A Six-Coordinate Peroxynitrite Low-Spin Iron(III) Porphyrinate Complex - The Product of the Reaction of Nitrogen Monoxide (·NO(g)) with a Ferric-Superoxide Species

Article abstract of DOI:10.1021/jacs.7b08468

Peroxynitrite (^-OON=O, PN) is a reactive nitrogen species (RNS) which can effect deleterious nitrative or oxidative (bio)chemistry. It may derive from reaction of superoxide anion (O₂^?-) with nitric oxide (·NO) and has bee

Full text of DOI:10.1021/jacs.7b08468

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Article
A Six-Coordinate Peroxynitrite Low-Spin Iron(III) Porphyrinate
Complex – The Product of the Reaction of Nitrogen
Monoxide (•NO(g)) with a Ferric-Superoxide Species
Savita K. Sharma, Andrew W. Schaefer, Hyeongtaek Lim, Hirotoshi Matsumura, Pierre
Moënne-Loccoz, Britt Hedman, Keith O. Hodgson, Edward I. Solomon, and Kenneth D. Karlin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08468 • Publication Date (Web): 01 Nov 2017
Downloaded from http://pubs.acs.org on November 2, 2017
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Journal of the American Chemical Society
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A Six-Coordinate Peroxynitrite Low-Spin Iron(III) Porphyrinate
Complex – The Product of the Reaction of Nitrogen Monoxide
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(
•NO ) with a Ferric-Superoxide Species
(
g)
†
‡
‡
§
§
Savita K. Sharma, Andrew W. Schaefer, Hyeongtaek Lim, Hirotoshi Matsumura, Pierre Moënne-Loccoz,
||
‡, ||
, ‡, ||
, †
Britt Hedman, Keith O. Hodgson, Edward I. Solomon,* and Kenneth D. Karlin*
†Johns Hopkins University, Baltimore, Maryland 21218, USA
‡Department of Chemistry, Stanford University, Stanford, California 94305, USA
§Division of Environmental & Biomolecular Systems, Oregon Health & Science University, Portland, OR 97239-3098, USA
||Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, California
9
4025, USA
KEYWORDS Ferric superoxo, Heme-peroxynitrite, Ferryl, Dioxygen, Nitrogen dioxide, Nitrogen monoxide.
–
ABSTRACT: Peroxynitrite ( OON=O, PN) is a reactive nitrogen species (RNS) which can effect deleterious nitrative or oxidative
•
–
(
bio)chemistry. It may derive from reaction of superoxide anion (O
unobserved bound heme-iron-PN intermediate in the catalytic cycle of Nitric Oxide Dioxygenase (NOD) enzymes, which facilitate a •NO
III –
2
) with nitric oxide (•NO) and has been suggested to form an as-yet
III
Im
homeostatic process, i.e., its oxidation to the nitrate anion. Here, a discrete six-coordinate low-spin porphyrinate-Fe complex [(P )Fe (
OON=O)] (3) (P ; a porphyrin moiety with a covalently tethered imidazole axial ‘base’ donor ligand) has been identified and characterized
Im
by various spectroscopies (UV-Vis, NMR, EPR, XAS, resonance Raman) and DFT calculations, following its formation at –80 °C by addition
Im
III
•-
of •NO(g) to the heme-superoxo species, [(P )Fe (O
2
)] (2). DFT calculations confirm that 3 is a six-coordinate low-spin species with the
PN ligand coordinated to iron via its terminal peroxidic anionic O-atom with the overall geometry being in a cis-configuration. Complex 3
Im
III
-
thermally transforms to its isomeric low-spin nitrato form [(P )Fe (NO
3
)] (4a). While previous (bio)chemical studies show that phenolic
substrates undergo nitration in the presence of PN or PN-metal complexes, in the present system, addition of 2,4-di-tert-butylphenol
2
,4
( DTBP) to complex 3 does not lead to nitrated phenol; the nitrate complex 4a still forms. DFT calculations reveal that the phenolic H-atom
approaches the terminal PN O-atom (farthest from the metal center and ring core), effecting O–O cleavage, giving nitrogen dioxide (•NO
2
)
Im
IV
Im
III
-
plus a ferryl compound [(P )Fe =O] (7); this rebounds to give [(P )Fe (NO )] (4a).The generation and characterization of the long
3
sought after ferriheme peroxynitrite complex has been accomplished.
INTRODUCTION
mediated chemistry (vide infra). The anionic form of peroxyni-
trite (O=NOO ) exists in equilibrium with its conjugate acid
peroxynitrous acid form (O=NOOH), pKa = 6.8. PN and perox-
ynitrous acid decay rapidly by homolysis leading to the formation
-
Nitric oxide (•NO; nitrogen monoxide) is an important regu-
latory molecule in mammalian biology functioning as an intracel-
lular messenger which mediates multiple physiological processes,
including regulation of blood pressure, neurotransmission, im-
mune response and platelet aggregation. It is generated enzymat-
of nitrogen dioxide (•NO
30% yield or by isomerization to nitrate (NO
Nitrogen dioxide (•NO ) is a more moderate oxidant as well as
a nitrating agent (R-H + 2 NO
PN and/or its “decay” radical products (e.g., NO
2
) and hydroxyl radicals (•OH) in
− 3
~
3
).
2
ically from L-arginine, NADPH and O
2
by nitric oxide synthases
NOS). Cellular overproduction of NO can lead to toxicological
processes via the formation of reactive nitrogen species (RNS),
4
1
2
––> R-NO
2
+ HNO
2
). Thus,
(
2
, OH) are capa-
–
ble of performing oxidation and/or nitration reactions, including
reactions with biomolecules such as tyrosine, thiols, unsaturated
including NO and peroxynitrite (ONOO , PN) [oxoperoxoni-
2
trate(-1)] that are typically more reactive and toxic than NO. PN
4d,
5
9
-
fatty–acid–containing lipids, and DNA.
These reactions can
can be generated by the diffusion controlled (k = 3.9–19 × 10 M
1
-1
.-
2
contribute to a number of toxicological processes that include
S ) reaction of superoxide (O
2
) and nitric oxide or by metal ion
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DNA damage, interference with critical cell–signaling functions,
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protein nitration leading to cell death, etc. Peroxynitrite is also
implicated in a number of neurodegenerative diseases including
amyotrophic lateral sclerosis (ALS), Parkinson’s and Alzheimer’s
Scheme 1. Proposed Catalytic Cycle of Hemoglobin (Hb) act-
ing as a Nitric Oxide Dioxygenase (NOD).
5
a
disease. Nitration of tyrosine residues in proteins can have sig-
nificant effects that interfere with enzyme active site activity, pro-
6
tein folding, and consequently, function; evidence for the for-
mation of 3–nitrotyrosine at stoichiometric levels has been asso-
7
ciated with substantial loss of enzyme activity in the aging heart.
Other metalloproteins such as manganese superoxide dismutase
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(MnSOD), and copper-zinc superoxide dismutase (CuZnSOD)
6
c, 6d, 8
are known targets of tyrosine nitration.
nitration/inactivation of MnSOD can lead to an O
environment, and thus enhanced peroxynitrite formation, leading
It was shown that
•–
2
enriched
6
b,9 ,10
to further damage.
Chart 1.
To avoid toxic NO levels, microbial heme proteins known as
nitric oxide dioxygenases (NODs) catalyze the dioxygenation of
NO to produce the biologically benign nitrate anion (NO ).
Because of the importance of PN (bio)chemistry,
H)OON=O has received the attention of many theoretical stud-
(
–
14
3
ies to understand its formation, structure and reactivity. The per-
–
Oxygenated heme proteins are major targets/sinks of NO in
mammalian biological systems, specifically hemoglobin (Hb) and
myoglobin (Mb) which also have known NOD activity (Scheme
oxynitrite anion (O=NOO ) may exist in two geometric forms,
1
1
12
the cis and the trans isomers (Chart 1). Tsai and co-workers
employed a variety of computational methods and found that cis-
14a-c
-
-
1). It is generally thought that this occurs via the formation of
(
O=NOO ) is more stable than trans-(O=NOO ) by 3-4
III
–
a coordinated ferric peroxynitrite intermediate Fe ( OON=O).
However, ambiguity remains regarding the lifetime of such in-
termediates and whether isomerization to nitrate is initiated by
homolytic, heterolytic, or concerted O–O cleavage.
It is believed that the first stage of the reaction (oxy-heme +
NO) leads to the formation of a coordinated heme-peroxynitrite
species, but at this time there is no strong experimental evidence
supporting this assignment. Kinetics studies by Herold and co-
workers using rapid scan UV−vis spectroscopy, in a reaction of
kcal/mol. A theoretical study by Silaghi-Dumitrescu and co-
1
3
workers on the nonheme models of active sites of superoxide
reductase (SOR) and iron superoxide dismutase (FeSOD) inves-
tigated the formation and decomposition of possible peroxyni-
trite isomers in their ferrous and ferric oxidation states. Based on
their results from DFT calculations, the iron-bound cis form of
PN is more stable than the trans form. These researchers also
-
found that all ferrous models of cis and trans Fe-( OON=O) lead
to the immediate heterolytic cleavage of the O–ONO bond to
-
IV
NO(g) with oxy-myoglobin (MbO
2
) and oxy-hemoglobin
give nitrite (NO
2
) and Fe =O ferryl products, while on the other
(
HbO ), resulted in the formation of transient species with milli-
2
hand, the cis and trans ferric adducts homolytically cleaved to give
1
5
14a
13b
second lifetimes. Subsequently, Olson and co-workers used
EPR to probe rapid freeze-quenched (RFQ) samples from the
nitrogen dioxide plus the ferryl product. The same author has
also carried out DFT calculations on peroxynitrite adducts of
ferrous and ferric histidine-ligated hemes, and explored linkage
isomerism and the effect of protonation in these adducts, provid-
ing insight into possible mechanisms employed by hemoproteins
for scavenging peroxynitrite in vivo.
NO(g) reaction with HbO , reported formation of a high-spin
2
Fe(III) species on this same time scale. These intermediates were
assigned as high-spin ferric heme peroxynitrite complexes on the
basis of their electronic spectra. However, Goldstein and co-
1
6
workers reported an intermediate with analogous spectral prop-
erties from the reaction of ferryl myoglobin with NO2(g) and con-
cluded that it was the nitrato analog Fe (ONO
resonance Raman spectroscopy, Moënne-Loccoz and coworkers
identified an intermediate formed in the reaction of NO(g) with
III
2
). Similarly, using
MbO
2
at 3 °C in alkaline solution and trapped by RFQ on the
millisecond time-scale as the nitrato, not the peroxynitrite, com-
1
7
plex. Ford and Kurtikyan also carried out the experiment to
III
-
probe the feasibility of Fe ( OON=O) intermediate using the
six-coordinate oxyheme model complex (NH )Fe(Por)(O ) and
reacting it with NO(g) at low temperature via a sublimation and IR
spectroscopy methodology. Surprisingly, even at 80–100 K, the
six coordinate nitrato complex was formed without spectral de-
3
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tection of any intermediate. However, using more kinetically
stabilized d cobalt(III) complexes, Kurtikyan and co-workers
showed that the oxy−coboglobin model compound
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Scheme 2. Reaction (solvent: THF at -80 ⁰C) sequence showing
the formation of complex 2 by bubbling O2(g) to a solution of
complex 1 followed by the addition of •NO(g) to generate com-
plex 3 which thermally decay to give complex 4a.
(NH
3
)Co(Por)(O
2
)/(Py)Co(Por)(O ) also promotes the NOD
2
reaction, and that they could unambiguously stabilize and charac-
terize (by FT-IR spectroscopy and DFT calculations) the bound
cobalt-peroxynitrite complex, also demonstrating that the reac-
tion sequence paralleled the proposed scheme for hemeproteins
1
9
(Scheme 1). However, so far clear experimental evidence con-
firming the formation of coordinated peroxynitrite at the initial
stage of the NOD reaction has not been accomplished for heme–
containing globins or synthetic iron–porphyrins.
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Chart 2.
Previously, our laboratory carried out a spectroscopic study to
III
-
probe the viability of a Fe ( OON=O) intermediate in the reac-
III
•- 20
tion of oxy-heme model complexes of (F
8
)Fe -(O
2
)
and
Py
III
•- 21
(
P )Fe -(O ) (Chart 2) with NO(g) at low temperature, both
2
EXPERIMENTAL SECTION
of which resulted in the formation of five coordinated nitrato
complex without spectral detection of any intermediates. Howev-
er, as the addition of a phenol resulted in its o-nitration, we in-
ferred that a short lived peroxynitrite or peroxynitrite-like species
must have formed, but in the absence of the phenolic substrate,
isomerization to the nitrato complex was quite fast.
Materials and Methods. All reagents and solvents purchased
and used were of commercially available analytical quality except
as noted. Dioxygen was dried by passing through a short column
of supported P
4
O10 (Aquasorb, Mallinkrodt). Nitrogen dioxide
(•NO ) was obtained from Sigma-Aldrich (≥ 99.5%, 500 mL
2
In this work, we further probed for this most puzzling species
expected to form upon heme-superoxo reaction with NO(g), i.e. a
lecture bottle, ~ 14.33 psi @ 20 ⁰C). Nitrogen monoxide (•NO)
gas was obtained from Matheson Gases (High Purity Grade, Full
cylinder ~500 psi @ 20 ⁰C) and purified as follows: it was first
passed through multiple columns containing Ascarite II (Thomas
Scientific) to remove higher nitrogen oxide impurities. Further
purification by distillation was completed by warming frozen
III
–
Fe -( OON=O) heme species, now employing the very robust
Im
II
Im
five-coordinate reduced heme complex (P )Fe (1) (P = por-
phyrin with chelated axial imidazole base) as starting material,
which forms a stable six coordinate superoxo complex
Im
III
•-
22
[(P )Fe (O
2
)] (2) at low temperature (Scheme 2). In fact,
NO(g) (as crystalline N
um trap to 193 K through use of an acetone/dry–ice (- 80 ⁰C)
bath, and collection in a second liquid N cooled evacuated vacu-
2
O
2
) from 78 K in a liquid N cooled vacu-
2
reaction of 2 with NO(g) under cryogenic conditions resulted in
the formation of a new species which was stable under these con-
ditions, which we assign as the long sought after peroxynitrite
2
um trap. This secondary flask was again warmed to -80 ⁰C and
the purified NO(g) was collected in an evacuated Schlenk flask
(typically 50 mL) closed with a rubber septum secured tightly by
copper wire. Using these procedures, as previously described in
Im
III –
ferriheme complex, [(P )Fe ( OON=O)] (3). This species was
stable enough to perform spectroscopic analysis before decaying
to the low-spin, six-coordinate nitrato compound
Im
III
20
[(P )Fe (NO
3
)] (4a), as shown in Scheme 2 and further to the
even more detail, NO(g) in the 50 mL Schlenk flask is sitting at
Im
III
-
high-spin complex [(P )Fe ](NO
3
) (4b) (see SI, Scheme S1).
pressures a bit above 1 atm. The NO2(g) was filled directly from
the lecture bottle in 10 mL Schlenk flask. Addition of NO(g),
NO2(g) and O2(g) to metal complex solutions was effected by trans-
fer via a three-way long needle syringe connected to a Schlenk
line. Preparation and handling of air–sensitive compounds were
performed under an argon atmosphere using standard Schlenk
techniques or in an MBraun Labmaster 130 inert atmosphere (<1
In this report, further details for these chemical transformations
are provided by spectroscopic characterizations, and Density
Functional Theory (DFT) calculations offer insights into the
structure of the peroxynitrite intermediate and the observed reac-
tivity.
ppm O
2
, <1 ppm H O) drybox filled with nitrogen gas. Solvents
2
were purged with Ar prior to use. THF and Pentane was distilled
over Na/benzophenone ketyl or calcium hydride. 2,4-di-tert-
2
,4
butylphenol ( DTBP) was purchased from Sigma–Aldrich and
purified by multiple recrystallizations in toluene under Ar. meta-
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chloroperbenzoic acid (mCPBA) was purchased from Sigma–
Aldrich and purified by washing an ethereal solution with buffer
(410 mL 0.1 M NaOH, 250 mL 0.2 M KH PO , diluted to 1 L,
pH 7.5) and then dried over anhyd. MgSO before drying in vac-
uo. All other reagents were used as received.
X-ray Absorption Spectroscopy. Fe K-edge X-ray absorption
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spectroscopy (XAS) was conducted at the Stanford Synchrotron
Radiation Lightsource (SSRL) on the unfocused 20-pole, 2.0 T
wiggler beam line 7-3 under storage ring parameters of 3 GeV and
~500 mA. A Rh-coated premonochromator mirror was used for
harmonic rejection and vertical collimation. A Si(220) double-
crystal monochromator was used for energy selection. The sam-
ples were loaded into delrin XAS cells with 38 μm Kapton win-
dows. The samples were maintained at a constant temperature of
~10 K during data collection using an Oxford Instruments CF
1208 continuous flow liquid helium cryostat. A Canberra solid-
state Ge 30-element array detector was used to collect Fe Kα
2
4
4
Benchtop UV-Vis measurements were carried out by using a
Hewlett Packard 8453 diode array spectrophotometer equipped
with HP Chemstation software and a Unisoku thermostated cell
holder for low temperature experiments. A 10 mm path quartz
cell assembly (with extended glass tube with female 14/19 joint
and stopcock) was used to perform all the experiments. UV-Vis
samples preparation: In a standard UV-Vis experiment, in the
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Im
II
-1
glovebox, 3.0 mL of a complex (P )Fe (1) (range: 0.1 mM to
.015 mM) solution in THF was placed in the cuvette assembly, a
fluorescence data to k = 16 Å . Internal energy calibration was
0
accomplished by simultaneous measurement of the transmission
of a Fe foil placed between two ionization chambers situated after
the sample. The first inflection point of the foil spectrum was
assigned to 7111.2 eV. For edge and pre-edge analysis, an average
of the first scans collected from physically separated measure-
stir bar was added and the assembly capped with a rubber sep-
tum. After transfer to the benchtop and spectrometer with cryo-
stat, the sample was cooled to - 80 ⁰C. Dioxygen gas was bubbled
through the solution using a gastight microsyring, until full for-
Im
III
·-
Im
III
·-
mation of the superoxo [(P )Fe (O
2
)] (2) complex occurred.
ment spots on the sample was used for complex [(P )Fe (O
2
)]
)] (4a), and
the first three-scan average was used for complex
Im
III
-
Im
III
-
Im
III
Similarly, complex [(P )Fe ( OON=O)] (3) was generated by
addition of NO(g) to complex 2. Excess O2(g)/NO(g) was removed
by several vacuum/Ar cycles.
(2), [(P )Fe ( OON=O)] (3), and [(P )Fe (NO
3
Im
III
[(P )Fe (OH)] (5). (see SI for the further method of XAS
analysis).
Im
II
Im
II
Im
III
Synthesis. (P )Fe /d -(P )Fe (1), [(P )Fe (OH)] (5),
Im IV
8
Im
III
[
(P )Fe ](SbF
6
) (6) and [(P )Fe =O] (7) were synthesized
NMR Spectroscopy. All NMR spectra were recorded in 7 inch, 5
2
2-23
2
as previously described.
The oxy-heme complex
)] (2) was generated by bubbling dioxygen to a
mm o.d. NMR tubes. Low–temperature H-NMR (Bruker 300
Im
III
·-
2
[
(P )Fe (O
solution of complex 1. Similarly, complex [(P )Fe ( OON=O)]
3) was generated by removing excess of O2(g) from complex 2 by
2
MHz (46.05 MHz for H NMR) spectroscopy was carried out
Im
III -
using the spectrometer equipped with a tunable deuterium probe
(
to enhance deuterium detection); measurements were performed
2
several cycles of vaccum/Ar purge, followed by addition of NO(g)
at –80 °C under a N
2
atmosphere. The H chemical shifts were
2
via gas tight syringe under Ar. (For the synthesis of authentic
calibrated to natural abundance deuterium solvent peaks. H-
NMR samples preparation: Same as rRaman experiment, 0.50
mL of a complex d -(P )Fe (1) (2 mM) solution in THF was
Im
III
-
[
(P )Fe ](NO
3
) (4b), see the SI).
Im
II
Resonance Raman Spectroscopy. Samples were excited at a vari-
8
+
ety of wavelengths, using either a Coherent I90C-K Kr ion laser,
placed in a 5 mm rubber septum capped NMR tube. After cooling
+
Liconix HeCd laser, a Coherent 25/7 Sabre Ar ion laser, or a
to –80 ⁰C (acetone/N2(liq) bath), dioxygen was bubbled through
Im
III
·-
Lighthouse Photonics Sprout-D pumped M-Squared SolsTiS
Ti:Saph laser while the sample was immersed in a liquid nitrogen
cooled (77 K) EPR finger dewar (Wilmad), or cooled at 110 K
through contacted with the liquid nitrogen cooled sample holder.
The laser power (< 2 mW) and sample spinning conditions
the solution mixture to form the complex [(P )Fe (O )] (2).
2
The NMR tube was transferred rapidly into the NMR instrument
Im
III
-
which was precooled to -85 ⁰C. Complexes [(P )Fe (
Im
III
-
OON=O)] (3) and [(P )Fe (NO
3
)] (4a) were prepared in a
similar manner.
were set such that the RR spectra obtain before and after
extended data acquisition showed no detectable differences.
Resonance Raman sample preparation: In a typical experiment,
Electron Paramagnetic Resonance (EPR) Spectroscopy. Electron
paramagnetic resonance (EPR) spectra for all frozen samples
were recorded on a Bruker EMX spectrometer equipped with a
Bruker ER 041 X G microwave bridge and a continuous-flow
liquid helium cryostat (ESR900) coupled to an Oxford Instru-
ments TC503 temperature controller. Spectra were obtained at
Im
II
0
.650 mL of a complex (P )Fe (1) (2 mM) solution in THF
were placed in a 9 mm economy rubber septum capped NMR
tube. After cooling the NMR tube to -80 ⁰C (acetone/N2(liq)
bath), O2(g) was bubbled through the solution mixture to form the
1
9
4 K under nonsaturating microwave power conditions (ν =
.4108 GHz, microwave power = 0.201 mW, modulation ampli-
Im
III
·-
complex [(P )Fe (O )] (2). Similar to the procedure used for
2
Im III –
UV-Vis experiments, complex [(P )Fe ( OON=O)] (3) was
prepared by removing excess of dioxygen by Vacuum/Ar cycles
from 2 and careful addition of NO(g) was effected using three-way
tude = 10 G, microwave frequency = 100 kHz, receiver gain =
3
5
.02 × 10 ). EPR samples preparation: All samples were prepared
in a similar way as NMR and rRaman samples.
Im
III
–
gas tight syringe. Complex [(P )Fe ](NO )] (4b) was pre-
3
DFT calculations. Density functional theory calculations were
performed using Gaussian 09, Revision D.01. All geometry opti-
mizations and energies were obtained using the spin-unrestricted
B3LYP functional and ultrafine integration grid. The basis sets
used were as follows: 6-311g* for Fe, peroxynitrite (N and O),
and all N atoms immediately bound to Fe; 6-31g for all other
pared by thermal decay of complex 3. After generation of all
complexes, tubes were frozen in N2(liq) and flame sealed. Isotopi-
1
8
15 18
cally labeled ( O
2
/ N O) samples were prepared in analogous
manner.
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Journal of the American Chemical Society
atoms in the molecule (C, F, H, and remaining N and O). All
calculations were performed using a THF solvent PCM.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
RESULTS AND DISCUSSION
Im
II
Interaction of (P )Fe (1) with O2(g)/NO(g)
.
(1) UV-Vis Spectroscopy. In THF solvent, ferrous heme
complex 1 (0.015 mM) is a six-coordinate low-spin complex (at
2
2
th
all temperatures) (THF as 6 ligand) [λmax = 417 nm (Soret), ε
-
1
-1
-1
-1
= 258 600 M cm , 525 nm, ε = 28 650 M cm , 554 nm (sh), ε =
-
1
-1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7 250 M cm ]. Bubbling with O2(g) generates superoxo species
[(P )Fe (O )] (2) [λmax = 423 nm, ε = 247 440 M cm , 533
Im
III
•-
-1
-1
2
-
1
-1 22
nm, ε = 23 037 M cm ]. After removing the excess of O2(g)
using vacuum/Ar cycles, addition of 250 μL from our standard
freshly purified NO(g) flask (see Experimental Section) to the
solution of complex 2 causes an immediate change in the absorp-
tion spectrum, with a blue shift in the Soret band from 423 nm to
417 nm and concomitant Q band red shift from 533 nm to 541
nm, leading to the formation of the putative peroxynitrite species
Figure 1. UV-Vis spectroscopy showing generation of superoxo
complex 2 (red spectrum) by bubbling O2(g) through the THF solu-
Im
II
tion of (P )Fe (1) (black) at –80 °C, followed by addition of
•NO(g) to generate peroxynitrite complex 3 (green) with the final
decay product (warming to RT) being nitrate complex 4b (grey)
(Inset; Soret band and Q-band for 1, 2, 3, and 4b)
Im
III
–
-1
-1
[(P )Fe ( OON=O)] (3) (λmax = 417 nm, ε = 220 700 M cm ,
5
-
1
-1
41 nm, ε = 15 287 M cm ) (Figure 1, Scheme 2). This is stable
at -80 °C for minutes before slowly transforming to complex 4a
-
1
-1
-1
[λmax = 414 (Soret), ε = 220 500 M cm , 544 nm, ε = 15 000 M
-1
4
06.7-nm Exc.
441.6-nm Exc.
1359
cm ], which is
a
six-coordinated nitrato species
1
359
1567
Im
III
[(P )Fe (NO )] 4a (Scheme 2).
3
Confirmation that 4a is a nitrate compound (nitrogen-
containing product derived from •NO(g)) was obtained using
QUNTAFIX nitrate/nitrite test paper (semi-quantitative) and by
1546
1568
1
624
1314
1
1546
1346
1624
1569
1
2
2
4
ion chromatography (see SI). The test was performed after
warming 4a to RT, where the UV-Vis spectrum is that of a typical
five-coordinate high-spin complex 4b (Scheme S1, Figure 1).
Thus, we postulate that the nitrato ligand is not bound but rather
1368
1569
1
369
2
3
1570
1
370
1
570
Im
III
serves as a counter-anion at RT, i.e., with [(P )Fe ](NO
3
) (4b)
1371
as the formulation [λmax = 413, 501, 527, 572 (sh), 634 (sh) nm],
n4
n2
n4
n2
3
THF
as is known for a well-characterized analog compound,
Im
III
23,25
[(P )Fe ](SbF
6
) (6). Further, because it is relevant for stud-
1300
1400
1500 -1
1600
1300
1400
Raman shift / cm
1500
1600
-1
ies described below, we note that superoxo compound 2 does
Raman shift / cm
thermally decay and at RT transforms to the high-spin hydroxy
Figure 2. rRaman spectroscopy collected at 406.7 nm (left) and
Im
III
22
complex [(P )Fe (OH)] (5). In our UV-vis experiment we
did not observe any ferric or ferrous nitrosyl species (see Figure
S9).
Im
II
441.6 (right) nm excitation for complexes [(P )Fe ] (1, black),
Im
III
-.
Im
III
-
[(P )Fe -(O2 )] (2, red), and [(P )Fe -( OON=O)] (3, green),
showing the oxidation state marker band (ν ) and spin state marker
band (ν ). Note that apparent peak maxima can vary with excitation
due to differences in relative enhancement of contributing species.
2) Resonance Raman Spectroscopy. Further properties of
4
2
(
the products of dioxygen and NO(g) (Scheme 2) were interrogat-
ed using resonance Raman (rR) spectroscopy. The high-
frequency rR spectra of 1, 2 and 3 obtained with a 406.7 nm exci-
tation show oxidation state marker band (ν
4
) at 1359, 1368, and
) at 1567, 1569 and
-1
1
1
371 cm , and spin-state marker band (ν
571 cm , respectively. DFT calculations (vide infra) support the
2
-1
-1
slight increase in ν
4
and ν
2
frequencies (~1 cm calc.) for 3 rela-
tive to 2, which derives from the more π acidic peroxynitrite lig-
and causing a decrease in backbonding from Fe into the porphy-
rin e
g
orbitals (resulting in higher energy ν
4
and ν
2
core modes in
26
3
). Residual signals from 1 are clearly observed in the rR spectra
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Journal of the American Chemical Society
Page 6 of 13
of 2 and 3, presumably due to some loss of the superoxo complex
during the degassing of excess O prior to exposure to NO, but
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
spectra obtained with 406.7, 413.1 nm and lower energy excita-
tions like 441.6 and 457.9 nm (Figure 2 and Figure S4) identify 2
and 3 as distinct six–coordinate low-spin Fe(III) complexes (Fig-
1
6
18
ure 2). Two O
2
/ O
2
isotopic sensitive shifts were observed for
Im
III
•-
-1
the superoxo complex [(P )Fe (O
2
)] (2), at 1180 cm
1
8
−1
-1
18
−1
[Δ( O
2
), -56 cm ] and 575 cm [Δ( O
2
), -23 cm ] correspond-
ing to the O-O and Fe-O stretches, respectively (Figure S4), as
2
2
previously observed. As a control, we collected rRaman data on
the ferrous-nitrosyl complex, which has an v(Fe–NO) stretch of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5
-1
570 (Δ N = -20) cm , (Figure S4); this mode is not observed in
Im
III –
the data for complex 3. Complex [(P )Fe ( OON=O)] (3) was
also excited at several wavelengths ranging from 406.7 nm to 900
1
5
nm, but no enhancement of an isotope-sensitive mode ( N or
1
8
O) was observed.
(3) X-ray Absorption Spectroscopy. Figure 3 shows the Fe
K-edge XAS spectra and the enlarged pre-edge region of 2, 3, 4a,
and 5. In comparing the data obtained for 2 and 3, the edge and
pre-edge features show notable changes. In addition, the spectral
features at 7130 – 7140 eV are different between 3 and 4a. These
indicate that 3 has distinctive properties compared to the super-
oxo complex 2 and the decay product 4a. Further information
about the spin state and the coordination number can be ob-
tained from the pre-edge region and the extended X-ray absorp-
tion fine structure (EXAFS) data. The Fe K pre-edge feature at
~
7112 eV originates from a 1s → 3d transition. This transition is
electric dipole forbidden, but gains weak intensity through a
quadrupole mechanism. For non-centrosymmetric complexes,
the intensity of this pre-edge feature increases due to metal 4p
mixing into the 3d orbitals. A systematic study using various Fe
model complexes showed that this feature is sensitive to oxida-
Figure 3. Fe K-edge XAS spectra (top) and enlarged pre-edge region
(bottom) for complex 2 (red), 3 (green), 4a (grey), and 5 (blue).
The inset shows smoothed second derivatives of the pre-edge region.
2
7
tion state, spin state, and site symmetry. The second derivative
of the pre-edge of 3 exhibits three features centered at 7111.3,
7
112.7, and 7113. 9 eV with intensities of 1.0, 3.6, and 1.2 units,
(4) NMR Spectroscopy. More structural information was ob-
2
respectively, for a total intensity of 5.8 units (Figure 3 and Table
S1). The shoulder at lower energy (~7111 eV), which was not
observed in the high-spin species of 5, is a key feature for low-spin
tained for these complexes from low temperature H-NMR spec-
troscopy (see Experimental Section). We previously reported on
Im
III
•–
22
complex d
8
-[(P )Fe (O
2
)] (2) (δ 10.0 ppm, low-spin,
2
7-28
5
Fe(III) model complexes including heme complexes.
The
Fe(III), d , diamagnetic) obtained by bubbling molecular oxygen
into a solution of complex 1 (δ 9.5 ppm, low-spin, Fe(II), d ),
total intensity of 5.8 units is typical for six-coordinate Fe(III)
synthetic complexes. The EXAFS data show that 3 is a six-
coordinate species with the first shell fit best using six Fe-N/O
contributions at 1.99 Å (Figures S10, S11, and Table S2). Taken
together, XAS suggests that peroxynitrite complex 3 is a six-
coordinate low-spin Fe(III) species.
6
shown in Figure 4 (top). Addition of NO(g) to complex 2, resulted
Im
III –
in the formation of complex [(P )Fe ( OON=O)] (3) with
pyrrolic-proton resonances occurring at three different frequen-
cies, (δ –1.0, 8.94 and 18.10 ppm) in a ratio of 1:2:1. Splitting in
deuterated pyrrolic-protons indicates the presence of low sym-
metry in the compound, which arises from the covalently teth-
ered imidazole group, as well as coordination of the peroxynitrite.
The small upfield shift of pyrrole-proton peak, here found at –1.0
ppm is common for low-spin S = ½ iron(III) six-coordinate com-
2
9
pounds. With thermal decay of peroxynitrite complex 3, a nitra-
Im
III
to product forms, [(P )Fe ](NO ) (4b), as presented in
3
Scheme 2 and Scheme S1) and this exhibits typical (for high
spin) downfield shifts in the pyrrole hydrogen resonances, at
2
5
2.0, 42.0 and 34.0 ppm. Therefore, the H-NMR spectroscopic
data agree with our analysis of the UV-Vis and rR spectropsopic
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Journal of the American Chemical Society
and low temperatures, saturating at ~50% 4a formation). Fur-
experiments, in further support our formulation of 3 as a peroxy-
nitrite complex [(P )Fe ( OON=O)].
Im
III –
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
thermore, the signal for decay product 4b matches the authenti-
cally synthesized nitrato complex (see SI, Figure S1 and Figures
S5-S7).
3
0
Thus, the overall chemistry sequence shown in Scheme 2 par-
allels that found in the enzymes, NOD activity, where NO(g) is
oxidized to the nitrate anion in the Fe-O
2
+ NO(g) reaction. We
note that there is no evidence that a nitrito complex
Im
III
[
(P )Fe (NO )] ever forms; we also independently generated
2
this species and it possesses markedly differing UV-vis and EPR
spectroscopic properties, see the SI, Figure S7. Under these con-
ditions, we also do not observe formation of any ferrous/ferric
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1
nitrosyl complex. As we have shown earlier the ferrous nitrosyl
complex has characteristic UV-vis and EPR spectroscopic fea-
tures, shown in Figure S9.
Scheme 3. Reactivity of complex 3 towards 2,4-di-tertbutyl phe-
2
,4
2,4
nol ( DTBP): (top) DTPB added to complex 3; (bottom)
2
,4
DTBP added to 2 prior to complex 3 formation, which results in
nitration of phenol.
Figure 4. 2H-NMR (top) in THF at –80 °C for complexes (1, 2, 3
and 4b). (*) is for solvent THF; (bottom) EPR of peroxynitrite
complex 3 (green) in THF at 14 K. Decay of complex 3 leads to the
Im
III
formation of [(P )Fe -(NO )] (4b) (grey); note that only partial
3
formation of 4b (and increase in the g = 5.96 signal intensity) is
shown. See text for details.
(5) EPR Spectroscopy. EPR experiments also reveal that
complex 3 exhibits a low-spin, rhombic signature that is typical
(
6) Reactivity Studies. To gain additional insight into the
Im III –
properties of [(P )Fe ( OON=O)] (3), we investigated the
addition of 2,4-di-tert-butylphenol ( DTBP) to in situ-generated
for six-coordinated ferri-hemes (features at g
x
= 2.70, g
y
= 2.25
2,4
2
9a, 29c
and g = 1.72, Figure 4, bottom).
z
There is also a signal at g =
3
. Such reactions are known to give o-nitration of phenol for
5.96 that represents a rather small amount (5-10%) of a high-spin
Fe(III) species arising from decomposition; see Figures S5-S6
with text. The EPR spectroscopic experiments also demonstrate
20-21
some heme containing systems
and for complexes of other
2,4
32
transition metals. However, addition of DTBP to 3 yielded no
immediate spectral changes (via UV-vis and rR), and formation
of complex 4a was still observed after minutes at -80 ⁰C (Scheme
Im
III –
that putative peroxynitrite compound [(P )Fe ( OON=O)]
(3) is not a nitrato species, as thermal decay leads to the for-
mation of product 4b, associated with the spin state change to
high-spin (S = 5/2) (control experiments involving nitrate addi-
3
, top). Warming to RT yielded a positive test for nitrate ion and
2,4
only unreacted DTBP. This result suggests that isomerization
of peroxynitrite ( OON=O) complex 3 to nitrate complex 4a is
–
Im
III +
tion to [(P )Fe ] showed that the low-spin nitrato species (4a)
faster than phenol nitration. As depicted in Scheme 1, isomeriza-
–
could only be observed in the presence of a large excess of NO
3
-
tion of heme-peroxynitrite ( OON=O) to heme-nitrate (O-NO
2
)
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involves homolysis of the O-O bond to give a ferryl-oxo species
plus •NO , whereupon rebound of NO and formation of a new
minutes at -80 ⁰C is likewise indistinguishable from the decay of 3
to 4a (although the UV-vis features are slightly broader for the
reaction where DTBP is added before NO(g)). We are as of yet
unable to fully explain the observed difference in reactivity based
on our current data and DFT calculations (vide infra) (also see
reference 33).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
2
,4
34
O-N bond gives nitrate as product. However, under the present
reaction conditions, monitoring either by UV-vis, rRaman, NMR,
or EPR spectroscopies, we do not detect any high-valent Cmpd
IV
II-like (Fe =O) species.
(7) DFT Calculations. To better understand the nature of the
Im
III –
peroxynitrite complex [(P )Fe ( OON=O)] (3), and explore
possible effects of phenol addition both before and after for-
mation of 3, we turned to DFT calculations. For peroxynitrite
binding to a metal ion, several possible binding modes and con-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
formations must be considered, including N-atom vs. O-atom
ligation, cis- vs. trans- isomers (Figure 6a and 6b, respectively),
3
1
and even O,O’-chelation. Peroxynitrite N-binding to a heme-
1
3
Fe(III) has been proposed based on theoretical calculations, but
peroxo anion O-atom binding is observed experimentally (and
supported by DFT calculations) for several porphyrinate co-
1
9a
balt(III)-peroxynitrite complexes.
a
b
Figure 5. (top) Reaction of Compound II with •NO2(g) leads to
ferric nitrate complex 4b (see text and SI for detail discussion). (bot-
tom left) UV-vis spectroscopy of in situ generated ferryl complex 7 by
reacting 1 with mCPBA; (bottom right) the UV-vis of insitu generat-
ed complex 4b by addition of •NO2(g) to 7.
c
d
To probe the plausibility of this fast isomerization reaction, we
Im
IV
23
first generated the Cmpd II-like [(P )Fe =O] (7) complex by
adding 1.5 eq of mCPBA (meta-chloroperoxybenzoic acid) to
complex 1 at -80 ⁰C in THF, monitoring the reaction by UV-vis
spectroscopy (Figure 5, bottom left). Following the formation of
complex 7, addition of NO2(g) (see Experimental Section) result-
ed in a very short-lived intermediate observed in a single spec-
trum at -80 °C using our benchtop UV-vis spectrometer, which
immediately converted into the final Fe(III)-nitrato product 4b,
confirmed by a positive nitrate test as well as characteristic UV-
Vis and EPR spectroscopic features (Figure 5, bottom right; see
also Figure S8 with explanations).
Figure 6. DFT optimized structures for possible orientations of in-
termediate 3, as discussed in the text.
Indeed, our calculations predict that the lowest energy struc-
ture of 3 is cis-peroxynitrite bound to Fe(III) through the termi-
nal peroxy O (Figure 6a), which is 3.7 kcal/mol (all energies
given herein are ΔG at -80°C unless otherwise noted) more stable
than the trans isomer (Figure 6b), in agreement with computa-
Interestingly, we do observe effective nitration chemistry when
DTBP (1.1 equiv) is added to 2 prior to NO(g) at -80 ⁰C
2,4
(
(
[
Scheme 3, bottom). In this case, workup of the reaction solution
1
1-13
tional results reported for other metal-peroxynitrite systems.
requiring warming to RT) reveals that the ferric product
Im
III
We also investigated the possibility of a cis-peroxynitrite binding
via the O-atom of the N=O moiety (Figure 6d), but this is found
to be much higher in energy (~20 kcal/mol) than the peroxo O-
bound isomers shown in Figure 6a,b. Geometry optimization of
an N-bound peroxynitrite (Figure 6c and Table S3), which could
(P )Fe (OH )] forms along with high yields (>85%) of 2,4-di-
2
tert-butyl-6-nitrophenol (2-NO
well as a negative test for NO
2
-DTBP) (GC analysis; see SI), as
-
-
,33
3
/NO
2
ions (see SI). The nitrated
phenol product is only observed after stirring the reaction mix-
ture for hours at RT, however analysis of the solution immediate-
III
–
2
,4
form by attack of dioxygen on an Fe -( NO) complex, results in
ly after warming yields only 10-15% of unreacted DTBT, indi-
cating that the nitration reaction occurs prior to stirring at RT.
Interestingly, the species formed upon NO(g) addition at -80 ⁰C
cleavage of the interior N-O bond and dissociation of molecular
III
-
O
2
. It is interesting to note that a comparison of {Fe ( NO) + O
2
III
•–
2
,4
and {Fe (O
2
) + NO}, where the metal complex and gas mole-
cule are non-interacting species, shows the {Fe ( NO) + O } to
(i.e. 2 + DTBP + NO(g)) is spectrally indistinguishable (via UV-
Vis, rR, EPR, and NMR) from that of 3 generated without
III
-
2
2
,4
be 7.0 kcal/mol lower in energy. However, the cis-PN structure
DTBP present (as in Scheme 2), suggesting that a product
comparable to 3 has formed. Furthermore, the decay after several
III
•–
given for 3 is 8.5 kcal/mol lower than the separated {Fe (O
2
) +
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NO} (15.2 kcal/mol lower in ΔH, but this neglects the entropic
cost of NO association), and therefore formation of Fe (-
OONO) is thermodynamically favored. This is in agreement with
case without phenol present, rebound of NO
2
to form nitrate has
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
III
‡
a very low barrier (ΔG = 3.2 kcal/mol) and proceeds to form the
III
–
Fe (NO ) product 4a (ΔG = –25.9 kcal/mol) shown in Figure
3
IV
our experimental data demonstrating that 3 is in fact distinguish-
8c. In contrast, we note that H-atom abstraction by the Fe =O
III
31, 35
III
•– 22
III
•
able from Fe (NO)
and Fe (O
2
) .
(Figure 8b) to yield {Fe -OH + PhO + NO } is thermodynami-
2
III
-
cally uphill by 4.1 kcal/mol, greater even than the barrier for
isomerization to nitrate. Taken together, these results are in good
agreement with our observation that addition of DTBP to 3 still
Relative to Fe ( OONO) (Figure 6a), the geometry optimized
structure of Fe (NO
III
-
3
) is lower in energy by 31.5 kcal/mol for
2,4
the LS (S=1/2) and 28.5 kcal/mol for the HS (S=5/2) surfaces.
yields decay to the nitrate product 4a (at a rate comparable to
The preference for the LS state is likely due to the 6C structure, in
2
,4
2,4
–
that observed without DTBP), along with unreacted DTBP.
which NO
3
ion remains bound. Nevertheless, our calculations
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
support that the nitrato complex is the decay product of 3.
Taking the optimized structures for 3 and 4a, we evaluated a
reaction coordinate between them, and found that O–O cleavage
a
b
‡
was the lowest energy barrier (ΔG = 9.3 kcal/mol). Elongating
the O–O bond past the barrier, an intermediate is found that is
4
.1 kcal/mol lower energy than 3, and consists of an Fe(IV)=O
and •NO , where the NO molecule has reoriented such that the
N-atom is pointed toward the Fe-bound O-atom (Figure 7a).
2
2
The NO
2
can then rebound onto the Fe(IV)=O with a negligible
‡
barrier (ΔG = 2.5 kcal/mol) to form the N–O bond and yield the
nitrate product shown in Figure 7b, which is 31.5 kcal/mol below
the bound peroxynitrite structure (vide supra).
c
d
a
b
Figure 8. DFT-optimized structures for phenol-bound (a)
Im
Im
)Fe ( OONO) (3), (b) {(P )FeIV=O
III
–
Im
(
(
P
P
+
•NO }, (c)
2
III
–
Im
III
•-
)Fe (NO
3
) (4a), and (d) (P )Fe (O ) (2).
2
IV
Finally, we examined the case in which phenol is added prior to
Figure 7. DFT optimized structures for the (a) Fe =O + •NO
2
III
–
NO(g) to consider whether the observed change in nitration reac-
tivity could be attributable to a different peroxynitrite structure
forming initially, caused by the interaction with phenol. Phenol
binding to the Fe-superoxo species 2 is thermodynamically favor-
intermediate and (b) Fe (NO
3
) (4a) product following decay of
III
–
the Fe ( OONO) 3 complex.
We next sought to understand the difference in reactivity ob-
served for phenol addition before vs. after NO(g), where only the
latter produces effective phenol nitration. Since our data indicate
that a peroxynitrite species forms initially in both cases (vide su-
pra), we first considered the interaction of phenol with a fully
formed peroxynitrite to understand how it might impact the de-
cay process. Exploring several approach vectors of phenol onto
able by ΔE = –9.5 kcal/mol and ΔG = –1.2 kcal/mol, depicted in
Figure 8d. With the phenol bound to the terminal O, reaction
with NO initially yields a cis-peroxynitrite structure where the
phenol is H-bonded to O(2) that is downhill to form by 3.0
kcal/mol (see SI, Figure S12). However, this can easily isomerize
(with a barrier lower than that of intra-peroxynitrite cleavage) to
the O(1)-bound structure shown in Figure 8a, lowering the ener-
gy an additional 2.6 kcal/mol and yielding the same peroxynitrite-
phenol adduct as in the case of phenol addition to an already-
III
-
the optimized cis Fe ( OONO) (Figure 6a), it was found that
phenol binding to the O-atom directly bound to Fe(O(1)) is
most favorable, while the next-lowest energy binding mode
Im
III –
formed [(P )Fe ( OONO)] (3). This supports our data sug-
(
which is on the O(2) atom) is 2.6 kcal/mol higher. Relative to
III
gesting that Fe -bound peroxynitrite forms independent of the
the fully separated species, the O(1)-bound adduct has an energy
of ΔE = –7.3 kcal/mol and ΔG = +0.5 kcal/mol, indicating an
essentially thermoneutral process. Phenol (bound to O(1)) is
calculated to have a minimal impact on the barrier to O-O cleav-
age (ΔG = 9.4 kcal/mol), and yields similar thermodynamics for
formation of the {Fe =O + •NO
2,4
sequence of NO(g)
/
DTBP addition.
Thus, our calculations modelling the formation and subse-
Im
III –
quent decay (via O-O cleavage) of [(P )Fe ( OONO)] (3)
with and without phenol present indicate that phenol would have
a minimal impact on either process. However, our current com-
putations do not adequately explain the observation of nitrated
phenol upon warming to RT. A simple calculation of
‡
IV
2
} intermediate (ΔG = –4.6
kcal/mol), where phenol ends up H-bonded to the ferryl-oxo
Figure 8b). Note that O–O cleavage is again the more favorable
process, as Fe–O cleavage gives a much higher barrier. As was the
Im
III
–
Im
III
–
(
{(P )Fe (NO
3
) + PhOH} to {(P )Fe (OH ) + 2-
NO PhOH} shows that this process is favorable by 15 kcal/mol
2
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Journal of the American Chemical Society
Page 10 of 13
in ΔG at 298 K, but given that observation of 2-NO PhOH re-
2
this research is supported by the USA National Institutes of
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
quires warming to room temperature, the nature of the active
Health (GM040392). Use of the Stanford Synchrotron Radia-
tion Lightsource, SLAC National Accelerator Laboratory, is
supported by the U.S. Department of Energy, Office of Sci-
ence, Office of Basic Sciences under Contract No. DE-AC02-
33
nitrating species remains uncertain.
CONCLUSION
7
6SF00515. The SSRL Structural Molecular Biology Program
In summary, for the first time we were able to observe an in-
termediate in a reaction of heme-superoxo complex with NO(g)
is supported by the DOE Office of Biological and Environ-
mental Research, and by the National Institutes of Health,
National Institute of General Medical Sciences
(P41GM103393). For P.M-L., this research was supported by
the USA National Institutes of Health (GM074785). The
contents of this publication are solely the responsibility of the
authors and do not necessarily represent the official views of
NIGMS or NIH.
Im
II
using a synthetically designed heme (P )Fe complex and were
able to characterize the long sought after peroxynitrite ferriheme
Im
III –
species [(P )Fe ( OON=O)] (3), using a number of spectro-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
scopic techniques. The results led to the formulation of this com-
III
plex as a low–spin, six-coordinate Fe -PN species. Employing
DFT calculations, we determined that the cis conformation of
complex 3 is more stable than the trans. The addition of phenol to
3
leads to homolysis of the O-O bond resulting in fast isomeriza-
tion of PN to nitrate, rather than the nitration of phenol that has
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karlin@jhu.edu
edward.solomon@stanford.edu
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directly detected in activated macrophages), and logic in a
chemical perspective, very strongly implicate peroxynitrite in
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III
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Im
III
-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
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3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(P )Fe -(O ) (2) is not a good proton abstracter (even 10
2
equiv. of phenol shows no reaction); also, proton transfer from
phenol to the Fe-superoxo moiety is calculated from DFT to be
a highly endergonic process. Therefore, we do not believe we
are generating HO2.
(
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SYNOPSIS TOC
Journal of the American Chemical Society
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