Volume profile analysis for the reversible binding of superoxide to form iron(II)-superoxo/iron(III)-Peroxo porphyrin complexes

Article abstract of DOI:10.1021/ic102092h

The one-electron reduced iron(II)-dioxygen adduct, {Fe^II-O ₂}^-, is known to be an important intermediate in the catalytic cycle of heme (mono)oxygenases. The same type of species, considered as Fe^III-peroxo, can be formed in a direct reaction between a Fe ^II center and superoxide. In a unique high-pressure study of the reaction between superoxide and the Fe^II complex of a crown ether porphyrin conjugate in dimethylsulfoxide (DMSO), the overall Fe ^II-superoxide interaction mechanism could be visualized and the nature of all species that occur along the reaction coordinate could be clarified. The equilibrium between the low-spin and high-spin forms of the starting Fe^II complex was quantified, which turns out to be the actual activation step toward substitution and subsequent inner-sphere electron transfer reactions. The constructed reaction volume profile demonstrates that the reaction product consists of Fe^III-peroxo and Fe ^II-superoxo species that exist in equilibrium, which can better account for the versatile reactivity of {Fe^II-O₂} ^- adducts toward different substrates.

Full text of DOI:10.1021/ic102092h

11254 Inorg. Chem. 2010, 49, 11254–11260
DOI: 10.1021/ic102092h
Volume Profile Analysis for the Reversible Binding of Superoxide
to Form Iron(II)-Superoxo/Iron(III)-Peroxo Porphyrin Complexes
€
ꢀ
ꢀ
Katharina Durr, Norbert Jux, Achim Zahl, Rudi van Eldik,* and Ivana Ivanovic-Burmazovic*
Department of Chemistry and Pharmacy, University of Erlangen-Nu€rnberg,
Egerlandstrasse 1, 91058 Erlangen, Germany
Received October 15, 2010
The one-electron reduced iron(II)-dioxygen adduct, {FeII-O2}-, is known to be an important intermediate in the
catalytic cycle of heme (mono)oxygenases. The same type of species, considered as FeIII-peroxo, can be formed in a
direct reaction between a FeII center and superoxide. In a unique high-pressure study of the reaction between
superoxide and the FeII complex of a crown ether porphyrin conjugate in dimethylsulfoxide (DMSO), the overall
FeII-superoxide interaction mechanism could be visualized and the nature of all species that occur along the reaction
coordinate could be clarified. The equilibrium between the low-spin and high-spin forms of the starting FeII complex
was quantified, which turns out to be the actual activation step toward substitution and subsequent inner-sphere
electron transfer reactions. The constructed reaction volume profile demonstrates that the reaction product consists of
FeIII-peroxo and FeII-superoxo species that exist in equilibrium, which can better account for the versatile reactivity of
{FeII-O2}- adducts toward different substrates.
Introduction
By using an iron complex of a crown ether porphyrin
conjugate (H2Porph, see Chart 1 below), we were able for the
first time to quantify the kinetics and thermodynamics of
superoxide coordination,5 and to gain more insight into the
nature of the product species.6 This was possible because the
product of the reaction of our FeII complex with KO2 was
stabilized by a positively charged moiety (crown ether che-
lated potassium) in close proximity to the coordinated super-
Superoxide is the one-electron reduction product of molec-
ular oxygen that arises during numerous oxidation reactions
in both living and non-living systems. The major targets of
the superoxide anion radical as reactive oxygen species under
biological conditions are metal ion centers. Upon coordina-
tion to a metal ion (Mnþ), superoxide can be stabilized by
forming metal-superoxo species (Mnþ-O2-) that can subse-
quently undergo inner-sphere electron transfer, resulting in
either metal reduction and oxygen release (M(n-1)þ þ O2) or
formation of metal-peroxo species (M(nþ1)þ-O22-).1 Metal-
(su)peroxo species are important intermediates in the cata-
lytic cycles of metalloproteins, and in particular heme-
proteins that are responsible for the activation of dioxygen
and hydrogen peroxide, and subsequently lead to versatile
oxidation processes of different substrates.2-4 However,
quantitative investigations of the kinetics and thermo-
dynamics of reactions between superoxide and heme metal
centers yielding metal-(su)peroxo species are almost unknown
because of the instability of such product species.
•-
oxo/peroxo group. We showed in that study that O2 can
react with a metal center in a reversible manner to form a
quite stable iron-(su)peroxo species that can release super-
oxide to form the starting FeII complex by fine-tuning of the
proton concentration.5 Such reaction behavior is of general
chemical as well as biological importance, since it shows that
upon proton addition, the iron-(su)peroxo adduct does not
necessarily dissociate to hydrogen peroxide and an FeIII
species (as in the case of SOD active enzymatic and mimetic
systems)7,8 or undergoes O-O bond cleavage to form a high
valent oxo-iron species (as in the case of cytochrome P450).
In addition, although the product of the reaction between the
FeII porphyrin and superoxide was accepted to be a high-spin,
*To whom correspondence should be addressed. E-mail: vaneldik@
chemie.uni-erlangen.de (R.v.E.), ivana.ivanovic@chemie.uni-erlangen.de
(I.I.-B.).
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(5) Durr, K.; Macpherson, B. P.; Warratz, R.; Hampel, F.; Tuczek, F.;
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(3) Schlichting, I. J.; Berendzen, J.; Chu, K.; Stock, A. M.; Maves, S. A.;
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r
pubs.acs.org/IC
Published on Web 11/08/2010
2010 American Chemical Society

Article
Inorganic Chemistry, Vol. 49, No. 23, 2010 11255
Chart 1
in the elucidation of the nature of its transition state and pro-
duct species. In particular, our volume profile analysis pro-
vides evidence for the existence of an intriguing FeII-superoxo
complex in the product solution as a discrete isomer of the
coexisting FeIII-peroxo complex.
Experimental Section
Materials. Reagents and solvents were obtained from com-
mercial sources (Aldrich and Acros) and were of reagent quality
unless otherwise stated. DMSO was purchased as extra-dry
solvent and kept under protective gas. All chemicals, except
2,4,6-tri-(t-butyl)phenol (TBPh), were used as received without
further purification. TBPh was recrystallized from MeOH prior
to use. The synthesis of the [FeIII(Porph)Cl] complex and the
preparation of the superoxide solutions have been previously
reported.5 The corresponding [FeII(Porph)(DMSO)n] (n = 1, 2)
complex was obtained by either chemical (with Na2S2O4) or
electrochemical reduction (bulk electrolysis).
side-on FeIII-peroxo complex,9,10 we recently provided evi-
dence from spectroscopic and density functional theory (DFT)
studies that in DMSO solution it coexists with its isomeric
(redox-tautomer) low-spin, end-on FeII-superoxo form.6 The
same species can be obtained by an one-electron reduction of a
FeII-dioxygen adduct leading to {FeII-O2}-,11-13 which is the
usual pathway of its generation within catalytic cycles of the
corresponding heme enzymes responsible for versatile oxidation
reactions in biological systems. It seems that the real nature
and reactivity of this important intermediate can be better
accounted for in terms of the coexistence of two isomeric forms
in equilibrium, namely, FeII-superoxide and FeIII-peroxide.
In this work it was our goal to elucidate the mechanism of
the formation of this interesting iron-(su)peroxo adduct and
to obtain further information on its nature by applying high-
pressure thermodynamic and kinetic techniques. Studies on
the effect of pressure on forward and backward reactions, as
well as on the overall reaction equilibrium, enable the con-
struction of volume profiles that can reveal crucial mechan-
istic information by assisting the visualization of possible
intermediates and/or transition states on the basis of partial
molar volume changes that occur along the reaction coordi-
nate.14-16 Volume profiles have been extremely helpful to
clarify some of the most essential mechanistic aspects and to
elucidate the contribution of spin and oxidation state changes
during activation processes of small molecules by metallo-
enzymes and model complexes.14,16 We have, therefore,
performed high-pressure NMR, UV/vis, and stopped-flow
measurements to be able to construct a volume profile for the
reaction of our FeII complex with superoxide. These results
enlighten, for the first time in the literature, mechanistic
aspects of the metal-superoxide interaction, which resulted
NMR Studies. Sample preparations were done in an Ar
MBraun glovebox. Solutions of [FeII(Porph)(DMSO)n] (n =
1, 2) were prepared by addition of an excess of Na2S2O4 to a
2 mM (10 mM in case of the pressure dependent measurements)
solution of [FeIII(Porph)Cl] in dry DMSO-d6. The suspension
1
was stirred for 30 min. H NMR spectra were recorded after
filtration.
Temperature dependent NMR spectra in DMSO-d6 were
measured on a Bruker Avance 300 or Bruker AVANCE DRX
400WB instrument. All spectra were recorded in 5 mm outer
diameter (o.d.) NMR tubes, and chemical shifts were reported
as δ (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.17 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 test 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 an argon atmo-
sphere. A safe subsequent transfer to the high-pressure probe is
assured. The pressure was applied to the high-pressure probe
via a perfluorated hydrocarbon pressure medium (hexafluoro-
propyleneoxide, Hostinert 175, Hoechst) and measured by a
VDO gauge with an accuracy of 1%. Temperature was adjusted
with circulating, thermostatted 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.18 Temperature was chosen to be 320 K and
kept constant, since at lower temperatures DMSO can freeze
upon increasing the pressure.
Effect of Temperature and Pressure on the Equilibrium Con-
stant K1. In analogy to FeIII porphyrins with spin admixed states,19
the percentage of the low-spin species is calculated according to
eq 1 (δis the chemical shift of the pyrrole protons of the equilibrium
mixture; the limiting value for the low-spin species is about 9 ppm20
and for the high-spin species about 60 ppm).21
(9) McCandlish, E.; Miksztal, A. R.; Nappa, M.; Sprenger, A. Q.;
Valentine, J. S.; Strong, J. D.; Spiro, T. G. J. Am. Chem. Soc. 1980, 102,
4268–4271.
(10) Selke, M.; Valentine, J. S. J. Am. Chem. Soc. 1998, 120, 2652–2653.
(11) Davydov, R.; Satterlee, J. D.; Fujii, H.; Sauer-Masarwa, A.; Busch,
D. H.; Hoffman, B. M. J. Am. Chem. Soc. 2003, 125, 16340–16346.
(12) Hersleth, H.-P.; Hsiao, Y.-W.; Ryde, U.; Goerbitz, C. H.; Andersson,
K. K. Biochem. J. 2008, 412, 257–264.
Intð%Þ ¼ ½ð60 - δÞ=51ꢀꢁ100%
ð1Þ
(17) Zahl, A.; Neubrand, A.; Aygen, S.; van Eldik, R. Rev. Sci. Instrum.
1994, 65, 882.
(13) Unno, M.; Chen, H.; Kusama, S.; Shaik, S.; Ikeda-Saito, M. J. Am.
Chem. Soc. 2007, 129, 13394–13395.
(14) Franke, A.; Hessenauer-Ilicheva, N.; Meyer, D.; Stochel, G.;
Woggon, W. D.; van Eldik, R. J. Am. Chem. Soc. 2006, 128, 13611–13624.
(15) Maigut, J.; Meier, R.; Zahl, A.; van Eldik, R. J. Am. Chem. Soc. 2008,
130, 14556–14569.
(18) Maigut, J.; Meier, R.; Zahl, A.; van Eldik, R. Inorg. Chem. 2007, 46,
5361–5371.
(19) Ikezaki, A.; Nakamura, M. Inorg. Chem. 2002, 41, 6225–6236.
(20) (a) Shirazi, A.; Goff, H. M. J. Am. Chem. Soc. 1982, 104, 6318–6322.
(b) Walker, F. A. The porphyrin handbook; Academic Press: New York, 2003;
Vol. 5, pp 81-182.
(21) (a) Parmely, R. C.; Goff, H. M. J. Inorg. Biochem. 1980, 12, 269–280.
(b) Song, B.; Park, B.; Han, C. Bull. Korean Chem. Soc. 2002, 23, 119–122.
(16) Hubbard, C. D.; van Eldik, R. In Physical Inorganic Chemistry.
Methods and Techniques; Bakac, A., Ed.; Wiley: New York, 2010; pp 269-365.

€
Durr et al.
11256 Inorganic Chemistry, Vol. 49, No. 23, 2010
Figure 1. (a) 1H NMRspectrumof [FeII(Porph)(DMSO)n] (n =1, 2)at320 K anddifferentpressures;(b)plotofln(K1) versuspressure. The pressure range
was limited to 60 MPa because of the interference of the paramagnetic signal at higher pressures with a signal of the high pressure medium.
The species distribution at different temperatures and pres-
sures is summarized in Supporting Information, Table S1. From
these results the equilibrium constant K1 (in which the activity of
solvent is considered to be 1) can be calculated according to eq 2.
On the basis of the temperature and pressure dependent data for
K1 the corresponding thermodynamic parameters can be esti-
mated according to eqs 3 and 4, respectively.22
the electrolysis the time-resolved UV/vis spectra were recorded
simultaneously in situ. Electrochemical reduction was performed
under nitrogen at a Pt gauze 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.
Potential or current were controlled with an Autolab instrument
with PGSTAT 30 potentiostat. The experiment was stopped when
no change in the UV/vis spectra was observed anymore. Time-
resolved UV/vis spectra were recorded by use of a Hellma 661.502-
QX quartz Suprasil immersion probe attached via optical cables to
a 150 W Xe lamp and a multiwavelength J & M detector.
A solution of K[FeIII(Porph)(O22-)] to monitor the “off”
reaction was prepared as described above for the high pressure
UV/vis measurements.
K1 ¼ ½HSꢀ=½LSꢀ
ð2Þ
where [HS] is the high-spin mono-DMSO complex concentra-
tion, and [LS] is the low-spin bis-DMSO complex concentration
lnðK1Þ ¼ - ΔH°=RT þ ΔS°=R
½D lnðK1Þ=DPꢀT ¼ - ΔV°=RT
ð3Þ
ð4Þ
All kinetic measurements were carried out under pseudo-first-
order conditions at a complex concentration of 5 ꢁ 10-6 M and
superoxide concentration of 10-4 M. The superoxide concen-
tration was selected on the basis of earlier concentration depen-
dent measurements, which resulted in a linear dependence of
The reaction enthalpy ΔH° was obtained from the slope and
the reaction entropy ΔS° from the intercept of the plot of ln(K1)
versus 1/T (see Supporting Information, Figure S3). The reac-
tion volume ΔV° results from the slope of the plot of ln(K1)
versus pressure (see Figure 1).
k
obs on the superoxide concentration, with a negligible intercept,
for the “on” reaction.5 Measurements under high pressure were
carried out using a homemade high-pressure stopped-flow
instrument,24 for which Isolast O-rings were also used for all
syringe seals. The stopped-flow instrument was thermostatted at
35 °C. The reactions were monitored at 440 and 430 nm, which
correspond to the absorption maxima of the product and reactant
complex, respectively (see Figures 9 and 11 in reference 5).
Values of ΔVq were calculated from the slope of plots of ln(k)
versus pressure (Supporting Information, Figures S4 and S5) in
the usual manner based on eq 5. For monitoring the “on”
reaction, DMSO solutions of [FeII(Porph)(DMSO)n] (n = 1, 2)
and KO2 were mixed in the high pressure stopped-flow. For the
measurement of kobs for the “on” reaction as a function of
pressure, fresh solutions of superoxide and the reactant complex
were prepared under ambient conditions and thermostatted for
20 min at elevated pressure in the stopped-flow, before mixing
them to measure kobs (Supporting Information, Table S2). For
monitoring the “off” reaction at each pressure, fresh solutions of
K[FeIII(Porph)(O22-)] and a moderate acid, TBPh, were pre-
pared and thermostatted for 20 min at elevated pressure in the
stopped-flow, before mixing them to determine kobs (Supporting
Information, Table S3).
High Pressure UV/vis Measurements. Sample preparation
was done in an Ar MBraun glovebox. A sample of K[FeIII
-
(Porph)(O22-)] was prepared by addition of an excess of KO2 to
a 10-5 M solution of [FeIII(Porph)Cl] in a dry DMSO solution of
0.1 M (n-Bu)4NPF6 (tetrabutylammonium hexafluorophosphate).
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 spectrophoto-
meter using a homemade high-pressure cell.23 The high-pressure
pump was purchased from NOVA SWISS (Nova Werke AG,
CH-8307 Effretikon, Vogelsangstrasse); it allows measurements
up to 150 MPa.
High Pressure Stopped-Flow Kinetics and Data Processing.
The preparation of KO2 solutions in DMSO and determination
of superoxide concentrations have previously been reported.5
The presence of (n-Bu)4NPF6, which was used to provide the
constant ionic strength (0.1 M) in the kinetic measurements, in
general increases the superoxide solution stability.
Solutions of [FeII(Porph)(DMSO)n] (n = 1, 2) were prepared
by bulk electrolysis of 10-5 M solutions of the respective iron(III)
porphyrin in DMSO at -0.2 V versus Ag/AgCl. 0.1 M of
Bu4NPF6 was also present in solution as electrolyte. During
½D lnðkÞ=DPꢀT ¼ - ΔVq=RT
ð5Þ
Determination of kobs for the “on” reaction, which depends
linearly on [O2-],5 is complicated by the fact that superoxide
(22) Wilkins, R. G. Kinetics and Mechanisms of Reactions of Transition
Metal Complexes; VCH: New York, 1991.
(23) (a) Spitzer, M.; Gartig, F.; van Eldik, R. Rev. Sci. Instrum. 1988, 59,
2092–2093. (b) Fleischmann, K. F.; Conze, G. E.; Stranks, R. D.; Kelm, H. Rev.
Sci. Instrum. 1974, 45, 1427.
(24) (a) van Eldik, R.; Palmer, D. A.; Schmidt, R.; Kelm, H. Inorg. Chim.
Acta 1981, 50, 131–135. (b) van Eldik, R.; Gaede, W.; Wieland, S.; Kraft, J.;
Spitzer, M.; Palmer, D. A. Rev. Sci. Instrum. 1993, 64, 1355–1357.

Article
Inorganic Chemistry, Vol. 49, No. 23, 2010 11257
Scheme 1. Reaction Scheme Summarizing the Existing Equilibria and
Table 1. Thermodynamic Parameters for the Equilibrium Constant K1
Superoxide Reaction with the FeII Porphyrin in DMSOa
K1 at 298.2 K
0.030 ( 0.001
þ48 ( 1
ΔH°/kJ mol-1
ΔS°/J K -1 mol-1
ΔG°/kJ mol-1 at 298.2 K
ΔV°/cm3 mol-1 at 320 K
þ133 ( 3
þ9 ( 2
þ26 ( 2
broad signal around 11 ppm (see Supporting Informa-
tion, Figure S1) belongs to the resonance of the pyrrole
protons of the paramagnetic [FeII(Porph)(DMSO)] spe-
cies. The position and line broadening of the pyrrole
protons of a porphyrin system are generally used to
determine the spin state of the central metal.20b The
remaining paramagnetic resonances of [FeII(porphyrin)-
(DMSO)] cannot be observed, as they are merged with the
diamagnetic ones.
On increasing the temperature the paramagnetic signal
shifts strongly to lower field (see Supporting Information,
Figure S2), whereas the rest of the spectrum remains
almost unchanged. The line broadening increases only
moderately. The reason for this behavior is a shift of K1 in
Scheme 1 in favor of the paramagnetic [FeII(Porph)-
(DMSO)] complex. After allowing the sample to cool
down, the starting spectrum is regained as the equilibrium
is restored. This demonstrates the existence of a reversible
low-spin/high-spin equilibrium, where the contribution
of the high-spin species increases with increasing temperature.
Since the low-spin/high-spin equilibrium constant K1
should also be pressure sensitive, we performed pressure
dependent 1H NMR measurements (Figure 1). Increasing
pressure results in a shift of the equilibrium to the
diamagnetic low-spin species. This is expected as pressure
favors DMSO coordination and the formation of the low-
spin species. On decreasing pressure the larger contribu-
tion of the high-spin species (Figure 1a) is restored as in
the starting state of the equilibrium.
a The reactant exists in a spin state equilibrium K1 (here: low-spin = LS,
S = 0; high-spin = HS, S = 2). The reaction of KO2 with this
equilibrium mixture and the dissociation of (su)peroxide are character-
ized by the rate constants k2 and k-2, respectively. The product complex
exists in a spin/redox state equilibrium K3 (here: FeII low-spin = LS,
S = 0; FeIII high-spin = HS, S = 5/2).
undergoes disproportionation, that is, spontaneous decomposi-
tion, during the time required to thermostat the solutions at
elevated pressure. The disproportionation of superoxide is a
second order process and expected to be accelerated by increas-
ing pressure. As a consequence, the superoxide concentration
after thermostatting the solutions at elevated pressure, before
mixing them to determine kobs, will be somewhat lower than
prepared at ambient pressure, which will lower the apparent kobs
value. Therefore, the apparent activation volume needs to be
corrected for this effect according to eq 6 to obtain the actual
ΔVq(kobs) value for the “on” reaction.
ΔVqðappÞ ¼ ΔVqðkobsÞ þ ΔVqðcorrÞ
ð6Þ
To determine the value of ΔVq(corr) we measured the time
dependence of kobs at three different pressures (Supporting
Information, Table S4).
The slopes obtained from the linear time dependence of
ln(kobs) (Supporting Information, Figure S6) were plotted ver-
sus pressure (Supporting Information, Figure S7, Table S5), and
from the corresponding slope, [∂ ln(kobs)/∂t)]/∂P = -(1.07 (
0.06) ꢁ 10-4 cm3 J-1 min-1, ΔVq(corr) was obtained according to
eq 7 (for the calculation see Supporting Information).
The temperature and pressure dependent measure-
ments enable the quantification of the thermodynamic
parameters for the equilibrium K1 in Figure 1, which are
summarized in Table 1 (for details see Experimental
Section and Supporting Information, Table S1).
ΔVqðcorrÞ ¼ - ½D lnðkobsÞ=DtÞꢀ=DPꢁDt ꢁ RT
ð7Þ
Results and Discussion
Pressure and Temperature Dependent NMR Study of
the Complex [FeII(Porph)(DMSO)n] (n = 1, 2). Solvation
of iron(II) tetraphenylporphyrins in DMSO leads to
an equilibrium between five- and six-coordinate species
with one or two coordinated DMSO molecules, respec-
tively.5,20,21 Since such an equilibrium (K1 in Scheme 1)
also exists in solution for the reactant complex, [FeII-
(Porph)(DMSO)n] (n = 1, 2), we performed temperature
and pressure dependent 1H NMR measurements to
study the thermodynamics of the underlying equilibrium
process.
The temperature dependence plot of ln(K1) (see Sup-
porting Information, Figure S3) was found to be linear
and resulted in significantly positive values for the thermo-
dynamic parameters ΔH° and ΔS° (Table 1). They are in
agreement with the endothermic and dissociative char-
acter of the underlying low-spin to high-spin transition.
The values of K1 and corresponding species distribution
(see Supporting Information, Table S1) show that in
DMSO solution mostly the low-spin bis-DMSO complex
exists (97% at 298 K and 0.1 MPa). The pressure depen-
dence of ln(K1) also gave a straight line (Figure 1b) and
resulted in ΔV° = þ26 ( 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. It
is known that the low-spin/high-spin transformation in
iron model complexes and enzymatic (cytochrome P450)
systems is accompanied by a volume increase of between
Literature data on NMR spectra of [FeII(TPP)] (TPP =
dianion of tetraphenylporphin) in DMSO show that the
complex exists in a mixture of low-spin and high-spin
species,20,21 with a moderately broad paramagnetic signal
of the pyrrole protons around 12 ppm for the high-spin
species. This finding corresponds directly to the 1H NMR
spectrum of the investigated [FeII(Porph)(DMSO)n] (n=1,2)
system (see Supporting Information, Figure S1).
In the range from 0 to 10 ppm, there are signals with
only minor line broadening that correspond to the signals
of the diamagnetic [FeII(Porph)(DMSO)2] species. The
12 and 15 cm3 mol-1 14
suggesting that the additional
,
volume expansion of 11-14 cm3 mol-1 found in the pres-
ent case can be ascribed to the dissociation of DMSO. From
a comparison with other similar processes on iron systems,14
it seems that the volume change of ∼30 cm3 mol-1 is in

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Durr et al.
11258 Inorganic Chemistry, Vol. 49, No. 23, 2010
Figure 2. Volume profile for the reversible binding of superoxide to the Fe(II) porphyrin. (All depicted volume changes along the reaction coordinate are
given in cm3 mol-1 and discussed in the manuscript.).
general characteristic for the change in coordination number
on the iron center coupled to a spin-state transformation.
Pressure Dependence of the Reversible Reaction with
Superoxide. Since we have now quantified the effect of
pressure on the equilibrium between the two [FeII(Porph)-
(DMSO)n] (n = 1, 2) species in DMSO solution (vide
supra), we further performed high-pressure stopped-flow
studies on the reaction of these complexes with KO2. We
have previously shown that this is a reversible reaction
(Scheme 1) and have quantified the corresponding overall
superoxide binding constant (K), both thermodynami-
cally (UV-vis titration) and kinetically (by determining
the kon and koff rate constants, K = kon/koff, vide infra), as
well as the activation parameters (ΔHq and ΔSq) for the
forward, “on”, reaction.5
of 2,4,6-tri-(t-butyl)phenol in the high-pressure stopped-
flow, the activation volume for the “off” reaction could be
obtained, namely, ΔVq(koff) = -7.2 ( 0.5 cm3 mol-1
,
from the slope of the linear dependence of ln(koff) on
pressure (see Supporting Information, Figure S5).
Volume Profile Analysis for the Reversible Binding of
Superoxide. On the basis of the above presented results,
the volume profile for the overall reaction can be depicted
as in Figure 2.
As a result of the fact that the overall reaction mecha-
nism involves a pre-equilibrium between low-spin and
high-spin [FeII(Porph)(DMSO)n] (n = 1, 2) species, the
observed rate constant for the reaction with superoxide
can be expressed by eq 8, where k2 is the forward rate
constant for the coordination of O2-, K1 is the pre-
equilibrium constant, and koff = k-2/(1 þ K3) is the
overall reverse rate constant (Scheme 1).
By following the coordination of superoxide to the
iron(II) complex by high-pressure UV-vis stopped-flow
technique, we obtained the pressure dependence of kon.
From the linear pressure dependence of ln(kobs) for the
“on” reaction (see Supporting Information, Figure S4)
and eq 5 the apparent activation volume was found to
be þ32 ( 2 cm3 mol-1. Since the spontaneous decom-
position of superoxide is a second order reaction, it is
somewhat accelerated with pressure and results in an
intrinsically lower superoxide concentration present in
solution at elevated pressures. This leads to an additional
decrease of the kobs values for the “on” reaction (kobs
depends linearly on [O2-])5 with increasing pressure and
positive volume contribution (þ5.5 ( 0.3 cm3 mol-1, see
Experimental Section and Supporting Information).
Thus, after correction for this effect the actual activation
volume for the “on” reaction was found to be ΔVq(kon) =
(þ32 - (þ6)) ( 2 = þ26 ( 2 cm3/mol.
-
kobs ¼ fK1k2½O2 ꢀ=ð1 þ K1Þg þ k- 2=ð1 þ K3Þ
ð8Þ
Equation 8 can be simplified to kobs = K1k2[O2-] þ
k-2/K3 since K1 is 0.03 M at 298.2 K, that is, K1 , 1 (see
Table 1), and spectroscopic observations showed that the
reaction product predominantly exists in the peroxo
]
form,5,6,9,10 that is, K3 . 1. It follows that kobs = kon[O2
-
þ koff, with the second-order rate constant for superoxide
binding kon = K1k2 and first-order rate constant for
superoxide release koff = k-2/K3. Thus, the obtained
activation volume for the “on” reaction, ΔVq(kon), in-
volves contributions from the effect of pressure on the
equilibrium between six- and five-coordinate iron(II)
species, ΔV°(K1), and on the rate constant for the binding
of superoxide k2, ΔVq(k2) (see Scheme 1 and Figure 2).
Interestingly, the experimental values of ΔV°(K1) and
ΔVq(kon) are identical and consequently it follows that
k2 for the rate-determining step must be almost pressure-
independent, that is, ΔVq(k2) ∼ 0 cm3 mol-1. This allows
us to visualize the transition state for the binding of super-
oxide as “early“ and reactant-like in terms of volume
changes, that is, it has the nature of the five-coordinate
mono-DMSO high-spin [FeII(Porph)(DMSO)] complex.
We have previously reported ΔHq(kon) = 61.2 ( 0.9 kJ mol-1
and ΔSq(kon) = þ48 ( 3 J K-1 mol-1 for the overall
binding process,5 and based on the herein determined
The backward “off” reaction can be followed by addi-
tion of a controlled concentration of a proton source to
the product mixture, to decompose the excess of super-
-
oxide present in solution (2O2 þ 2Hþ f O2 þ H2O2) and
to shift the reaction equilibrium back to the starting
iron(II) species (Scheme 1).5 2,4,6-tri-(t-butyl)phenol,
TBPh, was used as a moderate acid. We have previously
shown that the rate of the “off” reaction (release of
superoxide) does not depend on [TBPh] which behaves
as a trapping agent.5 By following the formation of iron(II)
species upon mixing the reaction product with a solution

Article
Inorganic Chemistry, Vol. 49, No. 23, 2010 11259
ΔH°(K1) and ΔS°(K1) values (Table 1) it follows that
ΔHq(k2) = ΔHq(kon) - ΔH°(K1) = 13 kJ mol-1 and
ΔSq(k2) = ΔSq(kon) - ΔS°(K1) = -85 J K-1 mol-1
.
These parameters further clarify the nature of the transi-
tion state and activation process. The low value of
ΔHq(k2) confirms the existence of the “early” transition
state. The significantly negative ΔSq(k2) can be accounted
for in terms of chelation of the weakly solvated Kþ in
DMSO by the dangling crown ether and the entrance of
O2- into the pocket between the porphyrin and the crown
ether moiety (Figure 2). The volume neutrality of the
activation process is a result of the opposite effects of the
intrinsic volume decrease caused by chelation of Kþ and
-
uptake of free O2 within the coordination pocket, and
the volume increase caused by the desolvation of free
Kþ and O2 on entering the crown ether and coordina-
-
tion pocket, respectively.
Along the reaction coordinate between an FeII species
and KO2 it is expected that the iron(II)-superoxo complex
appears either as a transition state, intermediate, or
as a product species. The value of the activation volume
for the “off” reaction, namely, ΔVq(koff) = -7.2 (
0.5 cm3 mol-1, offers valuable information to elucidate
the role of this species in the overall reaction mechanism.
Our previous studies have shown that such a species in
DMSO exists as the six-coordinate, low-spin K[FeII-
(Porph)(DMSO)(O2-)] complex,6 requiring a large nega-
tive value for ΔVq(k2) of approximate -26 cm3 mol-1
(which is clearly not the case, vide supra) if it would act as
the transition state. On the basis of the principle of
microscopic reversibility, the reaction must pass through
the same transition state in the rate-determining step,
approaching it from either the forward or the reverse
direction. Therefore, if the iron(II)-superoxo species
would exist as an intermediate between the FeII and
FeIII-peroxo complexes, the activation process in the
back direction would involve transformation from this
Figure 3. Pressure dependent UV/vis spectra of the iron-(su)peroxo
product complex. UV/vis spectra of the reaction product (10-5 M) as a
function of pressure at 35 °C in the presence of 0.1 M (n-Bu)4NPF6 were
obtained by use of a pill-box cuvette combined with a high-pressure
optical cell (see Experimental Section).
six-coordinate component of the product mixture. This is in
agreement with what has been observed on studying tem-
perature dependent spectra of liver microsomal cytochrome
P450 in the presence of different substrates.25 On releasing
pressure the starting spectrum was obtained, which demon-
strates the reversibility of the pressure induced change in the
equilibrium position. The quantification of the equilibrium
constant K3 could not be achieved since the spectra of the
pure iron(II)-superoxo or pure iron(III)-peroxo form 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 either the one or the other direction to independently
characterize these two species.
-
low-spin, six-coordinate FeII-O2 intermediate to the
Conclusions
high-spin, five-coordinate transition state (vide supra).
This process would require a quite positive ΔVq(koff),
which wouldcorrespondtoΔVq(k-2)depictedinFigure2.
The experimentally obtained moderately negative value
of ΔVq(koff) can only be accounted for if the involvement
of an equilibrium between the low-spin FeII-superoxo
and high-spin FeIII-peroxo species in the product solution
is taken into consideration. This supports our suggestion
that the high-spin FeIII-peroxo complex must coexist in
equilibrium with its redox-tautomer, low-spin FeII-superoxo
form, and therefore ΔVq(koff) is the sum of two contribu-
tions, namely, ΔVq(koff) = ΔVq(k-2) - ΔV°(K3) since
koff = k-2/K3 (see Scheme 1 and Figure 2). The value
of ΔV°(K3) is expected to be very similar to that of
ΔV°(K1) because of the similarity of the overall reactions
involved, that is, about þ26 cm3 mol-1, which leads to
the overall negative value found for ΔVq(koff), namely,
In this paper, for the first time, the thermodynamics of the
iron(II) porphyrin low-spin/high-spin equilibrium and high-
pressure studies on the reversible binding of superoxide to a
metal center are reported. The obtained results elucidate the
overall metal-superoxide reaction mechanism and the nature
of all species that appear along the reaction coordinate. On
the basis of the overall volume profile presented in Figure 2,
the nature of the transition state is almost identical to that of
the high-spin five-coordinate complex in the reactant state
showing that the spin change on the iron(II) porphyrin center
is crucial for its reactivity and activates it toward substitution
and consequently inner-sphere electron transfer processes.
The only difference between the high-spin complex of the
reactant state and the transition state is that the latter
contains KO2 in the pocket between the porphyrin and the
covalently attached crown ether moiety so that the superoxide
is in a close proximity to the reactive iron center. The volume
profile reveals that the product solution contains an equilib-
rium mixture of the high-spin iron(III)-peroxo and low-spin
iron(II)-superoxo complexes. The pressure dependence of the
UV/vis spectrum of the product solution confirms the pres-
ence of this equilibrium. These results offer strong support
-7.2 ( 0.5 cm3 mol-1
.
Further confirmation for the existence of the high-
spin/low-spin equilibrium in the product state was
obtained by monitoring the UV-vis spectrum of the
product solution as a function of pressure. As expected
the increasing pressure causes a small but significant
red-shift of the Soret absorption band (see Figure 3),
indicating a shift of the equilibrium toward the low-spin
€
(25) Rein, H.; Ristau, O.; Friedrich, J.; Janig, G.-R.; Ruckpaul, K. FEBS
Lett. 1977, 75, 19–22.

€
Durr et al.
11260 Inorganic Chemistry, Vol. 49, No. 23, 2010
for the existence of the iron(II)-superoxo species not as a
resonance form of the iron(III)-peroxo but as a discrete stable
complex in solution, which is in perfect agreement with our
the effects of the solvent, a trans ligand, and possible
involvement of coordinated peroxide/superoxide in hydro-
gen bonding or electrostaticinteractions, on the structure and
electronic properties of the heme iron-superoxide adducts
and the peroxo/superoxo equilibrium, as well as their reac-
tivity toward different organic substrates of biological and
synthetic interest. To reveal the effect of the nearby positive
charge, we are currently studying the analogous iron por-
phyrin complex without covalently attached crown ether.
€
recent report on the corresponding Mossbauer-, EPR-, and
mass-spectra of the reaction product, as well as DFT
calculations.6
The existence of two different forms of FeII-superoxide
adducts, that is, the reduced FeII-dioxygen adduct, is very
intriguing because it can better account for their versatile
reactivity toward different substrates.26 In general, superoxo
species are prone to abstract a hydrogen atom,27 whereas
peroxo species exhibit rather nucleophilic reactivity.10,28
Thus, future work should be directed toward understanding
Acknowledgment. This work was supported by the
Deutsche Forschungsgemeinschaft through SFB 583
“Redox-active metal complexes”.
(26) Selke, M.; Sisemore, M. F.; Valentine, J. S. J. Am. Chem. Soc. 1996,
118, 2008–2012.
Supporting Information Available: Temperature dependent
NMR spectra of [FeII(Porph)(DMSO)n] (n = 1, 2), species
distribution and the values of K1 at different temperatures and
pressures, plot of ln(K1) versus 1/T, pressure dependent rate
constants for the “on” and “off” reaction, pressure dependence
of ∂ ln(kobs)/∂t for the “on” reaction. This material is available
free of charge via the Internet at http://pubs.acs.org.
(27) (a) Bollinger, J. M.; Krebs, C. Curr. Opin. Chem. Biol. 2007, 11, 151–
158. (b) Mukherjee, A.; Cranswick, M. A.; Chakrabarti, M.; Paine, T. K.;
Fujisawa, K.; M€unck, E.; Que, L., Jr. Inorg. Chem. 2010, 49, 3618–3628.
(28) (a) Park, M. J.; Lee, J.; Suh, Y.; Kim, J.; Nam, W. J. Am. Chem. Soc.
2006, 128, 2630–2634. (b) Seo, M. S.; Kim, J. Y.; Annaraj, J.; Kim, Y.; Lee, Y.-M.;
Kim, S.-J.; Kim, J.; Nam, W. Angew. Chem., Int. Ed. 2007, 46, 377–380.