-
ilar fashion to generate a thiaporphyrin derivative [36]. The reac-
tion was completed within 2 h to afford reddish brown
compound (FeOPDME). The ESI-TOF mass spectrum of FeOPDME
provides a peak at m/z = 594.169, which is consistent with the cal-
culated exact mass number for [M–Cl]+. This finding supports the
formation of a ferrous species in the macrocycle with consideration
of the monoanionic feature of the oxaporphyrin framework. This
assignment was also confirmed by measurement of the molar
vpMaraðm3=molÞ ¼ 3 ꢀ
D
d ꢀ 10ꢁ6=ð1000 ꢀ MÞ
ð1Þ
ð2Þ
ð3Þ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
leff
¼
vpMara ꢀ 3 ꢀ k ꢀ T=NA ꢀ l0
=lB
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
leff ¼ g SðS þ 1Þ
where
D
d is difference of chemical shift of CH2Cl2 in ppm in the
presence and absence of FeOPDME, M is concentration of FeOPDME
in mol/L, leff is the magnetic susceptibility, k is the Boltzmann con-
stant (1:38 ꢀ 10ꢁ23 (J/K)), T is temperature in K, NA is the Avogadro
constant ð6:02 ꢀ 1023ðmolꢁ1ÞÞ, l0 is the magnetic permeability of a
vacuum (4 ꢀ
p
ꢀ 10ꢁ7ðT2m3=JÞ), lB is the Bohr magneton
(9:27 ꢀ 10ꢁ24ðJ=TÞ), and g is the g value for electron spin (2:002).
2.5. Preparation of rMb(FeOP)
Horse heart Mb was purchased from Sigma-Aldrich and purified
by CM-Cellulose cation exchange column (Wako). Removal of the
heme molecule from Mb was performed by the reported procedure
[33]. An FeIIOP solution (60
l
L in total, 600
sium phosphate buffer containing 16.7 mM sodium dithionite) was
added dropwise to an apoMb solution (3 mL, 6 M in 100 mM
lM in 100 mM potas-
l
potassium phosphate buffer at pH 7.0) under a nitrogen atmo-
sphere. Excess sodium dithionite and FeOP were removed by gel
filtration chromatography using a HiTrap desalting column (5 mL,
GE Healthcare) with 100 mM potassium phosphate buffer at pH
7.0 under anaerobic conditions. The obtained protein solution
was stored anaerobically at 4 °C.
2.6. Oxidation of rMb(FeIIOP) with potassium ferricyanide
To a solution of rMb(FeIIOP) (400
sium phosphate buffer at pH 7.0), a potassium ferricyanide solu-
tion (8.8 L, 45 mM in 100 mM potassium phosphate buffer at
lL, 450 lM in 100 mM potas-
l
pH 7.0) was added under nitrogen atmosphere. UV–vis and CD
spectra were measured before and after addition of potassium
ferricyanide.
Fig. 3. UV–vis absorption spectra of oxaporphyrin 7 in methanol with 10 mM
hydrochloric acid (solid line) and 50 mM sodium hydroxide (dotted line) containing
1% water at 25 °C.
2.7. Ligand binding studies of rMb(FeIIOP)
To a freshly prepared rMb(FeIIOP) solution in 100 mM potas-
sium phosphate buffer at pH 7.0 (7.8 mM, 3 mL), potassium cyanide
(30 lL, 7.8 mM in 100 mM potassium phosphate buffer at pH 7.0)
was added anaerobically and UV–vis spectra were measured. The
kinetics of cyanide binding were further studied under anaerobic
conditions. The transient absorption spectra of the reaction of
rMb(FeIIOP) or aqua-met native myoglobin (nMb) (35
lM) with
potassium cyanide (350–1050
lM) were monitored using
a
stopped-flow rapid scanning apparatus in 100 mM potassium
phosphate buffer at pH 7.0 at 25 °C. The absorption changes were
fitted to a single exponential equation to determine apparent rate
constants kobs at various concentrations of cyanide anion. The asso-
ciation rate constants kon, dissociation constants koff, and affinities
for cyanide anion KCN- were determined with plots of kobs against
concentration of cyanide anion. The concentrations of cyanide
anion were determined by the following Henderson–Hasselbach
equation (Eq. (4)).
Fig. 4. UV–vis absorption spectrum of ferrous FeOPDME in methanol at 25 °C.
Please cite this article in press as: H. Meichin et al., Inorg. Chim. Acta (2017), http://dx.doi.org/10.1016/j.ica.2017.06.054

H. Meichin et al. / Inorganica Chimica Acta xxx (2017) xxx–xxx
5
susceptibility of FeOPDME using the Evans method [39]. According
to the difference of the chemical shift of reference dichloro-
methane in chloroform-d1 (Fig. S5), the molar susceptibility was
determined to be 1.25 ꢀ 10ꢁ7 m3molꢁ1, which is in agreement
with a complex with S = 2. The UV–vis absorption spectrum of
FeIIOPDME is shown in Fig. 4. The characteristic split Soret band,
two characteristic Q-bands and one shoulder-like Q-band are
observed and these are mostly consistent with those of
a
previously reported iron complex of an oxaporphyrin derivative
[37]. The CV measurements revealed two redox peaks at +0.31 V
and ꢁ1.1 V vs Ag|AgCl in acetonitrile (Fig. S6). The former redox
peak is assigned to the FeII/FeIII process, which is positively shifted
by 710 mV compared to that of heme b, indicating significant sta-
bilization of the ferrous species in oxaporphyrin. The positive shift
of the E1/2 value of the FeII/FeIII process in FeOPDME is similar to
the observation made in the previous report of a TPP-type oxapor-
phyrin iron complex [37]. The latter redox peak is attributed to the
formation of a one-electron reduced species of the macrocycle,
because oxaporphyrin 7 has a reversible redox peak in the CV mea-
surement at ꢁ1.1 V vs Ag|AgCl (Fig. S6).
The methyl ester moieties were successfully hydrolyzed under
basic conditions to yield FeOP. After the reaction, the split Soret
band and the characteristic Q-bands disappear in the UV–vis spec-
trum (Fig. 5). This finding suggests that the ferrous state of iron
oxaporphyrin is spontaneously oxidized to a ferric
l-oxo dimer
product under aerobic conditions [37]. The ESI-TOF mass spectrum
of the reaction mixture has a peak with m/z = 574.1349, in which
the isotope pattern indicates z = 2+ (Fig. S7). This value is consis-
tent with the calculated value of the ferric
(m/z = 574.1356 (z = 2+)). Interestingly, it was found that washing
the ferric -oxo dimer complex in dichloromethane with 0.1 M
l-oxo dimer complex
Fig. 5. UV–vis absorption spectra of the l-oxo dimer of ferric state of FeOP (solid
line) and monomer of the ferrous state of FeOP (dotted line) in methanol at 25 °C.
l
HCl removes the iron ion with recovery of the oxaporphyrin free-
base species. Instead of employing acidification, the monomeric
ferrous state of FeOP complex was obtained by reduction of the
l-oxo dimer complex upon the addition of an excess amount of
sodium dithionite. The formation of the ferrous species was con-
firmed by UV–vis spectroscopy (Fig. 5).
Next, insertion of the FeIIIOP
l-oxo dimer into the heme pocket
of Mb was attempted after removal of heme. However, a UV–vis
spectral change upon the addition of the dimer into the solution
of the apoprotein was not observed because of the highly stable
l
-oxo bridge structure. Thus, FeIIOP was inserted into apoMb in
the presence of sodium dithionite (Fig. 6). The titration curve in
Fig. 6a clearly shows the curvature at a 1:1 ratio, indicating stoi-
chiometric binding of FeIIOP with apoMb. Size exclusion chro-
matography suggests that the elution volume monitored at
400 nm and 280 nm for the obtained reconstituted protein is con-
sistent with that observed for the native protein (Fig. 7b). In addi-
tion, the ESI-TOF mass spectrum of the reconstituted protein
indicated a 9+ ionized form (calcd: 1947.451, found: 1947.351)
(Fig. S8), which confirms formation of rMb(FeOP).
Fig. 6. UV–vis spectrum of rMb(FeOP) in 100 mM potassium phosphate buffer at
pH 7.0 at 25 °C.
Fig. 7. (a) Plots of absorbance at 530 nm against the amount of added FeIIOP and (b) size exclusion chromatogram of rMb(FeOP) (black solid line: 280 nm, gray solid line:
400 nm) and nMb (black dotted line: 409 nm) in 100 mM potassium phosphate buffer at pH 7.0 at 4 °C.
Please cite this article in press as: H. Meichin et al., Inorg. Chim. Acta (2017), http://dx.doi.org/10.1016/j.ica.2017.06.054

6
H. Meichin et al. / Inorganica Chimica Acta xxx (2017) xxx–xxx
The autoxidation behavior of rMb(FeIIOP) was studied. UV–vis
spectral changes of rMb(FeIIOP) were observed with clear isos-
bestic points after exposure to air (Fig. 8a) and characteristic Q-
bands essentially disappear within 48 h. CD spectral changes
(Fig. 8b) suggest that FeOP is released from the heme pocket of
Mb, because the Cotton effect in the Soret region, which is derived
from the chiral environment of the heme pocket, obviously
decreases after the reaction. In contrast, the UV–vis spectrum of
rMb(FeIIOP) under a nitrogen atmosphere does not show any
changes over 12 h (Fig. S9), supporting our assumption that the
spectral changes observed under aerobic conditions are derived
from the autoxidation. Furthermore, the disappearance of the CD
signal in the Soret region after the addition of potassium ferri-
cyanide indicates that FeIIIOP is released from the Mb matrix
(Fig. 9). Despite a lot of researches on engineering of Mb, artificial
metal complex cofactors which possess dicationic charge are quite
limited. [23] It is proposed that the dicationic charged complex
FeIIIOP significantly decreases in the affinity for the Mb matrix
because the hydrophobic interaction between native heme and
Mb matrix accounts for the factor of 105–107 Mꢁ1 in the overall
binding affinity of 1 ꢀ 1014 Mꢁ1. [40] Similarly, oxidation of ferrous
heme to ferric state in soluble guanylyl cyclase, a hemoenzyme,
results in release of heme from protein matrix due to lack of
hydrophobic interaction [41].
Fig. 10. UV–vis spectrum of 7.8
presence (dotted line) of potassium cyanide in 100 mM potassium phosphate buffer
at pH 7.0 containing 10 mM sodium dithionite at 25 °C. [rMb(FeIIOP)] = 7.8
M.
[KCN] = 770 M.
l
M of rMb(FeIIOP) in the absence (solid line) and
l
l
induces a color change from brown to pink. UV–vis spectral
changes are observed with clear isosbestic points, indicating that
cyanide is capable of binding to the iron center of FeIIOP (Fig. 10)
A study of ligand binding to rMb(FeIIOP) was conducted. The
addition of potassium cyanide to freshly prepared rMb(FeIIOP)
with
a
binding constant of 1.2 ꢀ 104 M–1
, although native
Fig. 8. (a) UV–vis and (b) CD spectral changes of rMb(FeOP) over 48 h under aerobic conditions in 100 mM potassium phosphate buffer at pH 7.0 at 25 °C.
Fig. 9. (a) UV–vis and (b) CD spectra of rMb(FeOP) in the absence (solid line) and presence (dotted line) of potassium ferricyanide under anaerobic conditions in 100 mM
potassium phosphate buffer at pH 7.0 at 25 °C. [rMb(FeOP)] = 441 M. [K3Fe(CN)6] = 860 M.
l
l
Please cite this article in press as: H. Meichin et al., Inorg. Chim. Acta (2017), http://dx.doi.org/10.1016/j.ica.2017.06.054

H. Meichin et al. / Inorganica Chimica Acta xxx (2017) xxx–xxx
7
Fig. 11. (a) Differential transient absorption changes of rMb(FeIIOP) during the reaction with potassium cyanide. Spectra were measured every 0.05 s over 0.5 s (solid line:
0 s) and (b) plots of rate constants for the reaction of potassium cyanide with (solid line) rMb(FeIIOP) and (dotted line) nMb against various concentrations of cyanide ion in
100 mM potassium phosphate buffer at pH 7.0 at 25 °C. In the case of rMb(FeOP), the solution contained 10 mM sodium dithionite.
Table 1
is capable of binding cyanide as an axial ligand, which is sharply
different from nMb. These findings suggest that a metal oxapor-
phyrin complex has potential for providing new functions as an
artificial cofactor of hemoproteins. Characterization of the struc-
ture and enzymatic activity of rMb(FeIIOP) is now in progress.
Cyanide Binding Parameters for Native and Reconstituted Mb.
a
b
Protein
kon
(
l
Mꢁ1 sꢁ1
)
koff (sꢁ1
)
KCNꢁ (l )
Mꢁ1
d,e
ferric nMb
8.9 0.3 ꢀ 10ꢁ5
8.0 ꢀ 10ꢁ4
0.11
ferrous rMb(FeOP)c
1.3 0.1 ꢀ 10ꢁ2
1.1 0.6b
1.2 0.6 ꢀ 10ꢁ2
Reaction conditions: 100 mM phosphate buffer (pH 7.0) at 25 °C.
a
Acknowledgements
Association rate constants of cyanide ligand.
Dissociation rate constants of cyanide ligand.
in 100 mM potassium phosphate buffer containing 10 mM sodium dithionite.
in 110 mM sodium phosphate buffer at pH 6.6 at 25 °C, protein concentration of
b
c
This work was supported by Grants-in-Aid for Scientific
Research provided by JSPS KAKENHI Grant Numbers JP15H05804,
JP24655051, JP15H00944, JP22105013, and JP16H00837, and JST
PRESTO (JPMJPR15S2). H.M. acknowledges support from the Pro-
gram for Leading Graduate Schools for Osaka University: Interdis-
ciplinary Program for Biomedical Sciences (IPBS).
d
5.2
l
M.
e
Ref [42].
deoxymyoglobin does not interact with cyanide. Transient absorp-
tion spectra were obtained using stopped-flow techniques with
various concentrations of potassium cyanide (Fig. 11). Apparent
association and dissociation rate constants, kon and koff, were
determined as summarized in Table 1. The association of
cyanide with rMb(FeIIOP) is relatively fast with a kon value of
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2017.06.054.
1.3 ꢀ 10ꢁ2
l
Mꢁ1 sꢁ1, which is 150-fold faster than that of ferric
nMb. One of the possible reasons for the faster binding may be
attributed to the different position of the iron atom in the
porphyrinoid plane of FeIIOP, relative to its position in heme b
[20]. The iron atom in FeIIOP could be located in the porphyrinoid
plane without any deviation from the plane according to the
previous report [43,44], whereas the iron atom in heme b is out
of plane (with a distance of DMb = 0.29 Å from the protoheme
plane) [45]. In contrast, the affinity of cyanide for rMb(FeIIOP) is
9-fold lower than that of ferric nMb due to its fast dissociation
rate. An X-ray crystal structure analysis and/or theoretical study
will be needed to further understand the interesting ligand binding
behavior of rMb(FeIIOP).
References
[1] A. Ghosh, The Smallest Biomolecules: Diatomics and their Interactions with
Heme Proteins, Elsevier, 2008. pp. 3–17.
[2] B. Meunier, S.P. de Visser, S. Shaik, Chem. Rev. 104 (2004) 3947–3980.
[3] U.M. Zanger, M. Schwab, Pharmacol. Ther. 138 (2013) 103–141.
[4] S.J. Klebanoff, J. Leukoc. Biol. 77 (2005) 598–625.
[5] D. Ankel-Fuchs, R.K. Thauer, Eur. J. Biochem. 156 (1986) 171–177.
[6] M. Goubeaud, G. Schreiner, R.K. Thauer, Eur. J. Biochem. 243 (1997) 110–114.
[7] B. Kräutler, A. Sigel, Metal Ions in Life Science, Springer, 2009. pp. 1–51.
[8] K. Gruber, B. Puffer, B. Kräutler, Chem. Soc. Rev. 40 (2011) 4346–4363.
[9] K.L. Brown, Chem. Rev. 105 (2005) 2075–2150.
[10] C.L. Drennan, S. Huang, J.T. Drummond, R.G. Matthews, M.L. Lidwig, Science
266 (1994) 1669–1674.
[11] R.G. Matthews, Acc. Chem. Res. 34 (2001) 681–689.
[12] B.A. Springer, S.G. Sligar, J.S. Olson, G.N. Jr, Phillips, Chem. Rev. 94 (1994) 699–
714.
4. Conclusion
[13] F. Draghi, A.E. Miele, C. Travaglini-Allocatelli, B. Vallone, M. Brunori, Q.H.
Gibson, J.S. Olson, J. Biol. Chem. 277 (2002) 7509–7519.
[14] H. Sato, T. Hayashi, T. Ando, Y. Hisaeda, T. Ueno, Y. Watanabe, J. Am. Chem. Soc.
126 (2004) 436–437.
[15] S. Chakraborty, J. Reed, M. Ross, M.J. Nilges, I.D. Petrik, S. Ghosh, S. Hammes-
Schiffer, J.T. Sage, Y. Zhang, C.E. Schulz, Y. Lu, Angew. Chem. Int. Ed. 53 (2014)
2417–2421.
[16] M. Bröring, F. Brégier, O. Burghaus, C. Kleeberg, Z. Anorg, Allg. Chem. 636
(2010) 1760–1766.
[17] M. Bordeaux, V. Tyagi, R. Fasan, Angew. Chem. Int. Ed. 54 (2015) 1744–1748.
[18] T. Hayashi, Y. Hitomi, H. Ogoshi, J. Am. Chem. Soc. 120 (1998) 4910–4915.
[19] I. Hamachi, S. Shinkai, Eur. J. Org. Chem. 1999 (1999) 539–549.
[20] T. Matsuo, H. Dejima, S. Hirota, D. Murata, H. Sato, T. Ikegami, H. Hori, Y.
Hisaeda, T. Hayashi, J. Am. Chem. Soc. 126 (2004) 16007–16017.
This work is the first example of the use of iron oxaporphyrin
(FeOP) as a new myoglobin cofactor. The precursor FeOPDME pro-
vides a positively shifted FeII/FeIII redox potential compared to that
of heme, indicating that the oxaporphyrin framework clearly stabi-
lizes the ferrous species. The cofactor, FeIIOP, was then successfully
incorporated into apoMb to form the reconstituted protein in the
presence of dithionite. FeIIOP is stable and remains in the protein
pocket under a nitrogen atmosphere at room temperature for over
12 h, whereas under aerobic conditions rMb(FeIIOP) releases FeOP
from the heme pocket via autoxidation. Furthermore, rMb(FeIIOP)
Please cite this article in press as: H. Meichin et al., Inorg. Chim. Acta (2017), http://dx.doi.org/10.1016/j.ica.2017.06.054

8
H. Meichin et al. / Inorganica Chimica Acta xxx (2017) xxx–xxx
[21] K. Oohora, Y. Kihira, E. Mizohata, T. Inoue, T. Hayashi, J. Am. Chem. Soc. 135
(2013) 17282–17285.
[22] T. Matsuo, A. Hayashi, M. Abe, T. Matsuda, Y. Hisaeda, T. Hayashi, J. Am. Chem.
Soc. 131 (2009) 15124–15125.
[33] F.W.J. Teale, Biochem. Biophys. Acta 35 (1959) 543.
[34] R.M. Izatt, J.J. Christensen, R.T. Pack, R. Bench, Inorg. Chem. 1 (1962) 828–831.
[35] M.J. Broadhurst, R. Grigg, A.W. Johnson, J. Chem. Soc. C (1971) 3681–3690.
_
[36] L. Latos-Grazyn´ ski, J. Lisowski, M.M. Olmstead, A.L. Balch, Inorg. Chem. 28
[23] T. Hayashi, Y. Morita, E. Mizohata, K. Oohora, J. Ohbayashi, T. Inoue, Y. Hisaeda,
Chem. Commun. 50 (2014) 12560–12563.
(1989) 1183–1188.
[37] M. Pawlicki, L. Latos-Grazyn´ ski, Inorg. Chem. 41 (2002) 5866–5873.
_
[24] Y. Morita, K. Oohora, A. Sawada, K. Doitomi, J. Ohbayashi, T. Kamachi, K.
Yoshizawa, Y. Hisaeda, T. Hayashi, Dalton Trans. 45 (2016) 3277–3284.
[25] H.M. Key, P. Dydio, D.S. Clark, J.F. Hartwig, Nature 534 (2016) 534–537.
[26] M.W. Wolf, D.A. Vargas, N. Lehnert, Inorg. Chem. 56 (2017) 5623–5635.
[27] G. Sreenilayam, E.J. Moore, V. Steck, R. Fasan, Adv. Synth. Catal. 359 (2017)
2076–2089.
[38] S. Stute, L. Götzke, D. Meyer, M.L. Merroun, P. Rapta, O. Kataeva, W. Seichter, K.
Gloe, L. Dunsch, K. Gloe, Inorg. Chem. 52 (2013) 1515–1524.
[39] I. Bertini, C. Luchinat, P. Turano, G. Battaini, L. Casella, Chem. Eur. J. 9 (2003)
2316–2322.
[40] M.S. Hargrove, D. Barrick, J.S. Olson, Biochemistry 35 (1996) 11293–11299.
[41] B.G. Fritz, X. Hu, J.L. Brailey, R.E. Berry, F.A. Walker, W.R. Montfort,
Biochemistry 50 (2011) 5813–5815.
[28] T. Chatterjee, V.S. Shetti, R. Sharma, M. Ravikanth, Chem. Rev. 117 (2017)
3254–3328.
[42] R.P. Cox, M.R. Hollaway, Eur. J. Biochem. 74 (1977) 575–587.
_
[29] P.J. Chmielewski, L. Latos-Grazyn´ ski, M.M. Olmstead, A.L. Balch, Chem. Eur. J. 3
[43] In ferric heme b, spin density exists in dx2
orbital of iron atom and this
ꢁy2
(1997) 268–278.
causes overlap between 2p-orbitals of pyrrole nitrogen atoms. The electron in
orbital is accommodated in r⁄ orbital to weaken the Fe–nitrogen bond,
_
[30] E. Pacholska, P.J. Chmielewski, L. Latos-Grazyn´ ski, Inorg. Chim. Acta 273 (1998)
dx2
ꢁy2
_
184–190.
resulted in the out-of-plane position of iron in heme b. Latos-Grazyn´ ski et al.
[31] A.R. Battersby, M.G. Baker, H.A. Broadbent, C.J.R. Fookes, F.J. Leeper, J. Chem.
Soc. Perkin Trans. 1 (1987) 2027–2048.
[32] D.W. Yoon, D.E. Gross, V.M. Lynch, J.L. Sessler, B.P. Hay, C.H. Lee, Angew. Chem.
Int. Ed. 47 (2008) 5038–5042.
proposed unoccupied dx2
orbital for bisaryl adduct of Fe(II) oxaporphyrin
ꢁy2
complex. As a result, the iron atom is in the plane of oxaporphyrin.
_
´
[44] M. Pawlicki, L. Latos-Grazynski, Inorg. Chem. 43 (2004) 5564–5571.
[45] G.S. Kachalova, A.N. Popov, H.D. Bartunik, Science 284 (1999) 473–476.
Please cite this article in press as: H. Meichin et al., Inorg. Chim. Acta (2017), http://dx.doi.org/10.1016/j.ica.2017.06.054