Nitric oxide binding to cationic and anionic ferric porphyrins in aqueous solution: Reversible formation of ferrous NO species of the cationic porphyrin

Article abstract of DOI:10.1016/j.ica.2004.01.038

Two water-soluble ferric porphyrins, sodium 5α,10β,15α, 20β-tetrakis(2-(sulfonatoacetamido)phenyl)porphyrinatoiron(III) (Fe ^IIITanP) and 5α,10β,15α,20β-tetrakis(2-(N,N,N- trimethylammoniumacetamido)phenyl)porphyrinatoiron(III) chloride (Fe

Full text of DOI:10.1016/j.ica.2004.01.038

Inorganica Chimica Acta 357 (2004) 2503–2509
www.elsevier.com/locate/ica
Nitric oxide binding to cationic and anionic ferric porphyrins
in aqueous solution: reversible formation of ferrous NO species
of the cationic porphyrin
1
*
Hiroyasu Imai , Yuko Yamashita, Shigeo Nakagawa , Hiroki Munakata,
Yoshio Uemori
Faculty of Pharmaceutical Sciences, Hokuriku University, 3 Ho Kanagawa-Machi, Kanazawa 920-1181, Japan
Received 3 September 2003; accepted 21 January 2004
Available online 6 March 2004
Abstract
Two water-soluble ferric porphyrins, sodium 5a,10b,15a,20b-tetrakis(2-(sulfonatoacetamido)phenyl)porphyrinatoiron(III)
(FeIIITanP) and 5a,10b,15a,20b-tetrakis(2-(N,N,N-trimethylammoniumacetamido)phenyl)porphyrinatoiron(III) chloride (FeIIIT-
catP), were synthesized. The pKa values of the coordinated H2O of FeIIITanP and FeIIITcatP were evaluated to be 8.0 and 4.1,
respectively. Reactions of NO with the ferric porphyrins were examined spectrophotometrically in aqueous solution. Porphyrin
FeIIITanP binds NO reversibly to give the corresponding ferric NO species at pH 1.3 and pH 3.0, and FeIIITcatP reacts similarly
with NO at pH 1.3. The thermodynamic data for the NO binding were estimated from vanÕt Hoff plots. At pH 3.0, visible and ESR
spectral data indicated that FeIIITcatP binds NO reversibly to produce ferrous NO species depending on NO partial pressures.
These results were discussed based on through-space intramolecular interactions between the coordinated H2O or NO and the ionic
substituents of the porphyrins.
Ó 2004 Elsevier B.V. All rights reserved.
Keywords: Water-soluble porphyrin; Iron complex; Nitric oxide; Binding equilibrium
ðNOÞFeIIIPor N!O ðNOÞFeIIPor
ð2Þ
1. Introduction
For synthetic model porphyrins, treatment of
5,10,15,20-tetraphenylporphyrinatoiron(III) FeIIITPP
with NO in toluene containing a small amount of
methanol irreversibly affords (NO)FeIITPP [6]. In
aqueous solution, an analogous water-soluble porphyrin
5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinatoiron
(III) (FeIIITPPS) reacts reversibly with NO to produce
NO-bound ferric species, (NO)FeIIITPPS, but the cor-
responding reduced species, (NO)FeIITPPS, was not
observed [5]. A recent study has shown that the reaction
of FeIIITPPS and excess NO in aqueous solution gives
(NO)FeIITPPS very slowly and this is catalyzed by ni-
trite ion [7]. We have also reported that the reaction in
H2O-organic mixed solvents reversibly yields (NO)-
FeIITPPS as the dominant product [8]. These results,
observed for both natural and model compounds, sug-
gested that the microenvironments and/or solvents
Reactions of NO with iron porphyrins have received
much attention because of their relevance to biological
systems [1]. NO binding to ferric porphyrins (FeIIIPor) as
well as to ferrous porphyrins (FeIIPor) is thought to be
physiologically important as exemplified in the vasodi-
lation that occurs in response to the bite of blood sucking
insects [2]. Ferric porphyrins in hemeproteins reversibly
bind NO to form (NO)FeIIIPor species, as follows:
FeIIIPor þ NO ꢀ ðNOÞFeIIIPor
Some of them further react irreversibly with NO to
ð1Þ
give (NO)FeIIPor species [3–5].
* Corresponding author.
E-mail address: h-imai@hokuriku-u.ac.jp (H. Imai).
1
Present address: Hokuriku University Computing Services Center,
Taiyogaoka 1-1, Kanazawa 920-1180, Japan.
0020-1693/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2004.01.038

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H. Imai et al. / Inorganica Chimica Acta 357 (2004) 2503–2509
around the bound NO at the active site might play an
important role in the reduction behavior of the ferric
ion. Thus, studies using properly designed model com-
pounds in aqueous solution could be expected to pro-
vide useful information on such effects on NO binding.
However, as a result of the lipophilic nature of por-
phyrins, only limited metalloporphyrins for studying
axial-ligand binding in aqueous solutions have been
synthesized.
In earlier works, we reported [9,10] that microenvi-
ronments near the active site of synthetic metallopor-
phyrins can affect the thermodynamic parameters for the
binding of axial ligands on the basis of non-covalent
intramolecular interactions. In this report, to elucidate
the microenvironmental effects on NO binding, we have
designed and synthesized anionic (FeIIITanP) and cat-
ionic (FeIIITcatP) water-soluble ferric porphyrins
(Fig. 1). Since these porphyrins have binding pockets
that are sterically similar but electrostatically opposite,
the microenvironments around a bound, polar axial li-
gand would be expected to affect the binding properties
differently.
sures of NO (PNO) were adjusted by mixing NO with Ar
while monitoring with a mass-flow controller (Kojima
2503F) and a gas-flow meter (Kojima 3810). The mixing
gas was further purified by KOH pellets before intro-
duction into a cell for visible spectral measurements.
ESR spectra were measured on a JEOL JES-FE2X
spectrometer.
The pKa values of the coordinated H2O of the ferric
porphyrins were estimated from the mid-point pH in the
visible absorption spectral changes under various pHs.
The K values as the binding constant for reaction (1)
were evaluated from the visible absorption spectral
changes at various PNO, on the basis of the published
method by Collman et al. [11]. Thermodynamic data
were estimated from the temperature dependence of K at
temperatures ranging from 10 to 30 °C.
2.3. 5a,10b,15a,20b-Tetrakis(2-aminophenyl)porphyri-
natoiron(III) chloride (1)
5a,10b,15a,20b-Tetrakis(2-aminophenyl)porphyrin
(H2TamPP) [12] (121 mg, 0.179 mmol) was dissolved in
tetrahydrofuran (50 ml) containing 2,6-dimethylpyridine
(0.5 ml, 2.17 mol). After the solution was purged with
N2 over 30 min, iron(II) chloride (0.300 g, 2.37 mmol)
was added and the mixture was stirred at room tem-
perature for 3 h under N2 atmosphere. The achievement
of the metalation was confirmed by disappearance of red
fluorescence with a UV lamp (365 nm). After evapora-
tion of the mixture, the solid was dissolved in a small
amount of CHCl3/CH3OH (50/1), then purified on a dry
silica-gel column (ca. 200 mesh, £2.5 ꢀ 8 cm) and eluted
with CHCl3/CH3OH (25/1). The eluate was evaporated
to dryness and the solid was recrystallized from
CH3OH/ether, yielding 110 mg (86%). Rf value ¼ 0.63
(60F254; CHCl3/CH3OH ¼ 20/1). UV–vis [kmax nm in
benzene]: 417, 508.
2. Experimental
2.1. Materials
All reagents and solvents were of commercial reagent
quality and were used without further purification. Ni-
tric oxide gas (99.9% purity) for measurements was
purified by passage through KOH pellets. Precoated
silica-gel plates (Merck Kieselgel 60F254) or reversed
silica-gel plates (Merck Kieselgel PR-8 F254) were used
for TLC to determine Rf values.
2.2. Equilibrium procedures and instrumentation
Visible absorption spectra were obtained on a Hitachi
U-2000 spectrophotometer. The pH values for spectral
measurements were adjusted with HClO4. Partial pres-
2.4. Sodium 5a,10b,15a,20b-tetrakis(2-(sulfonatoacetam-
ido)phenyl)porphyrinatoiron(III) ((H2O)2FeIII TanP)
To a solution of isobutyl chlorocarbonate (0.300 ml,
2.31 mmol) and triethylamine (0.600 ml, 4.30 mmol) was
added sulfoacetic acid (0.410 g, 2.93 mmol). The mixture
was stirred for 30 min at room temperature. To this
solution was added a solution of 1 (90.0 mg, 0.126
mmol) in CH2Cl2 (70 ml). After the mixture had been
stirred at room temperature for 24 h, the solution was
evaporated to dryness. The resultant solid was dissolved
in CHCl3 (100 ml) and the organic layer was extracted
with aqueous NaOH (0.1 mol lꢁ1, 50 ml). After the
aqueous layer was washed with CHCl3 (100 ml ꢀ 5), the
solution was evaporated to dryness. The solid was dis-
solved in a small amount of ethanol and filtered off a salt
solid, then the filtrate was evaporated. The residual solid
O
-
+
O
O
S
O
N
O
O
S
-
+
O
O
N
NH
NH
N
O
O
NH
NH
N
N
N
Fe
N
Fe
N
N
HN
N
HN
O
O
+
NH
O
NH
S
O
-
N
O
O
-
O
S
+
O
O
N
O
FeIIITanP
FeIIITcatP
Fig. 1. Water-soluble ferric porphyrins. Axial ligands (H2O) and
counter ions Naþ for FeIIITanP and Clꢁ for FeIIITcatP are omitted for
clarity.

H. Imai et al. / Inorganica Chimica Acta 357 (2004) 2503–2509
2505
was purified by reversed phase chromatography (Cos-
3. Results and discussion
mosil 75C18-OPN (Nakarai), £3.5 ꢀ 8 cm, CH3OH/
H2O (1/1)). The eluate was evaporated to dryness and
the solid was recrystallized from CH3OH/ether, yielding
120 mg (79%). Rf value ¼ 0.37 (PR-8 F254; CHCl3/
CH3COOH/pyridine/CH3OH ¼ 2/2/1/1). UV–vis [kmax
nm in H2O at pH 3 (log e)]: 395 (5.17), 526 (4.17). Anal.
Calc. for C52H36N8O16S4Na4Fe ꢂ 3CH3OH ꢂ 6H2O ꢂ OH:
C, 43.28; H, 4.03; N, 7.34. Found: C, 43.30; H, 4.33; N,
7.22%. FAB-MS (magic bullet); m=z calculated most
abundant parent mass (M): 1304. Observed: 1304 (Mþ,
6%), 1305 (M + Hþ, 7%), 1327 (M + Naþ, 9%).
3.1. Synthesis of porphyrins
Metal insertion to tetrakis(ortho-substituted-
phenyl)porphyrins is usually not easy [13], then often
requires a high reaction temperature that leads to
atropisomerization. Contrary to this, iron was success-
fully inserted to H2TamPP under mild conditions in the
present work, in which the absence of isomerization was
confirmed by silica-gel TLC. The easy metalation of
H2TamPP could be attributed to a catalytic function of
the amino groups that present near the center of the
porphyrin core and might act as a metal-ion provider
[14].
2.5. 5a,10b,15a,20b-tetrakis(2-(N,N-dimethylaminoace-
tamido)phenyl)porphyrinatoiron(III) chloride (2)
To construct electrostatically different microenviron-
ments around the central metal ion in ferric porphyrin,
anionic sulfonato or cationic ammonium groups were
introduced using methods similar to those described in
the literature [9,10]. These ionic groups also make the
porphyrins water-soluble. Since these porphyrins are the
a,b,a,b-atropisomer, the substituents of the porphyrin
prevent self-aggregation in aqueous solution and the
porphyrins have no isomeric conformations for axial-
ligand bound species.
To a suspended mixture of N,N-dimethylaminoacetic
acid hydrogen chloride (1.60 g, 11.5 mmol) in CH2Cl2
(50 ml) was added dropwise oxalyl chloride (5.00 ml,
38.7 mmol). After stirring for 2 h at room temperature,
the mixture was evaporated to dryness, then the solid
was dissolved in CH2Cl2 (30 ml). To a cooled solution of
1 (500 mg, 0.659 mmol) in CH2Cl2 (150 ml) containing
triethylamine (3.00 ml, 21.5 mmol) was added the acid
chloride solution. After stirring for 5 h at room tem-
perature, the solution was washed with aqueous NaOH
(0.1 mol lꢁ1, 150 ml), dried over anhydrous Na2SO4, and
evaporated to dryness. The solid was purified on a silica-
gel column (CHCl3, £4 ꢀ 30 cm) and eluted with
CHCl3/CH3OH (200/1). The eluate was evaporated and
recrystallized from CH3OH/ether, yielding 382 mg
(53%). Rf value ¼ 0.42 (60F254; CHCl3/CH3OH ¼ 5/3).
UV–vis [kmax nm in CH3OH]: 413, 507 (sh).
3.2. Acidity of coordinated H2O
In aqueous solutions, the central iron(III) ion of
ferric porphyrins FeIIIPor is six-coordinated in which
the axial ligands are two H2O [(H2O)2FeIIIPor] or one
H2O and one OHꢁ [(H2O)(OH)FeIIIPor], depending on
pH. The two species are easily distinguishable by their
visible spectra. From the spectral changes at various
pHs as exemplified in Fig. 2, the pKa values of the co-
ordinated H2O for the ferric porphyrins prepared were
estimated (Table 1). The pKa values for simple cationic
and anionic water-soluble ferric porphyrins reported
thus far have been in the range of 4.1 and 7.0 [15,16].
Interestingly, the pKa values of 8.0 for (H2O)2FeIIITanP
and of 4.1 for (H2O)2FeIIITcatP are out of and terminal
2.6. 5a,10b,15a,20b-tetrakis(2-(N,N,N-trimethylammo-
niumacetamido)phenyl)porphyrinatoiron(III) chloride
((H2O)2FeIII TcatP)
To a solution of 2 (210 mg, 0.196 mmol) in N,N-
dimethylformamide (100 ml) was added methyl iodide
(0.250 ml, 3.21 mmol). After the solution was stirred for
24 h at room temperature, ether was added to precipi-
tate a solid. The solid was dissolved in a small amount of
CH3OH and the porphyrin iodide was converted to
chloride on an ion-exchange resin column (amberlist A-
21 chloride form, £3 ꢀ 30 cm) then eluted with
CH3OH. The eluate was evaporated and recrystallized
from CH3OH/ether, yielding 127 mg (57%). Rf
1
0.8
× 5
0.6
0.4
0.2
0
value ¼ 0.40 (60F254
;
CHCl3/CH3OH/H2O ¼ 11/7/2).
UV–vis [kmax nm in H2O at pH 3 (log e)]: 395 (5.08), 526
(4.02). Anal. Calc. for C64H72N12O4Cl5Fe ꢂ 6H2O: C,
54.34; H, 5.99; N, 11.88. Found: C, 54.30; H, 6.00; N,
11.83%. FAB-MS (magic bullet); m=z calculated most
abundant parent mass (M): 1305. Observed: 1235 (Mþ–
2Cl, 0.6%), 1198 (Mþ–3Cl, 1.2%).
350
400
450
500
550
600
Wavelength/nm
Fig. 2. Visible spectra of (H2O)2FeIIITanP at various pHs (6.00, 6.75,
7.59, 7.92, 8.00, 8.50, 8.62, 8.80, 9.15, 11.06) at 25 °C.

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H. Imai et al. / Inorganica Chimica Acta 357 (2004) 2503–2509
Table 1
pKa values of the coordinated H2O of ferric porphyrins (25 °C)
Compounda
Type
pKa
Reference
(H2O)2FeIIITanP
(H2O)2FeIIITPPS
(H2O)2FeIIITCPP
(H2O)2FeIIITCl2PPS
(H2O)2FeIIITMNP
(H2O)2FeIIITMpyP
(H2O)2FeIIITcatP
Anion
Anion
Anion
Anion
Cation
Cation
Cation
8.0
7.0
This workb
c
d
e
f
6.72
4.1
6.09
5.79
4.1
f
This workb
a The charge of the complexes is omitted. Abbreviations used for coordinated porphyrin dianions: TCPP: 5,10,15,20-tetrakis(4-carboxylatophe-
nyl)porphyrinato; TCl2PPS: 5,10,15,20-tetrakis((2,6-dichloro-3-sulfonato)phenyl)porphyrinato; TMNP: 5a,10b,15a,20 b-tetrakis(2-(N-methylnicot-
inamido)phenyl)porphyrinato; TmpyP: 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrinato.
b In 0.05 mol lꢁ1 NaClO4.
c A.D. El-Awady, P.C. Wilkins, R.G. Wilkins, Inorg. Chem. 24 (1985) 2053. In 0.1 mol lꢁ1 NaNO3.
d J.D. Strong, C.R. Hartzell, Bioinorg. Chem. 5 (1976) 219.
e S. Jeon, T.C. Bruice, Inorg. Chem. 31 (1992) 4843.
f Ref. [15].
for the range, respectively, in spite of the estimate that
electronic effects of the ionic groups on the central
metals through the many chemical bonds should be
negligible. The estimate is also supported by the fact
that the visible spectra of these ferric porphyrins are
very similar (see Section 2). In natural proteins, varia-
tion in pKa of amino acid residues resulting from dif-
ferences in polar microenvironments is commonly
observed [17]. In (H2O)2FeIIITanP, predicting from the
case of a similar zinc porphyrin, the coordinated H2O
must be stabilized by hydrogen bond(s) with the sulfo-
nato groups of the porphyrin [9b]. This weakens the
release of protons from (H2O)2FeIIITanP, leading to a
high pKa value (Chart 1). Further, the electrostatic re-
pulsion between the coordinated OHꢁ and the anionic
sulfonato groups in (H2O)(OH)FeIIITanP must also in-
crease the pKa. In contrast to this, the cationic ammo-
nium groups of (H2O)2FeIIITcatP should stabilize the
coordinated OHꢁ more than they stabilize the H2O,
based on Coulomb interaction as shown in Chart 1;
hence, the pKa value would become substantially small.
3.3. Binding of NO at pH 1.3
To examine NO binding to ferric porphyrins, the
experimental conditions were adjusted to be acidic
where the dominant species was determined from the
estimated pKa values to be (H2O)2FeIIIPor. Fig. 3 shows
the spectral changes of (H2O)2FeIIITcatP under various
NO pressures (PNO) at pH 1.3. The spectral changes
were found to be reversible: bubbling of Ar gas through
the solution under 708 Torr PNO resulted in re-formation
of the initial spectrum of (H2O)2FeIIITcatP. These
spectral changes with clear isosbestic points were similar
to those for FeIIITPPS [5] and corresponded to reaction
(1). The thermodynamic data for reaction (1) are sum-
marized in Table 2. It is generally accepted that NO-
bound ferric porphyrin species can be formulated as
linear Fe2þ–NOþ. In this case, the NO affinity of
(H2O)2FeIIITcatP
is
higher
than
that
of
(H2O)2FeIIITanP, due to the increased binding energy
(ꢁDH). This is in conflict with the prediction, based on
simple electrostatic interactions, that anionic groups
SO3
O3S
SO3
O3S
H
H
pKa 8.0
+ H+
O
OH
Fe
Fe
(H2O)2FeIIITanP
(
)
( )
N CH3
3
N CH3
(
)
(
)
3
N CH3
N CH3
3
3
pKa 4.1
H
H
+ H+
O
OH
Fe
Fe
(H2O)2FeIIITcatP
Chart 1.

H. Imai et al. / Inorganica Chimica Acta 357 (2004) 2503–2509
2507
1.2
0.8
0.4
TcatP (Chart 2). This explanation is also supported by a
recent report in which NO binding to ferric porphyrins
in aqueous solution was found to be accelerated by
labilizing the ligated H2O molecule [18].
3.4. Binding of NO at pH 3.0
The spectral changes of (H2O)2FeIIITanP under var-
ious PNO at pH 3.0 were almost identical to those at pH
1.3. The equilibrium constants were also the same,
within the range of experimental errors, at pHs between
1.3 and 3.0. However, as shown in Fig. 4, the spectral
changes of (H2O)2FeIIITcatP at pH 3.0 are quite dif-
ferent from those at pH 1.3. The spectral changes were
reversible, though isosbestic points were no longer ob-
served. Although the spectral changes under low PNO
may be similar to those at pH 1.3, a new species as
x 5
0.0
400
500
600
Wavelength/nm
Fig. 3. Visible spectra of (H2O)2FeIIITcatP under various PNO (0, 15.3,
29.1, 44.8, 59.2, 79.0, 152, 297, 506, 708 Torr) at pH 1.3 (25 °C).
the major product is apparently formed at higher PNO
.
Table 2
Thermodynamic data for NO binding to ferric porphyrins
The kmax values of 412 and 539 nm under PNO of 71.1
Torr are similar to those for ferrous NO species,
(NO)FeIITPPS. Next, for purposes of comparison, we
K/atmꢁ1c DH°/kJ molꢁ1
DS°/J molꢁ1 Kꢁ1
(H2O)2FeIIITanPa
(H2O)2FeIIITcatPa
(H2O)2FeIIITPPSb
a At pH 1.3.
b At pH 6.0; Ref. [8].
3.45
6.16
1.40
)55 ꢃ 1
)64 ꢃ 1
)62 ꢃ 2
)175 ꢃ 5
)200 ꢃ 4
)206 ꢃ 6
0.8
c Calculated at 25 °C from vanÕt Hoff plots.
should stabilize the bound NOþ and cationic groups
destabilize the NOþ. Our earlier study [9] on ligation of
amines to anionic zinc porphyrins containing sulfonato
groups indicated that upon amine binding, the release of
the coordinated H2O tightly bound by hydrogen bond-
ing decreases the amine affinity with increases in both
DH and DS. NO binding to the ferric porphyrins also
accompanies the release of the bound H2O. Therefore,
the release of the tightly bound H2O from (H2O)2
FeIIITanP provides some additional, positive DH and
DS, which may lead to the decreased NO affinity of
(H2O)2FeIIITanP as compared with that of (H2O)2FeIII-
0.4
x 5
0.0
400
500
600
Wavelength/nm
Fig. 4. Visible spectra of (H2O)2FeIIITcatP under various PNO (0, 6.6,
12.1, 17.3, 29.4, 36.6, 44.2, 51.4, 59.7, 67.2, 71.1 Torr) at pH 3.0
(25 °C).
SO3
O3S
SO3
O3S
Oδ
N
K = 3.45 atm-1
H
H
O
Fe
Fe
(H2O)2FeIIITanP
(
)
( )
N CH3
3
N CH3
(
)
( )
N CH3
3
N CH3
3
3
K = 6.16 atm-1
H
H
Oδ
N
O
Fe
Fe
(H2O)2FeIIITcatP
Chart 2.

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H. Imai et al. / Inorganica Chimica Acta 357 (2004) 2503–2509
Table 3
Visible spectral data for iron porphyrins
re-formed the initial high spin signal, indicating that the
reaction with NO is reversible. Therefore, together with
the visible spectral changes, the reaction of
(H2O)2FeIIITcatP with NO at pH 3.0 generates (NO)-
FeIITcatP reversibly. Notably, the oxidation state of the
central iron reversibly changes depending on PNO. The
reactions of (H2O)2FeIIITcatP with NO are explained
summary as follows:
Compound
pH
kmax/nm
(H2O)2FeIIITanP
(H2O)2FeIIITanP + NO
(H2O)2FeIIITcatP
1.3–3.0
1.3–3.0
1.3–3.0
395
422
395
422
412
420
412
526
535
526
535
539
532
540
(H2O)2FeIIITcatP + NO 1.3
(H2O)2FeIIITcatP + NO 3.0
(H2O)2FeIITcatPa
(H2O)2FeIITcatPa + NO 3.0
3.0
ðH2OÞ FeIIITcatP þꢀNO ðNOÞFeIIITcatP
a Prepared from (H2O)2FeIIITcatP with Na2S2O4 under Ar.
2
ꢁNO
þNO
ꢀ ðNOÞFeIITcatP þ NOþ
ð3Þ
ꢁNO
prepared (NO)FeIITcatP from NO and FeIITcatP that
had been obtained reductively from (H2O)2FeIIITcatP
with Na2S2O4. The kmax values for the porphyrins are
summarized in Table 3. These data suggested that re-
action of (H2O)2FeIIITcatP with NO at pH 3.0 affords
(NO)FeIITcatP reversibly.
Since the Fe(II)–NO bond is formulated as bent
Fe3þ–NOꢁ, the positively charged groups near the
bound NO should stabilize the (NO)FeIIPor, as shown
in Chart 3. The formation of (NO)FeIIPor in Eq. (3) is
thought to occur through liberation of NOþ from
(NO)FeIIIPor to produce labile FeIIPor species that
successively react with NO to give (NO)FeIIPor, where
H2O acts as the NOþ acceptor as exemplified by the
following equation [20]:
Use of ESR spectroscopy allowed us to distinguish
each possible compound of iron porphyrins (i.e.,
(H2O)2FeIIIPor, (NO)FeIIIPor, and (NO)FeIIPor spe-
cies). It is generally accepted that (NO)FeIIIPor is dia-
magnetic than ESR-silent, whereas (NO)FeIIPor shows
a characteristic ESR spectrum [6,19]. The ESR spectra
of the iron porphyrins were obtained at 77 K for frozen
aqueous solutions after treatment with NO or Ar gas at
room temperature. For (H2O)2FeIIITanP, the d5 high
spin signal (not shown) disappeared upon NO binding
and no signal was observed, which findings supported
the formation of (NO)FeIIITanP. In the case of
(H2O)2FeIIITcatP, as expected from visible spectral
changes, the ESR spectrum of the porphyrin treated
with NO at pH 3.0 was quite different from that of
(H2O)2FeIIITanP. Upon NO bubbling through
(H2O)2FeIIITcatP, the high spin spectrum disappeared
and the characteristic spectrum shown in Fig. 5
(g1 ¼ 2:10, g2 ¼ 2:01 (A ¼ 23 G), g3 ¼ 2:00) was ob-
served. The spectral feature and the spectral parameters
were very similar to those reported for five-coordinated
(NO)FeIIPor [19]. Further, purging the NO gas with Ar
NOþ þ H2O ꢀ HNO2 þ Hþ
ð4Þ
It is obvious that H2O cannot thermodynamically act
as the NOþ acceptor at a low pH. This is very reason-
ably correlated to the observation that the reaction of
(H2O)2FeIIITcatP with excess NO gives the reduced
2
(NO)FeIITcatP at pH 3.0 but not at pH 1.3. This re-
duction behavior may also be explained in terms of the
pH dependence of the half-cell potential between NO
ꢁ
and NO2 (or HNO2, NOþ); at low pH, the potential
acts such that the ferric NO species is predominant.
The reason for oxidation from (NO)FeIIPor to
(NO)FeIIIPor observed for FeTcatP but not for natural
hemeproteins is not yet clear. Taking into account that
the microenvironments around the central iron in the
hemeproteins must be hydrophobic, the oxidation will
correlate with the incompletely protected NO of (NO)-
FeIITcatP and/or with the acidic aqueous solution ex-
amined. One of the plausible routes of oxidation of
(NO)FeIIPor is through an attack of Hþ or NOþ, where
the latter may be formed in acidic aqueous solution as
3
follows:
NO þ NO2 þ 2Hþ ! 2NOþ þ H2O
ð5Þ
2
The pH dependence of NO-binding behavior of iron porphyrins
has been reported [7]. For FeTcatP, unfortunately, reaction with NO
at higher pH than 4 gave a precipitate that could not be examined
spectrophotometrically.
3
Although we further purified NO gas of high-purity grade, we
could not completely exclude the possibility that the NO gas contained
NO2 as an impurity. The importance of a trace amount of NO2 in NO
gas in the observed chemistry of iron porphyrins has been previously
pointed out [7,21].
Fig. 5. ESR spectrum of the NO-reacting product from (H2O)2
FeIIITcatP at 77 K in H2O; g1 ¼ 2:10, g2 ¼ 2:01 (A ¼ 23 G), g3 ¼ 2:00.

H. Imai et al. / Inorganica Chimica Acta 357 (2004) 2503–2509
2509
N+(CH3)3
Oδ
N+(CH3)3
N+(CH3)3
δ −O
N+(CH3)3
N
N
Fe
Fe
bent form (NO)FeIITcatP
linear form (NO)FeIIITcatP
Chart 3.
[5] M. Hoshino, K. Ozawa, H. Seki, P.C. Ford, J. Am. Chem. Soc.
115 (1993) 9568.
4. Concluding remarks
[6] (a) B.B. Wayland, L.W. Olson, J. Chem. Soc., Chem. Commun.
(1973) 897;
(b) B.B. Wayland, L.W. Olson, J. Am. Chem. Soc. 96 (1974) 6037.
The synthesized ferric porphyrins with negatively or
positively charged microenvironments near the central
metal provide some unique properties in equilibrium
and redox behavior. Both the pKa values of the coor-
dinated H2O and the NO affinities of these porphyrins
are reasonably explained on the basis of intramolecular
interactions. Further, it is likely that the positively
charged microenvironments near the central metal sta-
bilize the formed (NO)FeIIPor species. Thus, the present
work demonstrates that some change in microenviron-
ment near the active sites of both model and natural
compounds has the potential to give specific reactivity
for exogenous ligands.
ꢀ
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