ORIGINAL PAPER

Air-stable, heme-like water-soluble iron(II) porphyrin:in situ preparation and characterization

Robert Huszank Æ Gyorgy Lendvay Æ Otto Horvath

Received: 23 June 2006 / Accepted: 29 January 2007 / Published online: 28 February 2007

� SBIC 2007

Abstract Preparation of the water-soluble, kinetically

labile, high-spin iron(II) tetrakis(4-sulfonatophenyl)por-

phyrin, Fe(II)TPPS4–, has been realized in neutral or

weakly acidic solutions containing acetate buffer. The

buffer played a double role in these systems: it was used for

both adjusting pH and, via formation of an acetato com-

plex, trapping trace amounts of iron(III) ions, which would

convert the iron(II) porphyrins to the corresponding ir-

on(III) species. Fe(II)TPPS4– proved to be stable in these

solutions even after saturation with air or oxygen. In the

absence of acetate ions, however, iron(II) ions play a cat-

alytic role in the formation of iron(III) porphyrins. While

the kinetically inert iron(III) porphyrin, Fe(III)TPPS3–, is a

regular one with no emission and photoredox properties,

the corresponding iron(II) porphyrin displays photoinduced

features which are typical of sitting-atop complexes (red-

shifted Soret absorption and blueshifted emission and Q

absorption bands, photoinduced porphyrin ligand-to-metal

charge transfer, LMCT, reaction). In the photolysis of

Fe(II)TPPS4– the LMCT process is followed by detach-

ment of the reduced metal center and an irreversible ring-

opening of the porphyrin ligand, resulting in the degrada-

tion of the complex. Possible oxygen-binding ability of

Fe(II)TPPS4– (as a heme model) has been studied as well.

Density functional theory calculations revealed that in

solutions with high acetate concentration there is very little

chance for iron(II) porpyrin to bind and release O2, devi-

ating from heme in a hydrophobic microenvironment in

hemoglobin. In the presence of an iron(III)-trapping addi-

tive that is much less strongly coordinated to the iron(II)

center than the acetate ion, Fe(II)TPPS4– may function as a

heme model.

Keywords Iron porphyrins � Heme-model �Sitting atop � Catalysis � Photochemistry

Introduction

Heme proteins (containing iron porphyrin derivatives in

their active sites) play an essential role in living organisms

in oxygen transport and storage (hemoglobin, myoglobin)

and in electron transfer processes (cytochrome c, cyto-

chrome oxidase) [1, 2]. Hemoglobin contained within the

red blood cells holds four ferrous porphyrin type groups

which provide the active site for oxygen binding and are

responsible for the red color of blood. Simple compounds

that would bind oxygen reversibly for many cycles could

also be of value as emergency blood substitutes or for

fractionation of oxygen from air.

There have been many attempts to model the function of

hemoglobin and myoglobin (the oxygen–carbon dioxide

cycle) by use of some kind of oxygen carriers as blood

substitutes. In the last few decades, considerable efforts

were made to find compounds that are able to mimic the

hemoglobin function. Two classes of blood substi-

tutes—namely, perfluorocarbon-type derivatives [3–8] and

different human hemoglobin and bovine hemoglobin

R. Huszank � G. Lendvay � O. Horvath (&)

Department of General and Inorganic Chemistry,

University of Pannonia,

P.O. Box 158, Veszprem 8201, Hungary

e-mail: otto@vegic.vein.hu

G. Lendvay

Institute of Structural Chemistry,

Hungarian Academy of Sciences,

P.O. Box 17, Budapest 1525, Hungary

123

J Biol Inorg Chem (2007) 12:681–690

DOI 10.1007/s00775-007-0217-y

preparations [9–14]—have been tested in clinical therapy.

In spite of partial success, all of these compounds have

numerous disadvantages and show many side effects, so

they have not become widespread in medical practice. As

an alternative, preparation of the heme group [hydrophobic

or hydrophilic iron(II) porphyrin] synthetically as a possi-

ble blood substitute has also been investigated intensively

[15–22]. These studies were focused almost exclusively on

a nonaqueous environment (probably because of the

hydrophobic nature of the heme group), while the desired

simple oxygen carrier should be able to function in aqueous

media under physiological conditions.

Molecular oxygen has a triplet, paramagnetic ground

state and high electronegativity. These features make it

one of the most powerful oxidizing agents. Stable yet

reversible coordination of this strong oxidant to the ir-

on(II) central atom is rather difficult, because heme

compounds mostly react irreversibly with dioxygen. Al-

most all synthetically prepared iron(II) porphyrin com-

plexes in organic solvents were found to be very unstable

in air and are rapidly oxidized to ferric porphyrin, which is

biologically inactive [23]. This process is an irreversible

overall redox reaction:

4FeIIPþ O2 ! 2 FeIIIP� O� FeIIIP� �

: ð1Þ

The initial binding of molecular oxygen is followed by a

bimolecular redox process, which leads to the formation of

a dioxygen-bridged dimer complex. This l-peroxo dimer

can be converted via the formation of an oxo-ferryl inter-

mediate to the l-oxo dimer. This bridged species can fur-

ther decompose to iron(III) porphyrin.

In fact, the initial dimerization process takes place very

efficiently in organic media where the complexes are

not ionic, i.e., have no charge. The same reaction, how-

ever, is probably less favorable in aqueous—especially

dilute—solution, because the solubility of the complex is

generally ensured by ionic substituents on the porphyrin

ring, and then both iron porphyrin units have large positive

or negative charge and repel each other. Nevertheless, the

presence of water (even a trace amount) causes autoxida-

tion in most iron(II) porphyrins coordinating dioxygen

[24]. In aqueous systems, even denatured hemoglobin

proved to be extremely oxygen sensitive after losing the

hydrophobic microenvironment ensured by the globin

moiety for the iron(II) center. This phenomenon has been

attributed to the nucleophilic attack of water molecules at

the iron–oxygen coordinative bond, excluding the O2•–

species formed in inner-sphere electron transfer from the

iron(II) center. Accordingly, iron(II) porphyrins in aqueous

media have been mostly studied in supramolecular systems

of biological origin [24]. Quite recent investigations indi-

cated that a possible way to prepare iron(II) porphyrins in

aqueous systems is in situ reduction of a water-soluble

iron(III) porphyrin encapsulated in an O-methylated

b-cyclodextrin dimer ensuring a hydrophobic microenvi-

ronment for the metal center [25]. This system proved to be

relatively stable against autoxidation in the presence of

dioxygen. However, preparation of air-stable iron(II) por-

phyrin in a real hydrophilic environment required a dif-

ferent strategy [26, 27]. In addition, the conversion of

ferrous porphyrin to ferric porphyrin in aqueous solution

has not yet been explained.

Besides the significant biological function of ferrous

porphyrin derivatives, the description of thermal and pho-

toinduced properties of water-soluble complexes would

also be important in bioinorganic chemistry. Nowadays,

interest in metalloporphyrins keeps increasing, owing to

their importance in many fields, such as biochemistry,

medical research and catalysis. The photophysical and

photochemical properties of excited metalloporphyrins

have been utilized, e.g., in optical sensors [28–30], artificial

photosynthetic systems (light-harvesting dendrimers) [31]

and photodynamic therapy [32–34].

The main subject of this paper is a report on the prep-

aration of iron(II) tetrakis(4-sulfonatophenyl)porphyrin,

Fe(II)TPPS4–, in aqueous solution so that the complex

formed is stable and is not oxidized in the presence of

oxygen. Production of Fe(II)TPPS4– was realized by the

complexation reaction of free-base porphyrin (H2TPPS4–)

and iron(II) ions in the presence of iron(III)-trapping ace-

tate buffer. In the following, after summarizing the tech-

niques used, we present the preparation method, then the

equilibrium, photophysical and photochemical properties

of the complex, according to which the complex can be

classified as a sitting-atop metal porphyrin. Finally, we

discuss whether this complex can serve as a simple heme

model with respect to reaction with dissolved molecular

oxygen.

Materials and methods

Materials

All the solutions studied were made by using ion-

exchanged then distilled Milli-Q water. H2TPPS4– and

FeSO4�7H2O were purchased from Aldrich. The iron(III)

porphyrin [iron(III)meso-tetrakis(4-sulfonatophenyl)por-

phyrin] was from Frontier Scientific Europe. In order to

hinder the formation of iron(III) ions, argon was bubbled

and metallic iron was added into the iron(II) sulfate solu-

tion. The pH values of 6 and 7 were obtained by acetate

buffers made from CH3COONa and CH3COOH in the

appropriate ratio. The pH was measured with a pH meter

using a glass electrode. For the exclusion of oxygen,

682 J Biol Inorg Chem (2007) 12:681–690

123

99.996% argon gas and the Schlenck technique were used

where necessary.

[Fe(II)TPPS4–, 4Na+]

The water-soluble porphyrin was synthesized in the equi-

librium reaction of 1.5 · 10–4 M Fe(II) and 3 · 10–6 or

5 · 10–6 M free-base porphyrin in aqueous, argon-satu-

rated solution at pH 6 and 7. The solutions also contained

0.3 M acetate buffer to adjust the pH and to trap trace

amounts of iron(III) ions, and, in addition, 1 M NaCl at pH

7 to increase the ionic strength. In the first step of the

process, the iron(II) and the acetate solutions were mixed

and then immediately degassed with argon gas (for about

30 min). At pH 7, NaCl was also added in the first step.

Subsequently, the mixture was kept under argon for 4 h.

Then porphyrin was added, also under oxygen-free condi-

tions. The system was allowed to react for at least 2 days at

room temperature. The spectral change of the starting

system (containing H2TPPS4–) indicates the formation of

the complex (details in ‘‘Formation of Fe(III)TPPS3– and

Fe(II)TPPS4– in water’’).

Apparatus

All the absorption and emission spectra were recorded with

a Specord S100 UV–vis one-way spectrophotometer and

with a PerkinElmer LS50 spectrofluorimeter, respectively.

Nanosecond emission lifetimes were measured using

PicoQuant time-correlated single photon counting appara-

tus. For the assessments and data fitting, deconvolution

software was used (FluoFit from PicoQuant). For contin-

uous photolysis at different wavelengths an AMKO LTI

system (consisting of a 150-W high pressure Xe–Hg arc

lamp and a monochromator) was utilized. The light

intensity was determined with a thermopile calibrated by

ferrioxalate actinometry. Quantum yield measurements

were carried out with samples of nearly 100% light

absorption. The concentration of the known porphyrin

species in the solutions was determined by using their

molar absorption coefficients, utilizing a least-squares fit-

ting method on the 350–500-nm region of the spectra.

Theoretical methods

Density functional theory (DFT) was used for the theoret-

ical determination of the structures and energies of iron(II)

porphyrin and its derivatives containing other ligands in the

singlet, triplet and quintet states. We used the B3LYP

[35–37] combination of functionals as coded in Gaussian98

[38], in conjunction with the LANL2DZ [39–42] basis set.

This combination proved to be adequate in numerous

related applications in the literature [43]. We made tests

using other basis sets, including 3–21G* and 6–31G*, and

found that the computed structures remain essentially the

same. Where comparison was possible, our results agree

well with those of earlier calculations [43, 44]. The

molecular geometries presented are true minima as checked

by calculation of vibrational frequencies. For the compari-

son of the energies of complexes with various spin multi-

plicity, the DSCF method was used; i.e., the structures and

energies were calculated using the restricted self-consistent-

field (SCF) procedure (RB3LYP) for the singlet species and

the unrestricted method (UB3LYP) for the open-shell

molecules, and the energies were directly compared. The

convergence of both the SCF iteration and the geometry

optimization was very slow, so level shifting, enforcing and

relaxing symmetry and change of occupation of orbitals

were necessary to satisfy tight convergence criteria.

Results

Formation of Fe(III)TPPS3– and Fe(II)TPPS4– in water

The formation of the porphyrin complexes was detected by

spectrophotometry. Figure 1 shows the spectra of the

complexes formed observed in an aqueous solution con-

taining 3 · 10–6 M H2TPPS4– and 1.5 · 10–4 M iron(II)

sulfate under two different conditions: without any buffer

and with 0.3 M acetate buffer.

In the absence of acetate buffer, the observed spectrum

is identical with that of Fe(III)TPPS3– (which in aqueous

solution exhibits peaks at 393, 528 and 651 nm [26, 45]).

In the presence of acetate buffer we detected a different

spectrum which is not identical with that of either iron(III)

porphyrin or free-base porphyrin. The characteristics of

this new spectrum are in very good agreement with those

observed in the in situ reduction of iron(III) porphyrin [26].

That spectrum was assigned to iron(II) porphyrin. We are

convinced this complex is formed in our reaction because

there is no other metal ion coordinating to porphyrin in the

solution except Fe2+.

In the reaction of free-base porphyrin and any kind of

iron(II) salt, ferric porphyrin was the final product in all

cases reported so far. On the basis of the latter observa-

tions, it was believed that iron(II) porphyrin was formed in

the first step but it was immediately oxidized to iron(III)

porphyrin by molecular oxygen. However, when using the

same system as in the earlier experiments, we removed

even trace amounts of dissolved oxygen by the Schlenk

technique (at least five degassing cycles), and the pre-

dominant part of the complex formed was iron(III) por-

phyrin. We conclude from this that iron(III) porphyrin

cannot be the result of oxidation of iron(II) porphyrin by

dissolved oxygen.

J Biol Inorg Chem (2007) 12:681–690 683

123

Since we found that in all preparation procedures of

iron(III) porphyrins that have been reported in the literature

the starting materials were iron(II) compounds and free-

base porphyrin [26, 46, 47], we checked whether Fe3+ ions

react with free-base porphyrin at all. We did not observe

complexation between ferric ions and free-base porphyrin

even when they were allowed to react for 1 week; hence, a

reaction different from the reaction in Eq. 1, suggested in

the literature, must be responsible for the formation of

iron(III) porphyrin.

We think that Fe2+ ions catalyze the reaction of free-

base porphyrin and Fe3+ ions, trace amounts of which are

always present in iron(II) solutions. It is known that for-

mation of kinetically inert metalloporphyrins can be cata-

lyzed by metal ions such as Hg(II), Cd(II), Pb(II) and

Cu((II), via formation of kinetically labile sitting-atop

complexes [48]. In the case of the relatively large ionic

radius, the metal center cannot fit into the core of the

porphyrin; hence, it is localized out of the ligand plane,

distorting it. Such a distortion makes the porphyrin co-

ordinatively more active. In our case, iron(II) porphyrin is

formed in the first step and two new coordination positions

arise on the other side of the porphyrin plane (on two

diagonally situated nitrogen atoms) and become accessible

for another metal ion, e.g., iron(III). Following the coor-

dination of iron(III), it excludes the iron(II) ion bound more

weakly on the other side of the porphyrin, and occupies the

very center of the ligand. Scheme 1 is a simplified sketch

of this latter stage in the catalytic process of the formation

of iron(III) porphyrin (the distortion of the porphyrin ring

is deliberately exaggerated).

Since the order of magnitude for the concentration of the

porphyrin in our experiments was only 10–6 M, in the ab-

sence of acetate buffer even trace amounts of iron(III), as

impurities in the iron(II) starting material, proved to be

enough for the efficient formation of Fe(III)TPPS3–, which

is a kinetically inert complex.

In the presence of acetate buffer, however, most iron(III)

ions are captured via formation of very stable polynuclear

acetato complexes [49]. This is the main reason for the

formation and stability of Fe(II)TPPS4– in the solutions

buffered with acetate.

An analogous ‘‘self-catalytic’’ effect of copper(I) was

observed [50] in the case of the formation of a copper(II)

porphyrin, where addition of metallic copper accelerated

the synthesis by a factor of 104. In this case also a copper(I)

complex formed in a synproportionation reaction plays the

role of the catalyst [51].

An alternative route to the formation of iron(III) por-

phyrin could be an electron transfer from the iron(II) metal

center (in the metalloporphyrin) to an approaching iron(III)

ion from the bulk. Such a reaction cannot be excluded on

the basis of the standard redox potentials, E(FeIII(H2O)n/

FeII(H2O)n) = 0.77 V [52] and E([FeIII(H2O)TPPS]3–/

[FeII(H2O)TPPS]4–) = 0.01 V [26]. However, we found

that Fe(II)TPPS4– is not sensitive to oxygen in the presence

of acetate ions (see later). If O2, which is a much stronger

oxidizing agent than iron(III), cannot oxidize the iron(II)

complex, we think that electron transfer from Fe(II)TPPS4–

to Fe3+ is not favored either. The catalytic activity of larger

metal ions, which cannot undergo oxidation (e.g., Hg2+,

Cd2+) in the formation of regular metalloporphyrins (see

above) also seems to support the substitution mechanism

[48]. Accordingly, Hg2+ ion as a catalyst was successfully

utilized in the preparation of the iron(III) complex of meso-

BrTPPS6– [53].

It is worth noting that in the presence of a reducing agent,

such as ascorbic acid, we got the same spectrum as in

solutions containing acetate buffer, indicating the formation

of Fe(II)TPPS4–. This result, however, does not necessarily

mean that ascorbic acid reduces the iron(III) porphyrin

formed; rather, it reacts with the traces of iron(III) ions,

Scheme 1 Simplified demonstration of the formation of ferric

porphyrin from a temporarily existing ferrous porphyrin

Fig. 1 Absorption spectra of the iron(II) porphyrin complex in a

solution containing 0.3 M acetate buffer (red lines) compared with

those of the free-base porphyrin (black lines) and the iron(III)

porphyrin complex (blue lines). TPPS tetrakis(4-sulfonatophenyl)

porphyrin

684 J Biol Inorg Chem (2007) 12:681–690

123

forming a complex or reducing it to iron(II), and blocks

them in a similar way to acetate ions. It should also be

mentioned that in situ formation of iron(II) porphyrin was

observed in the presence of nitrogen monoxide [54].

The absorption spectrum of Fe(II)TPPS4– in aqueous

solution containing 0.3 M acetate buffer exhibits strong

peaks at 421, 557 and 598 nm (for more detailed charac-

terization see ‘‘Photophysical studies’’). This compound

proved to be kinetically labile. Its formation reaction is

described by Eq. 2. The apparent equilibrium constant (K¢)for this reaction can be expressed by the actual concen-

trations, according to the law of mass action (Eq. 3). Since

constant pH was applied in all solutions studied, the effect

of H+ concentration is not taken into account in this case.

Fe2þ þ H2TPPS4�$K Fe(II)TPPS4� þ 2Hþ ð2Þ

K 0 ¼ K

[Hþ]2¼ [Fe(II)TPPS4�]

[Fe2þ][H2TPPS4�]ð3Þ

a0 ¼[H2TPPS4�]

[H2TPPS4�]þ [Fe(II)TPPS4�]¼ 1

1þ K 0[Fe2þ]

ð4Þ

Thus, the partial molar ratio of the free-base porphyrin (a0)

can be given as a function of Fe2+ concentration (Eq. 4).

Hence, 1/a0 is in linear correlation with the Fe2+ concen-

tration, and the slope of this straight line is K¢. Accord-

ingly, K¢ can be estimated from the spectra of solutions

containing constant porphyrin concentrations and various

concentrations of iron(II), evaluating the absorbances in the

range of the Soret bands (Fig. 2). Since in these solutions,

in spite of the presence of the acetate buffer, also some

iron(III) porphyrin was formed, during the evaluation the

individual concentrations of H2TPPS4–, Fe(II)TPPS4–,

Fe(III)TPPS3– and Fe2+ were determined by nonlinear

least-squares fitting with the Solver procedure of a

Microsoft Excel program, using the known individual

molar absorbances at several wavelengths. Taking the

actual values of the concentrations of H2TPPS4–,

Fe(II)TPPS4– and Fe2+, we calculated 1/a0 for each solu-

tion and plotted it as a function of Fe2+ concentration. The

slope of the line fitted to this plot gave the equilibrium

constant, K¢ = 2.3±0.6 · 104 M–1.

Photophysical studies

The absorption spectra of the free-base ligand (H2TPPS4–)

and the ferric and the ferrous porphyrins are shown in

Fig. 1. In the case of porphyrins, the absorption bands with

very high molar absorption coefficient can be assigned to

p–p* transitions. It is known from the literature that the Q

bands (first singlet excited state, S1) of the free-base por-

phyrins are split into Qx and Qy bands because of the

symmetry reduction caused by the two protons on the

imido nitrogens [55]; hence, this kind of splitting is ex-

pected to disappear in metalloporphyrins. A more detailed

analysis of the absorption spectrum, however, indicates

that in iron porphyrins the splitting of the Q bands does not

totally disappear. This suggests that the four metal–nitro-

gen bonds are not completely identical.

The ferric porphyrin’s B(0,0), or Soret, band (second

singlet excited state, S2) is at 393 nm, the more intensive

Qy bands are at 528 and 498 nm and the Qx bands are at

650 and 686 nm. The Soret and Qx bands are blueshifted,

while the Qy peaks are redshifted compared with the

corresponding ones of the free-base porphyrin (kB ¼412 nm, kQy

¼ 516; 555 nm, kQx¼ 582, 635 nm): This

regular (coplanar) paramagnetic porphyrin complex does

not show any luminescence.

The ferrous porphyrin displays an intensive B(0,0)

absorption band at 421 nm and a weak B(1,0) band at

400 nm; the Q(0,0) and Q(1,0) bands are at 597 and

556 nm. Two further weak bands can be seen at about 520

and 480 nm which can be assigned as Qy bands. The Soret

band is redshifted, while the Q bands are blueshifted

compared with those of the free ligand. The absorption

spectrum of Fe(II)TPPS4– is very similar to that of the

deoxyhemoglobin [56], in which the central ion lies 0.4 A

above the porphyrin plane [52].

Deviating from the corresponding iron(III) complex, the

ferrous porphyrin shows characteristic emissions (Fig. 3).

The intensive fluorescence bands (S0 ‹ S1) at 608 and

656 nm can be assigned to the transitions to different

vibrational levels of the electronic ground state. Figure 4

shows the complete energy-level diagram of Fe(II)TPPS4–

we propose. Since this complex does not phosphoresce, the

diagram shows only estimated energy levels for the triplet

excited states.

Fig. 2 Absorption spectra of solutions containing 1.5 · 10–6 M

porphyrin, 0.3 M acetate buffer (pH 6) and various concentrations

of iron(II) (designated by the CFe(II)/CTPPS values). Insert: 1/a0 versus

Fe2+ concentration for the determination of K¢ (see the text for details)

J Biol Inorg Chem (2007) 12:681–690 685

123

The quantum yields of fluorescence (FS1) for Q band

and Soret (B) band excitations are shown in Table 1. The

S1 fluorescence rate (kS1) and the corresponding radiative

lifetime (sS1) for the Fe(II)TPPS4– and H2TPPS4– species

were calculated for each complex using the measured de-

cay time (sm) and quantum yield (FS1). It is seen that while

the measured fluorescence lifetime of the iron(II) porphyrin

complex decreases compared with that of H2TPPS4–, the

radiative lifetime of the S1 excited state increases. This

means that ferrous porphyrin can release the excitation

energy also in some other, nonradiative ways.

A weak emission was also observed at 433 nm upon

excitation at 390 nm. This band can be assigned to the

S0 ‹ S2 transition. This kind of emission is scarcely de-

tected but it was also seen for the hydrophobic Zn(II)TPP

and H2TPP species (H2TPP is tetraphenylporphyrin) [59],

as well as for water-soluble Tl(III)TPPS3– and H2TPPS4–

[57]. The quantum yield of the emission from the S2 excited

state (FS2) is extremely low owing to its very short lifetime.

The moderately high quantum yields for the S1 emis-

sion (fluorescence) of Fe(II)TPPS4– (FS1 = 9.5 · 10–3 and

7.0 · 10–3), in accordance with the relatively long life-

times (in the nanosecond range), may be surprising be-

cause metalloporphyrins with open-shell metal centers are

generally nonfluorescent or extremely weakly fluorescent

with quantum yields below 10–4 [60, 61]. However, even

paramagnetic metallaporphyrins display considerable flu-

orescence as the examples of manganese(II) [62] and

lanthanide(III) [63] porphyrins indicate (FS1 < 10–3). The

rather low fluorescence quantum yields in the case of

regular in-plane metalloporphyrins are attributed to a

partial overlap of the eg(dp) orbitals of the ligand with the

nd orbitals of the central metal ion, leading to increased

spin–orbit interaction, which decreases the lifetimes of the

excited states, promoting radiationless transitions [64]. In

the case of water-soluble out-of-plane metalloporphyrins,

however, owing to the larger distance between the ligand

and the metal center, the electrons of the latter cause only

a minor perturbation and the photophysical properties of

these complexes are determined essentially by the por-

phyrin ring’s p electrons and the distortion of the plane,

affecting the energy levels of the delocalized p and p*

frontier orbitals. This interpretation has been confirmed

by photophysical studies of several heavy-metal por-

phyrins, such as Pd(II)TPPS4–, Pt(II)TPPS4– [65],

Hg(II)TPPS4– [66] and Tl(I)2TPPS4– [58]. The last two

complexes are known to be characterized by a sitting-atop

structure, i.e., the metal atom is out of the porphyrin

plane. The fluorescence quantum yields for these com-

plexes are of the same order of magnitude as for the free-

base porphyrin. These water-soluble complexes have

similar absorption spectra and exactly the same type

of emission spectra as Fe(II)TPPS4– (the corresponding

emission bands are at about the same wavelengths),

Fig. 3 Uncorrected emission

spectra of H2TPPS4– (brokenline) and Fe(II)TPPS4–

(continuous lines) in 0.3 M

acetate buffer (pH 6)

Fig. 4 Energy-level diagram and electron transitions of Fe(II)TPPS4–

in water. The energies of the triplet states and the ligand-to-metal

charge transfer (LMCT) state are only estimated

686 J Biol Inorg Chem (2007) 12:681–690

123

indicating the similar structure of the latter. In accordance

with the interpretation above, for Tl(III)TPPS3– [57] the

fluorescence quantum yield is 2 orders of magnitude less

than for the previous complexes. In this case, as a con-

sequence of the smaller size of the metal center, it is

close to in-plane position, resulting in stronger spin-orbit

coupling.

Photolysis of Fe(II)TPPS4–

Irradiation of the iron(II) porphyrin at both the Soret band

(421 nm) and a Q band (556 nm) resulted in the degrada-

tion of the complex itself (Fig. 5), accompanied by the

formation of ring-opened tetrapyrrole derivatives (bile-type

pigments) as indicated by the new bands at 390 and

450 nm [67]. The same types of products were obtained in

photolyses of other sitting-atop porphyrin complexes of

Tl(I), Tl(III) and Hg(II) [58, 66, 68] as well as in the case

of photobleaching of H2TPPS4– [69]. The irradiated ferrous

porphyrin was transformed almost completely during a 40-

min illumination period.

The quantum yields for the degradation are summarized

in Table 1. The photochemistry of this complex is char-

acterized by a porphyrin ligand-to-metal charge transfer

(LMCT) reaction, similarly to the photoinduced reactions

in the case of sitting-atop metalloporphyrins with a metal

center other than iron(II) (Tl+, Hg2+) [57, 66]. Irradiation of

H2TPPS4– under the same conditions results in degrada-

tion, the quantum yield of which is 2 orders of magnitude

lower than that observed for the ferrous porphyrin

(Table 1). In the absence of a metal center, no LMCT

process can occur. In the case of the sitting-atop com-

plexes, the LMCT process takes place in an indirect way,

because appropriate absorption bands do not appear in the

absorption spectra. Thus, the excited electron must come

from one of the orbitals of the porphyrin ligand to the iron

center. Since the rate of the phototransformation does not

decrease significantly in the presence of an efficient triplet

Table 1 Dynamic photophysical parameters of H2TPPS4- and Fe(II)TPPS4-

Complexes FS1(10–2)a sm(ns) kS1(106s–1)b sS1(ns)c Fdegradation(10–4)d FS2(10–5)e Stokes shifts (cm–1)

H2TPPS4– 7.5 [57] 9.99 7.5 133 0.06 [58] –f 253.6

Fe(II)TPPS4– 0.95, 0.7 1.97 4.83 207 9.7, 7.8 6.3 322.4

TPPS tetrakis(4-sulfonatophenyl)porphyrina The quantum yields of emission from the S1 state were calculated using the emission quantum yield of a known reference material such as

Ru(2,2¢-bipyridyl)32+ complex. The excitation wavelengths for Fe(II)TPPS4– at both the Q band and the Soret band, in this order

b kS1 ¼ US1=sm

c sS1 ¼ kS1�1

d Calculated from the photoinduced disappearance of the complex at the wavelengths for Fe(II)TPPS4– at both the Q band and the Soret band,

respectivelye The quantum yields of emission from the S2 state were calculated using the quantum yield of the S1 emission (FS1) and the ratio of the two

measured peaks’ integral areaf Extremely weak S2 fluorescence for H2TPPS4– was also observed, but its quantum yield was not determined [57]

Fig. 5 Photodegradation of

Fe(II)TPPS4– (3 · 10–6 M)

upon irradiation in 0.3 M

acetate buffer in a period

of 40 min

J Biol Inorg Chem (2007) 12:681–690 687

123

quencher (dissolved molecular oxygen), the LMCT process

must occur through the S1 and S2 singlet excited states

(Fig. 4).

In contrast, irradiation of the iron(III) porphyrin, under

the same conditions, caused no permanent chemical change

in the system, even if a similar LMCT reaction may take

place in the primary photochemical step. The reason for

this is that the regular in-plane metalloporphyrins can be

reversibly oxidized [70], because the temporarily formed pcation is very stable and the electron promoted to the metal

center has enough time to be transferred back to the por-

phyrin ligand. In contrast, in the case of sitting-atop por-

phyrins, after the LMCT reaction, the reduced metal ion,

[Fe(I) in our case] can be easily detached because of the

kinetic lability of this complex, and, thus, the electron

cannot be transferred back.

Scheme 2 demonstrates the assumed key step of the

photoinduced reaction of the iron(II) porphyrin.

Fe(II)TPPS4– as a possible heme model

For Fe(II)TPPS4– to be useful as a water-soluble heme

analog, it should be able to bind and release molecular

oxygen. To see if this is possible, after the Fe(II)TPPS4–

complex had been formed, the solution was saturated with

oxygen. The ferrous species proved to be stable against

oxidation; no conversion to the ferric form was observed

even over weeks. Besides, after the saturation of the

solution with oxygen, the spectra of the iron(II) porphyrin

complex did not change. This means that either molecular

oxygen does not coordinate to the ferrous porphyrin under

such conditions, or this coordination does not cause spec-

tral changes.

A possible reason why O2 is not coordinated to

Fe(II)TPPS4– is that the latter is more strongly coordinated

by some other ligands present in the solution used for the

synthesis of Fe(II)TPPS4–, so molecular oxygen cannot be

connected to the iron ion. In order to see how strongly the

most abundant ligands in the solution are bound to

Fe(II)TPPS4–, we performed DFT calculations using ir-

on(II) porphyrin as a model compound, denoted in the

following as FeP. We explored in some cases how much

the energetics is influenced by the presence of the four

phenyl groups of TPP, and found that the changes are small

and we can rely on the data obtained with the bare por-

phyrin ring. We investigated the binding energy of H2O, O2

and CH3COO– (denoted as Ac) to FeP in the singlet, triplet

and quintet states. As the iron(II) porphyrin molecule is

assumed to have two unoccupied coordination sites, not

only its 1:1 complexes with each ligand were studied, but

also all possible combinations of them. We found that for

all complexes except FeP and FeP–Ac–O2 the quintet state

is the ground state. Selected binding energies are listed in

Table 2.

In most cases the binding energies in ‘‘binary’’ FeP–L

complexes are found to change monotonously with the spin

multiplicity of the system. The acetate ion was found to

form the strongest bond with the iron(II) center (its binding

energy is 45–36 kcal mol–1 to the singlet and the quintet

iron porphyrin, respectively). The binding energy of the

first H2O ligand is much smaller, between 27 and

15 kcal mol–1, depending on the multiplicity. The binding

energy of 3O2 to FeP is around 10 kcal mol–1. The second

ligand is always connected to the opposite side of the

porphyrin plane compared with the first ligand, and is

generally much less strongly connected to the iron(II)

center than in the absence of the other ligand. For example,

the binding energy of the acetate ion in FeP–H2O.Ac is

about 13 kcal mol–1 smaller than in FeP–Ac, in all three

electronic states. In the binary FeP–H2O complex in all

electronic states and in the singlet and triplet states of the

ternary FeP–Ac–H2O complex, the H2O molecule, as ex-

pected, is connected to the iron through one of the lone

pairs of the oxygen atom. In contrast, in the quintet state of

Scheme 2 Simplified demonstration of the mechanism for the photoinduced degradation of iron(II) porphyrin. For the sake of simplicity, the

ionic substituents are designated by dashed lines)

Table 2 The dissociation energy (kcal mol–1) obtained in density

functional theory calculations for binary complexes of iron(II) por-

phyrin (FeP) with H2O, CH3COO– (Ac) and 3O2 as well as the energy

needed to remove a H2O or 3O2 ligand from their ternary complexes

containing Ac. See the text for details of the calculations

FeP–H2O FeP–Ac FeP–O2 FeP–Ac–H2O FeP–Ac–O2

S 26.6 45.3 11.4 13.7 –9.2

T 19.7 31.5 8.6 8.9 7.2

Q 14.9 36.3 12.4 11.2 0.1

688 J Biol Inorg Chem (2007) 12:681–690

123

FeP–Ac–H2O the hydrogen atoms of the water molecule

are closer to the FeP unit than its oxygen atom, and point

towards two neighboring nitrogen atoms of the porphyrin

frame, forming two O–H–N hydrogen bonds instead of a

donor–acceptor bond with the iron. This indicates that in

this state the iron(II) center is coordinatively saturated by

the acetate ligand.

The most stable binary complex is definitely FeP–Ac.

We can assume that FeP is in the quintet ground state in

solution. The coordination of H2O and acetate to FeP will

very probably not change the spin multiplicity of the

FeP complex, and they are bound to FeP by 15 and

36 kcal mol–1, respectively. The oxygen molecule is in

the triplet state in solution, and it will not necessarily

form the thermodynamically most stable complex (the

quintet state). If 3O2 forms a triplet complex with FeP, its

binding energy will be about 9 kcal mol–1; if a quintet

complex is formed, about 13 kcal mol–1 will be released.

This suggests that most FeP will be complexed by acetate,

and the least probable first ligand is the oxygen molecule.

Should ternary complexes be formed, H2O as a ligand

will be preferred to O2, because it is much more strongly

bound to FeP–Ac, especially in the quintet state. This

means that in solutions with high acetate concentra-

tion—as in our case where it is used to mask the Fe3+

ions—there is very little chance that FeP, and very

probably also Fe(II)TPPS4–, will be able to bind and

release O2 as in heme. Fe(II)TPPS4– may be used as an

oxygen carrier if one is able to mask Fe3+ with an

additive that is much less strongly coordinated to the

iron(II) center in Fe(II)TPPS4– than the acetate ion.

Conclusion

Preparation of a water-soluble, kinetically labile iron(II)

porphyrin has been realized in neutral or weakly acidic

solutions. The role of this complex in the synthesis of the

corresponding iron(III) porphyrin has also been elucidated.

Besides photophysical and photochemical characterization

of Fe(II)TPPS4–, its possible oxygen-binding ability (as a

heme model) has been studied as well.

In the reaction of free-base porphyrin and iron(II) ions,

ferric porphyrin was the final product even in deoxygen-

ated solutions because from the temporarily formed, ki-

netically labile iron(II) porphyrins the Fe(II) centers were

excluded by iron(III) ions existing or formed in these

systems. Moreover, this is the only way for formation of

iron(III) porphyrin because no direct reaction between free-

base porphyrin and iron(III) occurs. From this one can also

infer that iron(II) ions catalyze the formation of iron(III)

porphyrins. The synthesis of the Fe(II)TPPS4– complex

could only be realized in the presence of acetate buffer

capturing iron(III) ions. In buffered solutions, Fe(II)TPPS4–

proved to be stable even in the presence of molecular

oxygen.

The photophysical properties of the iron(II) porphyrin

complex consist of typical ‘‘sitting-atop’’ behavior (red-

shifted Soret absorption and blueshifted emission and Q

absorption bands, irreversible photoinduced porphyrin

LMCT reaction), while the corresponding iron(III) por-

phyrin is a regular one with no emission and photoredox

properties.

DFT calculations indicated that in solutions with high

acetate concentration—as in our case where it is used to

mask the Fe3+ ions—there is very little chance for

iron(II) porphyrin to bind and release O2 as heme does

in a hydrophobic microenvironment in hemoglobin.

Fe(II)TPPS4– can probably be used to model heme in

the presence of an iron(III)-trapping additive that is much

less strongly coordinated to the iron(II) center than the

acetate ion.

Acknowledgement This work was supported by the Hungarian Re-

search Fund (OTKA K63494, T049257).

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