Formation of an end-on ferric peroxo intermediate upon one-electron reduction of a ferric superoxo heme

Article abstract of DOI:10.1021/ja1001955

Chemical equation presented The low-spin end-on ferric peroxo heme intermediate has been proposed as an alternative reactive intermediate involved in the catalytic cycles of enzymes such as nitric oxide synthase and cytochrome P450. This transient heme intermediate has never been captured using synthetic heme models. We demonstrate herein our success in the solution preparation of such an end-on ferric peroxo intermediate derived from a heme model, which features both a group hanging over the porphyrin macrocycle and a covalently appended axial imidazole ligand, through one-electron reduction of its ferric superoxo precursor. The obtained ferric peroxo intermediate was further transformed into the corresponding ferric hydroperoxo species upon protonation. This heme model compound provides a convenient system for sequential preparation of the important and biologically relevant superoxo/peroxo/hydroperoxo heme intermediates through an oxygenation/one- electron reduction/protonation process simiar to the mechanisms used by enzyme systems.

Full text of DOI:10.1021/ja1001955

Published on Web 03/02/2010
Formation of an End-On Ferric Peroxo Intermediate upon One-Electron
Reduction of a Ferric Superoxo Heme
Jin-Gang Liu, Yuta Shimizu, Takehiro Ohta, and Yoshinori Naruta*
Institute for Materials Chemistry and Engineering, Kyushu UniVersity, Higashi-ku, Fukuoka 812-8581, Japan
Received January 15, 2010; E-mail: naruta@ms.ifoc.kyushu-u.ac.jp
Scheme 1
Activation of molecular oxygen by heme-containing enzymes
such as cytochrome P450,1a nitric oxide synthase (NOS),1b and
heme oxygenase1c generally involves one-electron reduction of a
ferric superoxo adduct to produce a nucleophilic end-on ferric
peroxo intermediate. The first of two specific protonations generates
the ferric hydroperoxo intermediate (compound 0), while for
cytochrome P450 and NOS, the second protonation leads to scission
of the O-O bond and formation of a highly reactive oxoferryl
species (compound I). In addition to the wide recognition of the
oxoferryl species as the active oxidant, there is increasing evidence
to suggest that the ferric peroxo species works as an alternative
reactive intermediate in the catalytic cycles of NOS and cytochrome
P450.2,3 Because of its transient character, the isolation and
spectroscopic characterization of such ferric peroxo species in heme-
containing enzymes have only been achieved using γ-ray or
synchrotron X-ray cryoreduction techniques with samples frozen
at 77 K, where the first proton delivery is hindered.4,5
(λmax ) 425, 550, and 590 nm).7b The rR spectra of 2 display an
isotope-sensitive band only at 582 (16O)/556 (18O) cm-1 in the low-
frequency region (Figure S2), which is assignable to the Fe-O2
stretching vibration of a heme superoxide species. The observed
ν(Fe-O2) frequency is somewhat higher than those of hemoproteins
and other heme models (νFe-O ) 570-576 cm-1
)
10 but similar to
2
those of previously reported heme superoxide species generated
from twin-coronet porphyrin models.11
Ferric peroxo porphyrin model complexes have been the focus
of studies as mimics of the enzymatic ferric peroxo intermediate
of biological systems.6 However, all of the previously reported ferric
peroxo model compounds exhibit the common characteristics of a
high-spin state and a side-on binding configuration. These are in
sharp contrast to the low-spin end-on counterpart observed during
the catalytic cycles of heme enzymes. As part of our continuous
efforts to develop models of heme-containing enzymes, we have
reported a series of peroxo-bridged heme-(O22-)-copper perox-
ides7 and recently described the formation of a heme-hydroperoxo
compound [(TMPIm)FeIII-OOH] by proton-coupled electron re-
duction of the corresponding ferric superoxo precursor.8 However,
the preparation of the unprotonated low-spin end-on peroxo species
from the TMPIm model using the reduction method has been
unsuccessful. In order to further optimize the model compound,
we synthesized 1 containing a bulky xanthene substituent that hangs
over the porphyrin macrocycle (Scheme 1),9 which may provide
suitable steric hindrance to protect the “naked” peroxo moiety,
thereby stabilizing it in solution. In this report, we describe our
success in the preparation of an end-on low-spin ferric peroxo heme
intermediate in solution through a one-electron reduction of its ferric
superoxo precursor. The obtained ferric peroxo intermediate was
further transformed into the corresponding ferric hydroperoxo
species upon protonation, as characterized by UV-vis, electron
spin resonance (ESR), and resonance Raman (rR) spectroscopies.
This ferric peroxo heme intermediate is an “individual” end-on low-
spin ferric peroxide that is not coupled to other transition-metal
ions and not protonated. This species represents the first example
of this type of heme model reported to date.
After formation of ferric superoxo 2 in solution, excess O2 was
removed, and one-electron reduction was carried out. Addition of
1 equiv of cobaltocene (CoCp2) to the resultant solution of 2 at
-70 °C immediately produced a new species, 3. The electronic
absorption spectra of 3 exhibit absorption maxima at 430, 568, and
610 nm (Figure 1A). These UV-vis features are different from
those of side-on peroxo species, whose Soret bands appear at much
longer wavelengths,6c,8 but similar to those of peroxymyoglobin
(peroxy-Mb) generated by synchrotron X-ray-irradiated oxy-Mb
crystals (λmax ≈ 428 and 567 nm).5 In addition, the ESR spectra of
3 clearly reveal a low-spin ferric species with small dispersion of
g-tensor components (g ) [2.27, 2.16, 1.96]; Figure 1A inset). This
result is in good agreement with that of ESR measurements of end-
on ferric peroxo heme intermediates generated in enzymes by
cryoreduction methods (e.g., for hemoglobin, g ≈ [2.26, 2.14,
1.96])4b and sets it apart from ESR data for side-on ferric peroxo
compounds, which are characterized by a rhombic high-spin ferric
signal at g ≈ 4.2.6,8 The characterization of 3 was further
corroborated by rR spectroscopy. The rR spectra of 3 demonstrate
two sets of isotope-sensitive bands (Figure 1B). One band appears
at 808 cm-1 and shifts to 771 cm-1 upon 18O substitution. This
808 cm-1 band is assignable to the O-O stretching vibration of a
peroxo species. Another band at 585 (16O)/560 (18O) cm-1 in the
low-frequency region is then assigned to the Fe-O stretching
vibration of the corresponding peroxo species. The observed
ν(O-O) frequency is similar to that of the side-on peroxide species
[(TMPIm)FeIII(O22-)]- (νO-O ) 807 cm-1).8 The ν(Fe-O) fre-
quency, however, is significantly shifted upward from 475 cm-1
for the high-spin species to 585 cm-1 for the low-spin peroxo
compound. The observed ν(O-O) and ν(Fe-O) frequencies of 3
are close to those of our previously reported low-spin peroxo-
bridged heme-(O22-)-copper peroxide complexes (νO-O ≈ 803
and 787 cm-1; νFe-O ≈ 611 cm-1).7b On the basis of these
experimental results, it can be concluded that one-electron reduction
Introduction of O2 to a solution of 1 in 20% MeCN/THF at
-30 °C readily produced a dioxygen adduct 2 with UV-vis features
(λmax ) 428, 550, and 592 nm; Figure S1 in the Supporting
Information) resembling those of our previously reported ferric
superoxo intermediate derived from a cytochrome c oxidase model
9
3672 J. AM. CHEM. SOC. 2010, 132, 3672–3673
10.1021/ja1001955  2010 American Chemical Society

C O M M U N I C A T I O N S
straightforward with this model system. Nevertheless, the observed
ν(O-O) and ν(Fe-O) values are similar to those of a previously
reported [(TMPIm)FeIII-OOH)] complex (νO-O ) 810 cm-1; νFe-O
) 570 cm-1)8 and also comparable to those of the hydroperoxo
intermediate isolated from cytochrome P450 by cryoreduction (νO-O
) 799 cm-1; νFe-O ) 559 cm-1).12b
In conclusion, a transient end-on low-spin ferric peroxo heme
intermediate derived from a heme model featuring both a group
hanging over the porphyrin macrocycle and a covalently appended
axial imidazole ligand has been successfully captured in solution
for the first time. Steric hindrance provides stabilization of the
“naked” peroxy heme in a manner similar to a countercation or a
hydrogen-bonding interaction. This heme model provides a con-
venient system for sequential preparation of the important and
biologically relevant superoxo/peroxo/hydroperoxo heme intermedi-
ates through a one-electron reduction/protonation process similar
to the mechanisms used by enzyme systems. The results of these
proposed investigations will provide a benchmark for characteriza-
tion and assignment of important peroxo/hydroperoxo heme
intermediates.
Figure 1. (A) UV-vis spectra of 3 (red line) generated by addition of
cobaltocene to the solution of 2 (black line) in 20% MeCN/THF at -70 °C.
Inset: ESR spectrum of 3 at 77 K. (B) rR spectra (λex ) 413 nm, 77 K) of
3 containing (a) 16O and (b) 18O and (c) their difference spectrum (a-b).
Acknowledgment. This work was financially supported by
Grants-in-Aid for Scientific Research (S) (17105003 to Y.N.) and
Young Scientists (B) (27150173 to J.-G.L.) from JSPS and on
Priority Areas (19027044 to Y.N.) and Innovative Areas (20200050
to J.-G.L.) from MEXT as well as by the Elemental Science and
Technology Project of MEXT.
Figure 2. (A) UV-vis spectrum of 4 generated by addition of methanol
to the solution of 3 in 20% MeCN/THF at -70 °C. Inset: ESR spectrum of
4 at 77 K. (B) rR spectra (λex ) 413 nm, 77 K) of 4 containing (a) 16O and
(b) 18O and (c) their difference spectrum (a-b).
of ferric superoxo 2 in solution readily generates the corresponding
end-on low-spin ferric peroxo 3.
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://pubs.acs.org.
The assignment of 3 as a ferric peroxide was further supported
by its tendency to form the corresponding ferric hydroperoxo species
upon protonation. Addition of methanol (400 equiv) to a solution
of 3 in 20% MeCN/THF at -70 °C affords 4, which exhibits
UV-vis characteristics (λmax ) 429, 566, and 508 nm; Figure 2A)
similar to those of its precursor 3. The ESR spectrum of 4 (Figure
2A inset) reveals a minor increase in the spread of the g values (g
) [2.32, 2.19, 1.95]) relative to that of 3. These results agree well
with those of previously reported hydroperoxo heme intermediates
from both a model compound8 and hemoglobin enzymes (g ≈ [2.32,
2.19, 1.95]).4b Further characterization of 4 was provided by rR
spectroscopy. The rR spectra display isotope shifts of 807 (16O)/
766 (18O) cm-1 in the region near 800 cm-1 and 575 (16O)/550
(18O) cm-1 in the low-frequency region (Figure 2B). Deuterium
substitution of 4 using MeOD produced a 2 cm-1 upshift of the
ν(O-O) band and a 3 cm-1 downshift of the ν(Fe-O) band (Figure
S3). These H/D substitution shifts are in agreement with previously
reported rR data for metallohydroperoxo species and consistent with
the existence of hydrogen bonding with the -OOH moiety.12
Consequently, the vibrational modes at 807 and 575 cm-1 are
assigned to the ν(O-O) and ν(Fe-O) stretching vibrations of the
hydroperoxide species, respectively. Protonation of ferric peroxo
3 to yield ferric hydroperoxo 4 causes ν(Fe-O) to decrease by 10
cm-1, whereas ν(O-O) is not significantly affected. This could be
an indication that the added methanol molecules may associate with
both the -OOH and axial imidazole moieties by means of hydrogen
bonding. This hydrogen bonding may weaken the electron back-
donation from the axial imidazole to the antibonding oxygen π*
orbital of the hydroperoxide in 4. As a result, the generally expected
inverse correlation between an increase in the value of ν(Fe-O)
and a decrease in the value of ν(O-O) upon protonation of a peroxo
to yield the corresponding hydroperoxo intermediate becomes less
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