Solution behavior of iron(iii) and iron(ii) porphyrins in DMSO and reaction with superoxide. Effect of neighboring positive charge on thermodynamics, kinetics and nature of iron-(su)peroxo product

Article abstract of DOI:10.1039/c1dt11521a

The solution behavior of iron(iii) and iron(ii) complexes of 5 ⁴,10⁴,15⁴,20⁴-tetra-tert-butyl-5,10, 15,20-tetraphenylporphyrin (H₂tBuTPP) and the reaction with superoxide (KO₂) in DMSO have

Full text of DOI:10.1039/c1dt11521a

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PAPER
Solution behavior of iron(III) and iron(II) porphyrins in DMSO and reaction
with superoxide. Effect of neighboring positive charge on thermodynamics,
kinetics and nature of iron-(su)peroxo product†
K. Duerr,a O. Troeppner,a J. Olah,‡b J. Li,a A. Zahl,a T. Drewello,a N. Jux,a J. N. Harveyb and
I. Ivanovic´-Burmazovic´*a
Received 12th August 2011, Accepted 26th September 2011
DOI: 10.1039/c1dt11521a
The solution behavior of iron(III) and iron(II) complexes of 54,104,154,204-tetra-tert-butyl-5,10,15,20-
tetraphenylporphyrin (H2tBuTPP) and the reaction with superoxide (KO2) in DMSO have been studied
in detail. Applying temperature and pressure dependent NMR studies, the thermodynamics of the
low-spin/high-spin equilibrium between bis- and mono-DMSO FeII forms have been quantifi◦ ed
(KDMSO = 0.082 0.002 at 298.2 K, DH◦ = +36 1 kJ mol-1, DS◦ = +101 4 J K-1 mol-1, DV
=
+16 2 cm3 mol-1). This is a key activation step for substitution and inner-sphere electron transfer. The
superoxide binding constant to the iron(II) form of the studied porphyrin complex was found to be (9
0.5) ¥ 103 M-1, and does not change significantly in the presence of the externally added crown ether in
DMSO (11 4) ¥ 103 M-1. The rate constants for the superoxide binding (kon = (1.30 0.01) ¥ 105 M-1
s-1) and release (koff = 11.6 0.7 s-1) are not affected by the presence of the external crown ether in
solution. The resulting iron(II)-superoxide adduct has been characterized (mass spectrometry, EPR,
high-pressure UV/Vis spectroscopy) and upon controlled addition of a proton source it regenerates the
starting iron(II) complex. Based on DFT calculations, the reaction product without neighboring
positive charge has iron(II)-superoxo character in both high-spin side-on and low-spin end-on forms.
The results are compared to those obtained for the analogous complex with covalently attached crown
ether, and more general conclusions regarding the spin-state equilibrium of iron(II) porphyrins, their
reaction with superoxide and the electronic structure of the product species are drawn.
stability, its characterization could only be achieved at subzero
Introduction
temperatures, both in enzymatic and porphyrin model systems.2,3
Even more difficult is the trapping and characterization of a
monoprotonated iron(III)-hydroperoxo form, which is a short-
lived intermediate on a way to an iron(IV)-oxo species, which
results from O–O bond cleavage assisted by a second protonation
step involving the same (distal) oxygen atom. Preparation of
the iron(III)-hydroperoxo complex has been recently reported by
using a porphyrin system with covalently linked axial imidazole
(Im).4 The axial imidazole ligation seems to be important for
the formation of a metastable hydroperoxo species, because in
a concerted fashion with the proton transfer it assists in the spin
state and binding mode switch, from high-spin side-on to a low-
spin end-on configuration (pathway (a), Scheme 1).4
We have also observed that the axial ligation influences the
nature of a heme iron-dioxygen species and controls the mecha-
nism of its formation.5 These findings were obtained by studying
the reaction of superoxide in DMSO with an iron complex of a
crown ether-porphyrin conjugate (H2Porph), which is also able
to chelate the potassium cation.5 There we have shown that axial
coordination of DMSO alone causes not only a spin-state and
Activation of molecular oxygen by an iron(II) center within heme-
containing enzymes (cytochrome P450, NO synthase, cytochrome
oxidase)1 proceeds through the formation of the one-electron
-
reduced form of the iron(II)-dioxygen adduct, {Fe–O2} , which
is widely accepted as an iron(III)-peroxo species.2 The same
species can be obtained in the reaction between heme iron(II) and
-
superoxide (O2 ). Due to its transient character and inherent low
aDepartment of Chemistry and Pharmacy, University of Erlangen-Nu¨rnberg,
Egerlandstr. 1, 91058, Erlangen, Germany. E-mail: ivana.ivanovic@
chemie.uni-erlangen.de
bSchool of Chemistry and Centre for Computational Chemistry, University
of Bristol, Cantock’s Close, Bristol, UK, BS8 1TS
† Electronic supplementary information (ESI) available: Spectroelectro-
chemistry, effect of temperature and pressure on the equilibrium constant
K
DMSO, ESI mass spectrometry, EPR spectroscopy kinetics and thermody-
namics of superoxide binding, high-pressure UV/vis measurements, DFT
calculations. See DOI: 10.1039/c1dt11521a
‡ Present address: Department of Inorganic and Analytical Chemistry
and Materials Structure and Modeling Research Group of the Hungarian
Academy of Sciences, Budapest University of Technology and Economics,
P.O. Box 91, 1521 Budapest, Hungary.
546 | Dalton Trans., 2012, 41, 546–557
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form by decomposing the excess of superoxide present in solution
(pathway (b), Scheme 1).
The arising question is whether findings such as reversible
superoxide binding, high stability of the obtained iron-(su)peroxo
porphyrin, the equilibrium between Fe(II)-superoxo and Fe(III)-
peroxo species and generation of the Fe(II) form upon proton
addition are unique features of this particular complex, caused by
the presence of the nearby positive charge from the K+-crown ether
moiety. To answer this question the current study investigates the
iron complex of the corresponding porphyrin without covalently
attached crown ether moiety (H2tBuTPP) and the reaction of the
Fe(II) form with superoxide (KO2) in DMSO. The reaction has also
been studied in the presence of externally added crown ether (18-
crown-6) in order to compare its effects with those of covalently
attached crown ether. To obtain insight into the influence of the
positively charged surrounding on the nature of the product heme
iron-(su)peroxo species, computational DFT studies have been
performed as well.
Experimental
Materials and general considerations
Reagents and solvents were obtained from commercial sources
and were of reagent quality unless otherwise stated. DMSO and
acetonitrile were purchased as extra-dry solvents. All chemicals
were used as received without further purification. K18O2 was
prepared according to a published method;6 18O2 was obtained
from Aldrich.
Scheme 1 Products of the superoxide reaction with heme Fe(II) and
subsequent fine tuned protonation: (a) heme species with the covalently
linked axial imidazole ligand,4 (b) heme species with the covalently linked
potassium cation moiety.5
H2tBuTPP
binding-mode switch of heme peroxide but also a redox-state
switch, resulting in the co-existence of two isomeric forms in
equilibrium, viz. Fe(II)-superoxo and Fe(III)-peroxo (pathway (b),
Scheme 1).5b,c The presence of nearby K+ seemed to be crucial for
the remarkably high stability of the iron-(su)peroxo complex that
was stable even at room temperature. Aided by its stability we have
been able to demonstrate that binding of superoxide to the iron(II)
center is a reversible process and we could quantify the kinetics
The H2tBuTPP (54,104,154,204-tetra-tert-butyl-5,10,15,20-tetra-
phenylporphyrin) ligand was obtained as a side product in
the synthesis of a precursor of H2Porph (52-N-(4-aza-18-crown-
6)methyl-54 ,104 ,154 ,204 -tetra-tert-butyl-56 -methyl-5,10,15,20-
tetraphenylporphyrin).5a
[FeIII(tBuTPP)Cl]
-
and thermodynamics of O2 binding.5a,c Interestingly, addition of
a controlled amount of proton sources to the reaction mixture has
not resulted in either formation of Fe(III)-hydroperoxo or O–O
bond cleavage and formation of a high-valent oxo-iron species.
Instead, it pulled the reaction equilibrium back to the iron(II)
H2tBuTPP (300 mg, 0.357 mmol) was dissolved in 30 mL of
CHCl3. An FeCl2 solution (67.0 mg, 0.524 mmol) in 30 mL of
ethanol was added. Two drops of 2,6-lutidine were added and
the solution refluxed for 2 h. Completion of the reaction was
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followed by thin layer chromatography on Al2O3 plates, which is
indicated by disappearance of the fluorescence of the free base
porphyrin. After that, the mixture was filtered and worked up by
shaking with water and half concentrated HCl. The organic layer
was dried over anhydrous MgSO4 and the solvent removed. The
o-phenyl), 7.86 (s, bis-DMSO m-phenyl), 3.34 (s, bis-DMSO t-Bu),
1.60 (s, mono-DMSO t-Bu).
K[FeIII(tBuTPP)(O22-)]
KO2 was suspended in a solution of [FeIII(tBuTPP)Cl] in dry
DMSO. The solution was stirred until the color changed to green
and was filtered under argon atmosphere. UV/Vis (DMSO): l
[nm] (e [L mol-1 cm-1]): 422sh (36100), 439 (148000), 568sh (6900),
612 (3710).
1
product is a brown powder in 84% yield. H NMR (400 MHz,
CDCl3, 25◦ C): d [ppm] 80.9 (br s, 8H, b-pyrr.), 13.7 (s, 4H, m-
ArH), 12.5 (s, 4H, m-ArH), 7.50 (s, 4H,◦o-ArH), 2.51 (s, 36H,
Ar-t-Bu). 13C NMR (100 MHz, CDCl3, 25 C): d [ppm] 145.0 (4C,
m-ArCH), 140.7 (4C, m-ArCH), 37.7 (4C, t-BuCq), 29.6 (12C,
t-Bu-CH3). FAB-MS: m/z 892 (M+ - Cl). UV/Vis (CH2Cl2): l
[nm] (e [L cm-1 mol-1]) 381 (54100), 419 (114000), 512 (12400),
Equipment
-1
˜
585 (2470), 615 (5640). IR (KBr): n [cm ] 2960, 2926, 2866, 1495,
Elemental analysis was carried out on a HERAEUS CHN-
Mikroautomat, a Euro EA 3000 (Euro Vector) and an EA
1108 (Carlo Erba) instrument. L-SIMS (Cs+) mass spectra were
recorded with a Micromass ZABSpec mass spectrometer employ-
1461, 1395, 1363, 1333, 1267, 1202, 1109, 1069, 998, 805, 721.
Elemental analysis for C60H60ClFeN4·3H2O. Calc.: C, 73.35; H,
6.77; N, 5.70. Found: C, 73.30; H, 6.95; N, 5.40%.
1
ing m-NBA as the matrix. Standard H and 13C NMR spectra
[FeIII(tBuTPP))2O]
were measured on a Bruker Avance 300 spectrometer. Standard
IR spectra (KBr) were measured on a FT-IR IFS 88 spectrometer
(Bruker Analytische Messtechnik GmbH).
The m-oxo-dimer was prepared by shaking a solution of
[FeIII(tBuTPP)Cl] (100.0 mg, 0.119 mmol) in CH2Cl2 (20 mL)
with a 2 M solution of aqueous NaOH (20 mL). The organic
layer was separated and dried over anhydrous MgSO4, and the
solvent was evaporated. A brown solid was obtained in 79% yield.
1H NMR (400 MHz, CDCl3, 25◦ C): d [ppm] 13.3 (s, 16H, b-
pyrr.), 7.76 (s, 8H, m-ArH), 7.65 (s, 8H, o-ArH), 1.67 (s, 72H,
Ar-t-Bu). 13C NMR (100 MHz, CDCl3, 25◦ C): d [ppm] 150.3
(8C, p-ArCq), 144.2 (16C, a-pyrr.); 142.1 (8C, ArCq), 134.3 (16C,
o-ArCH), 131.7 (16C, b-pyrr.), 123.3 (16C, m-ArCH), 119.9 (8C,
meso-C), 34.5 (8C, t-BuCq), 31.8 (24C, t-Bu-CH3). FAB-MS: m/z
1802 (M+) 892 (M+ - O - FetBuTPP). UV/Vis (CH2Cl2): l [nm]
Spectroelectrochemistry
Electrochemical reduction was performed under nitrogen at a
Pt mesh working electrode with an Ag wire pseudo-reference
electrode and a platinated Ti auxiliary electrode separated from
the working electrode compartment by a glass frit. The experiment
was stopped when no further change in the UV/Vis spectra was
observed. Time-resolved UV/Vis spectra were taken by a UV/Vis
immersion probe (Hellma 661.502-QX quartz suprasil). It was
attached via optical cables to a 150 W Xe lamp. Recording of
the spectra was done by a multi-wavelength J & M detector. An
Autolab instrument with a PGSTAT 30 was used as potentiostat.
(e [l cm-1mol-1]) 418 (194000), 513 (11800), 575 (2980). UV/Vis
-1
˜
(DMSO): l [nm] 417, 560 sh, 574, 613. IR (KBr): n [cm ] 2962,
2926, 2867, 1496, 1461, 1396, 1363, 1340, 1267, 1203, 1109, 1070,
999, 895, 876, 812, 799, 719.
1H NMR investigation of [FeII(tBuTPP)(DMSO)n] (n = 1, 2)
Sample preparations were done in an Ar MBraun glovebox.
A sample of [FeII(tBuTPP)(DMSO)n] (n = 1, 2) was prepared
by addition of an excess of Na2S2O4 to a 2 mM solution
of [FeII(tBuTPP)(DMSO)n] (n = 1, 2) in dry DMSO-d6. The
suspension was stirred for 30 min. H NMR spectra were taken
after filtration.
[FeIII(tBuTPP)(DMSO)2]+
A solution of [FeIII(tBuTPP)Cl] in DMSO yields the bis-DMSO
complex of the iron porphyrin. UV/Vis (DMSO): l [nm] (e [L
mol-1 cm-1]) 404sh (15400), 419 (17600), 496sh (3850), 535 (4000),
719 (2095).
1
Temperature-dependent NMR spectra in DMSO-d6 were mea-
sured on a Bruker Avance 300 or Bruker AVANCE DRX 400WB
instrument. All spectra were recorded in 5 mm o.d. NMR tubes,
and chemical shifts were reported as d (ppm) values calibrated to
natural abundance deuterium solvent peaks (ppm).
A homemade high-pressure probe described in the literature was
used for the variable-pressure experiments.7 Pressure-dependent
measurements were performed in a standard 5 mm NMR tube
cut to a length of 50 mm. To enable pressure transmittance to
the solution, the NMR tube was closed with a moveable KEL-
S piston. The advantage of this method is that oxygen-sensitive
samples can be easily placed in the NMR tube and sealed with the
KEL-S piston under argon atmosphere. A safe subsequent transfer
to the high-pressure probe is assured. The pressure was applied to
the high-pressure probe via a perfluorinated hydrocarbon pressure
medium (hexafluoropropylene oxide, Hostinert 175, Hoechst)
and measured by a VDO gauge with an accuracy of 1%. The
[FeIII(tBuTPP)OH]
Addition of water or NaOH to a solution of [FeIII(tBuTPP)-
(DMSO)2]+ yields the hydroxo complex [FeIII(tBuTPP)OH].
UV/Vis (DMSO): l [nm] (e [L mol-1 cm-1]): 416sh (14900), 438
(30600), 540 (3100), 573 (1400).
[FeII(tBuTPP)(DMSO)1,2]
The reduced form of [FeIII(tBuTPP)Cl] could be obtained by
chemical reduction or bulk electrolysis. Chemical reduction in
dry DMSO was achieved by using sodium dithionite (saturated
solution) as reductant. UV/Vis (DMSO): l [nm] (e[L mol-1 cm-1]):
411sh (12500), 430 (69750), 533 (3100). 1H NMR (reduction with
sodium dithionite, 300 MHz, DMSO-d6, 25 ◦C): d [ppm] 13.0 (br s,
mono-DMSO pyrr.), 9.90 (s, bis-DMSO pyrr.), 8.13 (s, bis-DMSO
548 | Dalton Trans., 2012, 41, 546–557
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temperature was adjusted with circulating, thermostated water
(Colora thermostat WK 16) to 0.1 K of the desired value and
monitored before each measurement with an internal Pt-resistance
thermometer with an accuracy of 0.2 K.8 The temperature was
chosen to be 320 K and kept constant, since at lower temperatures
DMSO can freeze upon increasing the pressure.
Kinetic data were obtained by recording time-resolved UV/Vis
spectra using a modified mSFM-20 Bio-Logic stopped-flow mod-
ule combined with a Huber CC90 cryostat or a Biologic SFM-
400 four syringes stopped-flow system (using only the first three
syringes) and a high density mixer to minimize mixing effects in
DMSO solutions. The stopped-flow set-ups were equipped with a
J&M TIDAS high-speed diode array spectrometer with combined
deuterium and tungsten lamps (200–1015 nm wavelength range).
Isolast O-rings were used for all sealing purposes to enable
measurements in DMSO, and solutions were delivered from 10
mL gas-tight Hamilton syringes. The syringes were controlled
by separate drives, allowing for variation of the ratio of mixing
volumes used in the kinetic runs. Data were analyzed using
the integrated Bio-Kine software version 4.23 and also the
Specfit/32TM program. At least five kinetic runs were recorded
under all conditions, and the reported rate constants represent
the mean values. The stopped-flow instrument was thermostated
to the desired temperature 0.1 ◦C. Experiments at 25 ◦C were
performed in DMSO solution. All kinetic measurements were
carried out under pseudo-first-order conditions; i.e. superoxide
concentration was in large excess (complex concentration was
usually 5 ¥ 10-6 M). The reactions were studied at an ionic strength
of 0.1 M (Bu4NPF6).
ESI and cryospray mass spectrometry
Using a syringe pump at a flow rate of 240 mL h-1, the DMSO
solutions were infused into an orthogonal ESI source of an Esquire
6000 ion trap mass spectrometer (Bruker, Bremen, Germany).
Nitrogen was used as the nebulizing gas at a pressure of 10
psi and as the drying gas at a temperature of 300◦ C and
a flow rate of 5 L min-1. The ion trap was optimized for
the respective target mass of the ions under investigation. The
source voltages varied with this optimization. All experiments
were carried out in the positive-ion mode. The sample of the
starting complex [FeIII(tBuTPP)Cl] was prepared by making a
5 ¥ 10-4 M suspension in dry DMSO (stirred overnight) under
nitrogen and subsequent filtration. Other samples were prepared
by dissolving excess of either KO2 or K18O2 in an overnight-
prepared 5 ¥ 10-4 M suspension of [FeIII(tBuTPP)Cl] in dry DMSO
under nitrogen and subsequent filtration. High mass accuracy
ESI spectra were recorded on an ultra high-resolution ESI-Time-
Of-Flight MS, a Bruker Daltoniks (Bremen, Germany) Maxis,
which also was coupled to the Bruker cryospray. For the cryospray
measurements samples were dissolved in a MeCN–DMSO (70 : 30)
mixture.
For the rapid kinetic measurements, the Bio-Logic stopped-
flow module was upgraded to a submillisecond mixing stopped-
flow configuration by combining it with a microcuvette accessory
(with an optical pathlength of 0.8 mm) and a monochromator to
minimize the dead time of the instrument.10
High-pressure UV/Vis measurements
Sample preparation was carried out in an Ar MBraun glovebox.
A sample of [FeIII(tBuTPP)(O22-)]- was prepared by addition of an
excess of KO2 to a 10-5 M solution of [FeIII(tBuTPP)Cl] in a dry
DMSO solution of 0.1 M TBAP. The suspension was stirred for 30
min. High pressure UV/Vis spectra were recorded after filtration.
Spectral measurements at elevated pressure were performed in
a pill-box cuvette on a Shimadzu UV-2101-PC spectrophotometer
using a home-made high-pressure cell.11 The high-pressure pump
was purchased from NOVA SWISS (Nova Werke AG, CH-
8307 Effretikon, Vogelsangstrasse); it allows measurements up to
150 MPa.
Thermodynamics and kinetics
Solution preparations of KO2, [FeII(tBuTPP)(DMSO)n] (n =
1, 2) and [FeIII(tBuTPP)(O22-)]- in DMSO without 18-crown-
6 were done according to our previous study.5 Solutions of
[FeIII(tBuTPP)(O22-)]- in DMSO with 18-crown-6 were prepared
by addition of a ten-fold excess of 18-crown-6 (relative to
the porphyrin concentration) to a freshly prepared solution of
[FeII(tBuTPP)(DMSO)n] (n = 1, 2).
A Hewlett-Packard 8452A spectrophotometer was used for
qualitative UV/Vis measurements and measurement of the bind-
ing constant of superoxide to [FeII(tBuTPP)(DMSO)n] (n = 1, 2)
without and with added 18-crown-6 (10 times the concentration
of the porphyrin complex).
A spectrophotometric study of the binding of superoxide to
[FeII(tBuTPP)(DMSO)n] (n = 1, 2) was performed in a 1-cm
path length cuvette with a 25 mL reservoir under nitrogen. The
1.3 ¥ 10-5 M solution (5 mL) of electrochemically generated
[FeII(tBuTPP)(DMSO)n] (n = 1, 2) was titrated with aliquots of
a saturated KO2 solution in DMSO added from a Hamilton
microsyringe. The same experiment was performed with the Fe(II)
complex and added 18-crown-6 (1.3 ¥ 10-5 M and 1.3 ¥ 10-4 M,
respectively). Data were fitted according to following equation
(A0 and A• represent the absorbance at 0 and 100% of the
product formation, respectively, Ax represents the absorbance at
the respective measured superoxide concentration):9
DFT calculations
Geometry optimizations of the structures were performed with
the Jaguar 6.0 program package12 using the B3PW91 and B3LYP
functionals, and a restricted open shell formalism (ROKS). The
6-31G(d) basis set was used on all atoms with the exception of
the metal ions for which the standard Los Alamos effective core
potentials13 were used with the associated double-z, basis LACVP
on potassium and the triplet-z basis LACV3P14 on iron. The calcu-
lated total energies of the optimized structures in vacuo, together
with the total energy of the dissociated DMSO ligand, were used
to compare the stability of the side-on and end-on complexes.
Results and discussion
In a DMSO solution Fe(III) porphyrins exist as bis-DMSO
species.15 Similar to the [FeIII(Porph)(DMSO)2]+ complex
-
-
Ax = A0 + (A• - A0)K[O2 ]/(1 + K[O2 ])
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with the covalently attached crown ether moiety,5 the
[FeIII(tBuTPP)(DMSO)2]+ complex reacts with an excess
of KO2 resulting in an adduct commonly described as
[FeIII(tBuTPP)(O22-)]-.2 The reaction proceeds in two steps. In
the first step Fe(III) is reduced to Fe(II), which in the second
step binds superoxide yielding [FeIII(tBuTPP)(O22-)]-.2,5 In this
study we used the electrochemically generated Fe(II) form of
the complex as the reactant, i.e. we concentrated on the second
reaction step. As previously reported,5a the first reaction step,
reduction of the FeIII to the FeII porphyrin complex by KO2, could
not be quantitatively studied in detail because of interference of
the Fe(III)-hydroxo species formation by KOH, which is inevitably
present in commercial KO2. Details on the spectroelectrochemical
measurements and UV/vis characterization of different Fe(III)
forms of the studied complex are given in ESI† (Fig. S1–S4, Table
S1 and Scheme S1)
1H NMR investigation of [FeII(tBuTPP)(DMSO)n] (n = 1, 2)
Solvation of iron(II) tetraphenylporphyrins in DMSO leads
to an equilibrium between mono- and bis-DMSO complexes
(Scheme 2).5,16 In order to investigate the thermodynamics
of such equilibrium in the case of [FeII(tBuTPP)(DMSO)n],
temperature- and pressure-dependent 1H NMR studies were
performed.
Fig. 1 (a) 1H NMR spectrum of [FeII(tBuTPP)(DMSO)n] (n = 1, 2) at
325.2 K and different pressures; (b) plot of ln(KDMSO) vs. pressure.
Scheme 2
Literature data on NMR of [FeII(tpp)] in DMSO reveal the co-
existence of high-spin and low-spin species16 with a moderately
broad paramagnetic signal of the pyrrole protons at around
12 ppm. This finding corresponds to the NMR spectra of
investigated [FeII(tBuTPP)(DMSO)n] (n = 1, 2) (Fig. S5, ESI†).
In the region from 0 to 10 ppm, there are signals with only
minor line broadening which correspond to the signals of the
diamagnetic [FeII(tBuTPP)(DMSO)2] species. The broad signal at
around 13 ppm belongs to the resonances of the pyrrole protons of
the paramagnetic [FeII(tBuTPP)(DMSO)] species. The remaining
paramagnetic resonances cannot be observed as they are merged
with the diamagnetic ones (Fig. S5, ESI†). The position and line
broadening of the pyrrole protons of a porphyrin system are
generally used in order to determine the spin state of the central
metal.17
Upon increase of temperature the paramagnetic signal shifts
strongly to lower field (Fig. S6, ESI†). The remainder of the
spectrum remains almost unchanged. Line broadening increases
only moderately. After letting the sample cool down, the starting
spectrum is retrieved. This supports the existence of the high-
spin/low-spin equilibrium, with the share of the high spin species
increasing with the temperature increase.
coordination of the second DMSO molecule. Decrease of pressure
after the experiment shows again a larger share of the high-spin
species and confirms the reversibility of the observed pressure
dependent process.
The temperature- and pressure-dependent measurements enable
the quantification of the thermodynamic parameters for the
equilibrium KDMSO in Scheme 2 (HS = high-spin mono-DMSO
complex; LS = low-spin bis-DMSO complex; the activity of
solvent is considered to be 1). The parameters are summarized in
Table 1 (for details see Supplementary Information and Table S2,
ESI†).
The temperature dependence plot of ln(KDMSO) (Fig. S7, ESI†)
was found to be linear and resulted in significantly positive values
for the thermodynamic parameters DH◦ and DS◦ (Table 1). They
are in agreement with the endothermic and dissociative character
of the underlying low-spin to high-spin transition. Interestingly,
the spin-state equilibrium in the case of ferric cytochrome P450
is characterized within the corresponding temperature range by
similar thermodynamic parameters (DH◦ = +42 kJ mol-1 and
Table 1 Thermodynamic parameters for the equilibrium constant KDMSO
Since the low-spin/high-spin equilibrium constant (KDMSO
,
KDMSO at 298.2 K
0.082 0.002
DH◦/kJ mol-1
+36
+101
1
Scheme 2) should also be pressure sensitive, we performed
pressure-dependent 1H NMR measurements. A pressure increase
shows a shift of the equilibrium to the diamagnetic low-spin
species (Fig. 1). This is expected, since a higher pressure favors the
DS◦◦/J K -1 mol-1
4
DG◦/kJ mol-1 at 298.2 K
DV /cm3 mol-1 at 325.2 K
6
+16
1
2
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DS◦ = +152 J K-1 mol-1).18 A typical entropy change that is
considered to drive the spin transition from low- to high-spin state
is approximately 50 J K-1 mol-1, and in the case of an Fe(II) spin-
crossover compound is found to be 60 J K-1 mol-1.19 The observed
entropy change of ~101 J K-1 mol-1 (Table 1) is significantly larger
than that associated with the simple spin transition indicating that
the covalent bond breaking and an increase in translational and
rotational degrees of freedom as a consequence of a dissociative
process have significant contribution. By way of comparison, in the
opposite associative process accompanied by high-spin to low-spin
transition upon CO rebinding to the five-coordinate (L)FeII4SP
complexes (4SP = tetrakis(4-sulfonatophenyl)porphyrin, L = H2O
or 2-methylimidazole)) the reaction entropy were found to be
-117 J K-1 mol-1.20
ESI mass spectrometry in DMSO
The best correlation of mass spectra with the solution chemistry of
the corresponding coordination compounds is currently achieved
by soft electrospray ionization (ESI). Therefore, we have used ESI
for the characterization of the reactant and product species in
solution.
The mass spectrum of the starting Fe(III) complex (Fig. S8,
ESI†), [FeIII(tBuTPP)(DMSO)2]+, shows a single peak at m/z
970.3, corresponding to [FeIII(tBuTPP)(DMSO)]+. The isotopic
distribution (Fig. S9, ESI†) confirms this assignment. A collision
experiment with He gas (MS/MS or tandem mass spectrometry)
yields the porphyrin complex [FeIII(tBuTPP)]+ without axial
ligands at m/z 892.4 (inset in Fig. S8, ESI†).
The reaction mixture of [FeIII(tBuTPP)(DMSO)2]+ and KO2
was investigated. The ESI mass spectrum of the reaction product
shows the main ion peak at m/z 1002.3 corresponding to {K+ +
K[FeIII(tBuTPP)(O22-)]} (Fig. 2). In order to confirm the peak
at m/z 1002.3 as the superoxo {K+ + K[FeIII(tBuTPP)(O22-)]}
adduct, the same experiment was repeated with a mixture of
K16O2 and K18O2 (1 : 1) (inset in Fig. 2). The mass spectrum shows
again the peak at m/z 1002.3 with a new one at m/z 1006.3,
The values of KDMSO and the corresponding distribution of
species (see Table S2, ESI†) show that in DMSO solution mostly
the low-spin bis-DMSO complex exists (92% at 298 K and
0.1 MPa). The pressure dependence of ln(KDMSO) also gave a
straight line (Fig. 1b) and resulted in DV◦ = +16 2 cm3 mol-1. The
high positive reaction volume is a result of DMSO dissociation that
accompanies the spin-state change on the Fe center. The reaction
volume changes for spin transitions in Fe(II) complexes have been
reported to range between 5 and 19 cm3 mol-1,19,21 whereas the co-
valent bond breaking contributes to a volume change with roughly
5–10 cm3 mol-1.22 This implies that the change in coordination
number on the iron center coupled to a spin-state transformation is
characterised by volume changes of 10–30 cm3 mol-1. For example,
CO photodissociation accompanied by low-spin to high-spin
change is coupled to volume expansions of ~17 and ~12 cm3 mol-1
for the above mentioned (L)FeII4SP complex and FeIIMP-11 (MP-
11 = microperoxidase-11), respectively.20 Similar processes of other
heme model complexes exhibit an reaction volume of ~30 cm3
mol-1.23 On the other hand measurements of spin equilibria and
ligand binding/release in the case of heme proteins typically
yield volume changes of 10–50 cm3 mol-1.24 However, the case
of proteins may differ from model complexes in that the spin-
state transition caused by ligand binding/release can be coupled
with additional changes of the protein structure and prominent
solvent reorganisation due to (de)hydration of regions outside the
active site, resulting in very large volume changes (larger than
200 cm3 mol-1).24b Interestingly, our recent studies on the spin state
equilibrium (analogue to that in Scheme 2) of the iron(II) complex
of the crown ether-porphyrin conjugate,5c [FeII(Porph)(DMSO)n]
(n = 1, 2), have shown that the presence of the crown ether moiety
slightly shifts the KDMSO equilibrium into the direction of the low-
spin bis-DMSO species (KDMSO = 0.030 0.001, 97% of the low-
spin component at 298 K and 0.1 MPa). This strongly suggests
that the covalently attached crown ether makes dissociation of
the sixth ligand somewhat less favorable.5c The corresponding
reaction enthalpy (DH◦ = +48 1 kJ mol-1) is more positive
than that for [FeII(tBuTPP)(DMSO)n], supporting the fact that
the crown ether makes DMSO dissociation more endothermic.
Furthermore, the significantly larger reaction volume (DV◦ = +26
2 cm3 mol-1)5c in the case of [FeII(Porph)(DMSO)n] implies
that DMSO release causes additional structural changes on the
resulting mono-DMSO species, which involves a new orientation
between the covalently attached crown ether and porphyrin plane
(creating a more “open” structure) accompanied by additional
solvent perturbations.
shifted by 4 mass units as expected for the 18O2 adduct, and its
elemental composition is also confirmed by the corresponding
isotopic distributions (Fig. S10, ESI†).
-
Fig. 2 Mass spectrum of {K+ + K[FeIII(tBuTPP)(O22-)]}. Inset: Mass
spectrum of {K+
(tBuTPP)(18O22-)]}.
+
K[FeIII(tBuTPP)(16O22-)]} and {K+
+
K[FeIII
-
The corresponding crown ether containing species, {K+
+
K[FeIII(Porph)(O22-)]}, was observed previously using the crown
ether attached porphyrin complex.5b Both these ions show a
distinctly different dissociation behavior in MS/MS experiments,
which indicates different structural motifs. Upon collision with
He gas, {K+ + K[FeIII(Porph)(O22-)]} shows the fragmentation
into intact K[FeII(Porph)]+ by the straightforward loss of KO2.
Obviously, one K+ cation is coordinated with the O2 moiety
while the other K+ cation resides with the crown ether. The
covalently attached crown ether stabilizes the porphyrin complex
as a potassium chelating agent. The MS/MS experiment of
{K+ + K[FeIII(tBuTPP)(O22-)]} shows a more complex dissociation
behavior which clearly also involves the decomposition of the
porphyrin. Fragment ions are observed at m/z 984.1, 958.1, 946.2
and 825.1 (inset in Fig. S11, ESI†). These fragment ions are shifted
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by two mass units to higher masses when the 18O2-labeled {K+ +
K[FeIII(tBuTPP)(18O22-)]} precursor is studied as the parent ion.
Therefore, each fragment ion has lost one oxygen atom in the
dissociation. A tentative assignment of the fragment ions would
be H2O loss (m/z 984.1), followed by loss of CN or C2H2 (m/z
958.1), followed by the loss a tert-butylphenyl radical (m/z 825.1).
An MS3 experiment confirmed the sequence: m/z 1002 dissociates
via m/z 958 into m/z 825. The fragment ion at m/z 946.2 could
correspond to the loss of KOH. In summary, the dissociations of
{K+ + K[FeIII(tBuTPP)(O22-)]} show both incorporation of one
oxygen atom and fragmentation of the porphyrin moiety.
We also used a cryospray mass spectrometric technique25
coupled to the ultra-high-resolution time-of-flight detector to
allow efficient transfer of the intact species from solution-state
into the gas phase. Operation on low temperatures (at -30 ◦C
in the MeCN–DMSO (70 : 30) solvent mixture)5a reduces possible
decomposition processes and dissociation of weakly bound ligands
to a minimum. Also under these conditions, in the MS spectrum
of the starting Fe(III) complex, [FeIII(tBuTPP)(DMSO)]+ (peak at
m/z 970.4287) appears as a main species (Fig. S12, ESI†), whereas
the spectrum of the reaction product clearly shows existence of the
{K+ + K[FeIII(tBuTPP)(O22-)]} adduct (peak at m/z 1002.3337)
(Fig. S13, ESI†). This suggests that DMSO as the sixth ligand is
quite labile, at least under the conditions of MS experiments.
the covalently attached crown in the complex structure. The
reaction and its reversibility were further thermodynamically and
kinetically quantified.
The superoxide binding constant was determined by titration
experiments in analogy to the previous study (Fig. S15, ESI†).5a
The experimentally obtained value (K = (9 0.5) ¥ 103 M-1) for
the binding of superoxide to [FeII(tBuTPP)(DMSO)n] is approx-
imately one order of magnitude lower than the corresponding
value in the case of [FeII(Porph)(DMSO)n] (K = (1.7 0.2) ¥
105 M-1).5a This corresponds to our qualitative observations of
[FeIII(tBuTPP)(O22-)]- being less stable than its analogue with
covalently attached crown ether moiety. The same experiment
was done with addition of a ten-fold excess of 18-crown-6 related
over the porphyrin concentration (Fig. S16, ESI†). The binding
constant of K = (11 4) ¥ 103 M-1 is obtained and it is the same
(within experimental error) as in the case with no external crown
ether present in solution. This shows that the presence of external
crown ether in the solution does not exhibit a beneficial stabilizing
effect. In that way, a stabilizing effect of covalently attached crown
ether on the peroxo complex could be quantified.
UV/Vis stopped-flow measurements of the reaction of
[FeII(tBuTPP)(DMSO)n] (n = 1, 2) with saturated KO2 solution
in DMSO were performed. The experiment was carried out
according to our previous study.5a,25 Superoxide concentrations
were varied by using saturated KO2 solution in DMSO and varying
the mixing volume ratios. The time-resolved spectra were fitted
to a single-exponential function to give values for the observed
rate constants kobs (Fig. 3). The corresponding second-order rate
constant kon = (1.30 0.01) ¥ 105 M-1 s-1 is approximately one order
ofmagnitude higher than inthe case of the complexwith covalently
attached crown ether (kon = (3.65 0.05) ¥ 104 M-1 s-1).5a Such
kinetic effect is most probably due to steric reasons. Considering
the binding constants and the values for kon for both complexes,
it can be concluded that koff (koff = kon/K) is approximately two
Kinetics and thermodynamics
We have previously studied the kinetics and thermodynamics of
the superoxide binding to the [FeII(Porph)(DMSO)n] porphyrin
complex with covalently attached crown ether.5a In order to under-
standtheinfluenceof acrown ether moiety on the thermodynamics
and kinetics of the iron porphyrins interaction with superoxide in
DMSO, a comparative thermodynamic and kinetic study on the
porphyrin without a covalently attached crown ether moiety, was
carried out. Two different sets of experiments were performed:
one without any crown ether and another with a ten-fold excess of
deliberately added 8-crown-6 present in solution.
As mentioned above, the reaction between the iron(II) form
of the complex with KO2 was studied, which results in
[FeIII(tBuTPP)(O22-)]-. Qualitative experiments on the stability of
[FeIII(tBuTPP)(O22-)]- (lmax = 439 nm) were done by bubbling moist
air through the complex solution in presence of excess superoxide.
In analogy to the iron porphyrin complex with the covalently
attached crown ether,5a the iron(II) complex (lmax = 430 nm) is
formed (Fig. S14, ESI†) due to the superoxide decomposition
and shift of the equilibrium to the starting complex (Scheme 3).
This shows that reversible binding of superoxide to an iron(II)
porphyrin is a general feature independent of the presence of
orders of magnitude slower for [FeIII(Porph)(O2 )]- (koff = 0.21
-
-
0.01 s-1)5a than for [FeIII(tBuTPP)(O2 )]- (koff = 14.4 0.1 s-1).
By slowing down the superoxide dissociation from the metal
center the crown ether moiety with coordinated K+ stabilizes
Fig. 3 Plot of kobs vs. superoxide concentration for the reaction of 1 ¥
10-5 M [FeII(tBuTPP)(DMSO)n] (n = 1, 2) and KO2 at 25◦ C using different
mixing volume ratios (I = 0.1 M Bu4NPF6). Inset: Time resolved UV/Vis
spectra of the reaction of complex with 0.1 ¥ 10-3 M of KO2.
Scheme 3
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the superoxide adduct. The assumption that the crown ether side
chain prevents to some extent dissociation of the axial ligand is
in agreement with somewhat higher amount of bis-DMSO species
present in the KDMSO equilibrium mixture (Scheme 2) in the case
of [FeII(Porph)(DMSO)n] (97%; n = 1 or 2) than in the case of the
[FeII(tBuTPP)(DMSO)n] system (92%; vide supra).
The koff value have been also directly determined by adding
controlled amount of a proton source (HOTf acid) into the
[FeIII(tBuTPP)(O2 )]- product solution, where an excess of KO2
-
was also present. HOTf was chosen since it is known that strong
acids react with superoxide in aprotic solvents extremely rapidly
-
(k¢ > 1 ¥ 107 M-1 s-1)26 which makes them very efficient O2
scavengers even at low concentrations. As shown in Scheme 3,
rapid decomposition of superoxide shifts the equilibrium back
to the Fe(II) form. Upon subsequent addition of superoxide to
this solution the product [FeIII(tBuTPP)(O2 )]- is fully recovered.
-
To better visualize this process we performed experiments by
using two-mixer stopped-flow system. The [FeIII(tBuTPP)(O2 )]-
-
complex (5 ¥ 10-6 M) with excess of KO2 (0.1–1 mM) was in the
first syringe and mixed with the solution of HOTf (0.1–2 mM)
from the second syringe (off-reaction). To the obtained product
solution (after 1 s delay) additional superoxide (from the third
syringe) was introduced in the second mixer (on-reaction). The
corresponding time-resolved spectra, kinetic trace and speciation
spectra are shown in Fig. 4. Such experiments have revealed
that independent on the applied acid concentration or excess of
KO2 the formation of the Fe(II) complex proceeds in one step,
without formation of any intermediate Fe(III) specie, with the
corresponding first order rate constant kobs = koff = 11.6 0.7 s-1.
This value is in excellent agreement with that obtained based on
kon and the binding constant (koff = kon/K, vide supra).
The kinetic studies were also performed for the reaction of
[FeII(tBuTPP)(DMSO)n] (n = 1, 2) in the presence of a ten-fold
excess of 18-crown-6 in solution. The obtained second-order rate
constant (Fig. S17, Table S3, ESI†) for the formation of the peroxo
complex was kon = (1.6 0.2) ¥ 105 M-1 s-1, which corresponds
to the one obtained without added crown ether (kon = (1.30
0.01) ¥ 105 M-1 s-1). Having kon and koff (11.1 0.5 s-1) for the
reactions in the presence of external crown-ether, the reaction
equilibrium constant can be calculated K = kon/koff = (14 3) ¥
103 M-1. The obtained value is in excellent agreement with the
thermodynamically estimated one (K = (11 5) ¥ 103 M-1, vide
supra) and with that obtained without added crown ether present
in solution.
Fig. 4 (a) Time resolved UV/Vis spectra (from the experiment in the
two-mixer stopped-flow) of the reaction of 5 ¥ 10-6 M [FeIII(tBuTPP)(O2-)]-
(in the presence of 10-3 M KO2) with 1 mM HOTf (in the first mixer)
and subsequently with KO2 (in the second mixer after 1 s delay; in
DMSO, I = 0.1 M Bu4NPF6); inset: kinetic trace at 440 nm and a
two-exponential fit. (b) Spectra of the species observed along the reaction
path: 1 [FeIII(tBuTPP)O22-]-, 2 [FeII(tBuTPP)(DMSO)n] (n = 1, 2), 3
[FeIII(tBuTPP)O22-]-.
As commercially available Na2O2 contains up to 10% of NaO2,
and since “naked” peroxide is not stable in aprotic solution,27
we used it as a source of superoxide, which is released in
solution by stirring of the granules in DMSO. This enables
quantitative generation of iron (su)peroxo species without using
a high superoxide excess (the concentration of superoxide that is
present in the saturated DMSO solution of Na2O2 is estimated to
be (4 2) ¥ 10-5 M).5b The EPR spectrum at 12 K of the reaction
product between the studied iron porphyrin and Na2O2 in DMSO
(Fig. 5) shows only a weak signal of rhombic high-spin FeIII at
g = 4.20, characteristic for the side-on peroxo species.2b,5b Signals
of a byproduct FeIII hydroxo species (g = 7.24 and g = 5.34) are
also present in the spectrum, similar to what has been observed
in the case of [FeIII(Porph)]5b and other porphyrin systems.28 The
signals of the free solvated superoxide and superoxide bound to
the Na+ cation (g = 2.10, g = 2.00)5b are much more intense than
the signals of the iron species. Thus, although the EPR spectra
demonstrated the presence of both FeII–O2- and FeIII–O22- species
of the crown ether porphyrin conjugate,5b evidence for the possible
EPR
In order to shed light on the electronic nature of our FeII porphyrin
superoxide adduct we performed X-band EPR measurements.
However, in contrast to our previous investigations on the crown
ether porphyrin system ([Fe(Porph)Cl]),5b due to the very low
solubility of the studied [Fe(tBuTPP)Cl] complex in DMSO
we were not able to obtain detailed information from those
measurements. Usually the iron signals were near the detection
limit and superoxide signals dominated. Moreover, due to the
lower stability of the superoxo adduct of the complex without
covalently attached crown ether, signals of the reaction side
products, such as hydroxo and m-oxo complexes (vide infra),
interfered significantly.
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High-pressure UV/Vis spectroscopy
Since based on the EPR spectra we were not able to get information
about the possible co-existence of two isomeric forms in product
solution, viz. Fe(II)-superoxo and Fe(III)-peroxo, we performed
pressure-dependent UV/Vis measurements to probe the existence
of an equilibrium state. (Due to the low complex solubility, which
limited the quality of the EPR spectral data, the Mo¨ssbauer
spectra of the required resolution could not be obtained as well.)
The high-spin/low-spin equilibria are usually pressure sensitive
and as expected the increasing pressure in the DMSO solution
of the product (su)peroxide adduct causes a small but significant
red-shift of the Soret absorption band (Fig. 6), indicating a shift of
the equilibrium towards the low-spin six-coordinate component of
the product mixture. This is in agreement with observations made
when studying the temperature dependent spectra of liver micro-
somal cytochrome P450 in the presence of different substrates.32
On releasing pressure the starting spectrum was obtained, which
demonstrates the reversibility of the pressure-induced changes on
the equilibrium. The presence of the externally added crown ether
in solution, did not affect this pressure-dependent behaviour (Fig.
S20, ESI†). The same behaviour we observed in the case of the
complex with the covalently attached crown ether moiety, where
we were able to demonstrate the existence of the equilibrium
Fig. 5 X-Band EPR spectra of [FeIII(tBuTPP)Cl] with Na2O2 in DMSO
at 12 K, microwave frequency was 8.989384 GHz, P = 1 mW; modulation
width = 1.0 mT, sweep width 800 mT.
superoxide coordination to iron, FeII–O2 , could not be found in
-
the case of the [Fe(tBuTPP)] system. At 77 K iron signals could
not be observed at all, whereas the superoxo signals showed no
change in g values with temperature increase.
between the low-spin K[FeII(Porph)(DMSO)(O2 )] and high-
-
spin K[FeIII(Porph)(O22-)] species by means of different methods
(Mo¨ssbauer, EPR, IR spectroscopy, ESI mass spectrometry, high-
pressure kinetic measurements, DFT calculations).5b,c The quan-
tification of such equilibrium constant is not possible to achieve
since the spectra of the pure iron(II)-superoxo or pure iron(III)-
peroxo forms are yet unknown, and there is no corresponding data
available in the literature. Therefore, future studies will be required
to tune this equilibrium such that it can be pushed completely in
any of the two directions in order to independently characterize
these two species.
When KO2 was used, higher concentrations of superoxide and
hydroxide were present in solution compared to the Na2O2 ex-
periment. The corresponding EPR spectrum of [FeIII(tBuTPP)Cl]
with KO2 in DMSO at 12 K (Fig. S18, ESI†), besides the signals
of rhombic iron(III)-peroxo (g = 4.20) and hydroxo species (g =
5.46, g = 7.32), showed a large paramagnetic signal around g =
1.99 mixed with the signal of free superoxide. It has the nature of a
system with one unpaired electron.29 It should be mentioned that
m-oxo species of iron(III) are EPR silent due to antiferromagnetic
coupling.29,30 The observed signal could be related to the formation
of an iron(II)–iron(III) m-oxo porphyrin dimer, [[FeII(tBuTPP)]–O–
[FeIII(tBuTPP)]]-, upon decomposition of the (su)peroxo species.30
As expected for an iron species, it is only observed at temperatures
below 77 K.30 Kadish et al.30 proposed an analogous species with
the TPP system and described it as a m-oxo complex with a single
electron added. The EPR spectrum of this species is very similar
to the one we observe, showing one asymmetric line with g = 1.95.
One can just speculate whether this species might be a hint for the
existence of both iron(III)-peroxo and iron(II)-superoxo forms in
solution. In the case of the complex with the covalently attached
crown ether such species have not been observed, in agreement
with the fact that the crown ether sterically hinders the formation
of dimer complexes. At 77 K only a signal of free superoxide is
observed and the other signals have vanished due to their low
concentration (inset in Fig. S18, ESI†).
Fig. 6 UV/Vis spectra of K[FeIII(tBuTPP)(O22-)] at different pressures in
By using a ten-fold excess of 18-crown-6 over [FeIII(tBuTPP)-
(DMSO)2]+ a huge signal at g = 2 is observed (Fig. S19, ESI†),
which upon temperature variation changes only its intensity but
not the shape. The signal corresponds to superoxide bound to
the potassium cation inside the crown ether and is influenced
by moisture in solution.31 In the case of iron signals only the
general rhombic signal of the peroxo species around g = 4.2 can
be observed (Fig. S19, ESI†).
the presence of 0.1 M TBAP.
DFT calculations and correlations with experiments
To understand the possible influence of a nearby positive charge on
the molecular and electronic structure of heme iron (su)peroxide
adducts we performed DFT studies. The same B3LYP and
554 | Dalton Trans., 2012, 41, 546–557
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∑
-
Table 2 Calculated B3PW91 (and B3LYP) relative energies (in kcal mol-1) for end-on and side-on {FeII(porphyrin) O2
}
and
{FeII(porphyrin) O2 ∑DMSO}
∑
-
∑
∑
{FeII(porphyrin) O2
}
{FeII(porphyrin) O2 ∑DMSO}
-
-
Multiplicity
End-on
Side-on
2
4
6
12.6 (8.2)
16.7 (14.0)
13.7 (13.9)
8.9 (3.5)a
17.8a
b
b
b
b
2
4
6
21.9 (18.5)
11.8 (12.9)
0.0 (0.0)
a Compared to the energy of the free side-on sextet structure and a free DMSO ligand. b No complex with DMSO bound to the axial position was found
on the PES.
B3PW91 levels of theory were used as in our previous study
of the species with the crown ether moiety,5b for consistency.
The optimized geometries of our computational models include
the iron porphyrin core structure without the phenyl groups in
meso positions, and the structures with and without coordinated
DMSO are considered. Several different spin states (doublet,
quartet and sextet) were considered for each structure (Table 2).
Only the doublet and quartet states of the end-on structure
bind DMSO with the doublet end-on structure being lowest in
energy.
Scheme 4 Comparison of selected atomic distances and spin densities
(red) for the calculated DMSO free sextet side-on porphyrin and the
DMSO bound doublet end-on species of porphyrin (this work) and
the model system with the covalently attached K+-crown-ether moiety.5b
Results were obtained at the B3PW91/BSI level of theory.
As in the previous study, the lowest energy structure found is
the sextet high-spin side-on structure, in which the coordination
site at the other side of the heme ring is vacant. The calculations
suggest considerable superoxo character, as shown by the large
spin densities on the both oxygen atoms (0.55, Scheme 4). The
study5b (reduced spin-density on the oxygen atoms 0.30, larger O–
˚
O bond distance 1.45 A). Our thermodynamic and kinetic studies
whichshow the greater stability of the (su)peroxoadductin thecase
of the complex with the K+-crown ether moiety are in agreement
with the shortening of the Fe–O bonds caused by nearby positive
charge. Additional stabilizing effect comes from the fact that the
dissociation of axial ligands is to some extent sterically hindered
by the bulkiness of the covalently linked crown ether (vide supra).
It should be mentioned that based on our experiments, cations
that are present in solution when KO2 or Na2O2 are used as
source of superoxide do not contribute to the product stability
like the covalently attached cation moiety, which due to the steric
proximity directly interacts with bound (su)peroxide inducing a
stronger effect.
The end-on structure, which binds DMSO, is higher in energy
than the side-on structure by 8.9 kcal mol-1 with B3PW91 or by 3.5
kcal mol-1 with B3LYP (Table 2). It has a doublet low-spin ground
state with superoxo character. The spin density on the O atom
nearer to Fe is 0.39 while on the distal one it is 0.55. The shortest
˚
calculated O–O distance is 1.34 A and the oxygen–oxygen bond
lies above one of the N–N diagonals of the porphyrin ring. This is
a typical superoxo O–O distance,33 consistent with the calculated
spin densities, though most experimental data on superoxide
adducts of iron porphyrins strongly suggest that they have peroxo
character2 (an exception is an earlier ESR study describing such
a species as Fe(II)-superoxo34). The Fe–O bond is somewhat
elongated (Scheme 4), and the calculated structure of this side-on
form can be understood in terms of weakly coordinated superoxide
to high-spin FeII. Our previous DFT studies on the model system
with the covalently attached crown ether including the chelated
K+ cation5b showed a larger peroxo character of the side-on sextet
structure, with decreased spin-density on the oxygen atoms, longer
O–O bond and shorter Fe–O bonds (Scheme 4). It seems that the
nearby positive charge has a significant influence on the electronic
structure of the side-on adduct. Indeed, a calculation on the simple
model used here, but with an added ‘naked’ K+ ion (no crown
ether moiety) leads to results more similar to those in the previous
˚
˚
Fe–O distance is 1.88 A, and the O–O distance is 1.30 A, similar
to the distance of free superoxide.27,33a The Fe–O bond is signif-
icantly shorter than in the case of side-on structure (Scheme 4).
The effect of the K+-crown ether side chain on this end-on form
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is negligible and does not influence its electronic structure, which
remains to have low-spin Fe(II)-superoxo character (Scheme 4).
The energy difference between the side-on and end-on species
suggests that the side-on structure is more favorable, but does
not exclude the existence of equilibrium between them (3.5 kcal
mol-1 energy difference obtained with B3LYP, Table 2). In the case
of the model complex with covalently attached K+-crown ether
there was a smaller energy difference between these two isomeric
forms.5b Our high-pressure experiments have shown the existence
of the equilibrium in the product solution (vide supra). We should
consider that the presence of the K+ cations under experimental
conditions in solution (from KO2) lowers the energy difference
between two forms, although not to that extent as in the case of
the system with covalently attached K+-crown ether side chain,
but still favors their coexistence.
ligand dissociation covalently attached crown ether slows down the
superoxide release from the product (su)peroxo species (resulting
in a decrease of koff by two orders of magnitude), (f) the more
prominent effect of attached crown ether on the superoxide
dissociation (koff) than on the DMSO dissociation (kon) suggests
that the positive charge of chelated K+ additionally prevents
the superoxide release, (g) these effects of the crown ether side
chain on kon and koff result in the ten times larger superoxide
binding constant and make the product (su)peroxo complex
significantly more stable, whereas external addition of crown ether
does not bring any stabilization, (h) due to this higher stability,
detailed spectroscopic studies on the (su)peroxo complex with
the crown ether side chain could be performed, demonstrating
the existence of the redox tautomerism between the end-on FeII-
superoxo and side-on FeIII-peroxo forms, which is supported by
DFT calculations, (i) the lower solubility and stability of the
(su)peroxo complex without crown ether moiety did not allow
detailed spectroscopic characterization of its nature, however,
DFT calculations demonstrate that without nearby positive charge
the reaction product has the FeII-superoxo character in both side-
on high-spin as well as end-on low-spin forms. These findings
are important for understanding the real nature of the corre-
sponding biologically relevant (su)peroxo intermediaries of heme-
containing enzymes involved in activation of molecular oxygen
and hydrogen peroxide, because they show that the nature and
electronic structure of these intermediates can be tuned not only by
axial ligand coordination but also by the charge of a surrounding
of an iron heme center in the active site of an enzyme. Our kinetic
and thermodynamic studies quantify the effect of semi-confined
environment around iron center, caused by the covalently attached
crown-ether, on its reactivity toward axial ligands in general, and
superoxide in particular. The results obtained in this investigation
represent a motivating foundation for future studies towards the
understanding of the effects that might be caused by the solvent, a
trans ligand, and possible involvement of coordinated (su)peroxide
in hydrogen bonding or electrostatic interactions, on the structure
and electronic properties of the heme iron-superoxide adducts and
their reactivity towards different organic substrates of biological
and synthetic importance.
Collectively, the DFT data suggest that without nearby positive
charge the reaction product has a character of a FeII-superoxo
species in both side-on high-spin as well as end-on low-spin
forms. The presence of the positive charge stabilizes the side-on
high-spin adduct and changes its character into the FeIII-peroxo
species, whereas the FeII-superoxo character ofthe end-on low-spin
isomer remains unchanged. One can speculate that if superoxide
adduct would be produced in an experimental setup with no
cations available in solution it would predominantly exhibit
superoxo character (increased spin-density on the oxygen atoms,
stronger O–O bond), but at the same time significantly decreased
stability (weaker Fe–O bond). Such experimental conditions can
be achieved by in situ radiolytic cryoreduction of heme FeII–
-
O2 adducts.3 Interestingly, such {Fe–O2} products have usually
been described as end-on peroxo species, although based on the
observed O–O distance3b,c and quantum chemical calculations
they are best interpreted as FeII–O2 .33a,35 Only Davydov et al.
-
clearly classified the primary reduction product of monomeric
oxy-hemoglobin (oxy-GMH3) from Glycera dibranchiata as FeII-
superoxo based on EPR/ENDOR experiments.3a They also
suggest that the internal redox transition to peroxo/hydroperoxo-
ferric intermediates is driven by H-bonding/proton donation by
the environment,3a which, based on our findings, can be interpreted
as peroxo form stabilization by adjacent positive charge.
Conclusion
Acknowledgements
Based on our investigations of the superoxide reactions with
FeII porphyrins with and without covalently attached crown
ether moiety and with deliberately added crown ether we can
conclude the following: (a) superoxide binds reversibly to FeII
porphyrins in DMSO independently on the general existence of
crown ether, (b) in DMSO solution with an excess of superoxide,
the product (su)peroxo species generates the starting FeII complex
upon controlled addition of a proton source, also independent on
the presence of the crown ether moiety, (c) to a certain extent, the
crown ether side chain favors the low-spin six-coordinate form
of the FeII porphyrin in the solution, (d) since generation of
the high-spin five-coordinate FeII species is the actual activation
step towards substitution and subsequent inner-sphere electron
transfer, the superoxide binding (kon) is approximately one order
of magnitude slower when crown ether is covalently attached
to porphyrin, whereas externally added crown ether does not
influence the rate determining step, (e) by preventing the axial
This work was supported by the Deutsche Forschungsgemein-
schaft through SFB 583 “Redox-active metal complexes” (K. D.,
A. Z., N. J., I. I. B). J. O. acknowledges receipt of an EU Marie
Curie Fellowship (Project “Modelling CYPs”).
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