Tuning the Geometric and Electronic Structure of Synthetic High-Valent Heme Iron(IV)-Oxo Models in the Presence of a Lewis Acid and Various Axial Ligands

Article abstract of DOI:10.1021/jacs.9b00795

High-valent ferryl species (e.g., (Por)Fe^IV=O, Cmpd-II) are observed or proposed key oxidizing intermediates in the catalytic cycles of heme-containing enzymes (P-450s, peroxidases, catalases, and cytochrome c oxidase) involved in biological respiration and oxidative metabolism. Herein, various axially ligated iron(IV)-oxo complexes were prepared to examine the influence of the identity of the base. These were generated by addition of various axial ligands (1,5-dicyclohexylimidazole (DCHIm), a tethered-imidazole system, and sodium derivatives of 3,5-dimethoxyphenolate and imidazolate). Characterization was carried out via UV-vis, electron paramagnetic resonance (EPR), ⁵⁷Fe M?ssbauer, Fe X-ray absorption (XAS), and ^54/57Fe resonance Raman (rR) spectroscopies to confirm their formation and compare the axial ligand perturbation on the electronic and geometric structures of these heme iron(IV)-oxo species. M?ssbauer studies confirmed that the axially ligated derivatives were iron(IV) and six-coordinate complexes. XAS and ^54/57Fe rR data correlated with slight elongation of the iron-oxo bond with increasing donation from the axial ligands. The first reported synthetic H-bonded iron(IV)-oxo heme systems were made in the presence of the protic Lewis acid, 2,6-lutidinium triflate (LutH⁺), with (or without) DCHIm. M?ssbauer, rR, and XAS spectroscopic data indicated the formation of molecular Lewis acid ferryl adducts (rather than full protonation). The reduction potentials of these novel Lewis acid adducts were bracketed through addition of outer-sphere reductants. The oxidizing capabilities of the ferryl species with or without Lewis acid vary drastically; addition of LutH⁺ to F₈Cmpd-II (F₈ = tetrakis(2,6-difluorophenyl)porphyrinate) increased its reduction potential by more than 890 mV, experimentally confirming that H-bonding interactions can increase the reactivity of ferryl species.

Full text of DOI:10.1021/jacs.9b00795

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Article
Tuning the Geometric and Electronic Structure of
Synthetic High-Valent Iron(IV)-Oxo Models in the
Presence of a Lewis Acid and Various Axial Ligands
Melanie A. Ehudin, Leland Bruce Gee, Sinan Sabuncu, Augustin Braun, 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.9b00795 • Publication Date (Web): 12 Mar 2019
Downloaded from http://pubs.acs.org on March 12, 2019
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Journal of the American Chemical Society
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Tuning the Geometric and Electronic Structure of Synthetic High-Va-
lent Iron(IV)-Oxo Models in the Presence of a Lewis Acid and Various
Axial Ligands
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Melanie A. Ehudin,†,¶ Leland Bruce Gee,‡,¶ Sinan Sabuncu,§ Augustin Braun,‡ Pierre Moënne-Loccoz,§
⊥
Britt Hedman, Keith O. Hodgson,‡,⊥ Edward I. Solomon,*,‡,⊥ and Kenneth D. Karlin*,†
†Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, United States
‡Department of Chemistry, Stanford University, Stanford, California 94305, United States
§Department of Biochemistry & Molecular Biology, Oregon Health & Science University, Portland, OR, 97239-3098, United
States
⊥
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, Cali-
fornia 94025, United States
¶These authors contributed equally to this work.
KEYWORDS. ferryl, Compound-II, Lewis-acids, axial ligands, synthetic model, iron porphyrins
ABSTRACT: High-valent ferryl species (e.g., (Por)FeIV=O, Cmpd-II) are observed or proposed key oxidizing intermediates in
the catalytic cycles of heme-containing enzymes (P-450s, peroxidases, catalases, and cytochrome c oxidase) involved in bio-
logical respiration and oxidative metabolism. Herein, various axially ligated iron(IV) oxo complexes were prepared to exam-
ine the influence of the identity of the base. These were generated by addition of various axial ligands (1,5-dicyclohexylimid-
azole (DCHIm), a tethered-imidazole system, and sodium derivatives of 3,5-dimethoxyphenolate and imidazolate). Charac-
terization was carried out via UV-vis, EPR, 57Fe Mössbauer, Fe X-ray absorption (XAS), and 54/57Fe resonance Raman (rR)
spectroscopies to confirm their formation and compare the axial ligand perturbation on the electronic and geometric struc-
tures of these heme iron(IV) oxo species. Mössbauer studies confirmed axially ligated derivatives were iron(IV) and six-coor-
dinate complexes. XAS and 54/57Fe rR data correlated with slight elongation of the iron-oxo bond with increasing donation
from the axial ligands. The first reported synthetic H-bonded iron(IV)-oxo heme systems were made in the presence of the
protic Lewis acid, 2,6-lutidinium triflate (LutH+), with (or without) DCHIm. Mössbauer, rR, and XAS spectroscopic data indi-
cated the formation of a molecular Lewis acid ferryl adducts (rather than full protonation). The reduction potentials of these
novel Lewis acid adducts were bracketed through addition of outer-sphere reductants. The oxidizing capabilities of the ferryl
species with or without Lewis acid vary drastically; addition of LutH+ to F8Cmpd-II (F8 = tetrakis(2,6-difluorophenyl)porphy-
rinate) increased its reduction potential by more than 890 mV, experimentally confirming that H-bonding interactions can
increase the reactivity of ferryl species.
INTRODUCTION
intermediate ferryl species have been the focus of many
studies, but due to the transient nature of these intermedi-
ates, synthetic iron porphyrin models have played an essen-
tial role in understanding the biological intermediates in di-
oxygen activation and oxygen atom transfer reactions in na-
ture.3,12
Heme metalloenzymes activate dioxygen or hydrogen
peroxide to generally form two intermediates: the highly
oxidizing oxoiron(IV) porphyrin cation radical formally
known as Compound-I (Cmpd-I) and the one-electron re-
duced ferryl porphyrin Compound-II (Cmpd-II). These high-
valent intermediates can be found in peroxidases, catalases,
oxidases, P450s, and globins.1–3 Many of these metalloen-
zymes have various proximal axial bases that allow the for-
mation of a protonated ferryl derivative or a valence tauto-
mer of Compound I ((Por•+)FeIV=O) or II as a key reactive
intermediate (Figure 1). High-valent oxoiron(IV) complexes
are key oxidative reactive intermediates in heme and non-
heme iron enzymes, catalyzing various essential biochemi-
cal processes (e.g., catabolism, angiogenesis, respiration,
and apoptosis).1,3–11 The oxidative and oxygenation reac-
tions catalyzed by heme metalloenzymes and the nature of
Figure 1. Heme FeIV=O enzyme intermediates with various ax-
ial ligands. The “push” from strong electron donating axial ba-
ses with anionic character found in cytochrome P450 and cata-
lase results in the greater “pull” on an H-atom so that their
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Page 2 of 23
anionic character on the histidine), Cmpd-II is an FeIV=O
Cmpd-II species are formally protonated, an iron(IV)-
hydroxide.13 Due to hydrogen bonding interactions with a
nearby proximal residue, the axial histidine ligand in most pe-
roxidases has partial anionic character which may result in an
FeIV-OH (APX); without such H-bonding (HRP, CcO), an FeIV=O
is observed.
species, even at pH’s as low as 3.49 Collectively, this indicates
not only the identity of the axial ligand – but also that its
secondary coordination sphere tunes the FeIV=O/OH unit.
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In spite of these important enzymatic studies, there still
remains a need for enzymatic and synthetic model studies
to examine the influence of axial ligation on facilitating pro-
tonation, verify the protonation state of Cmpd-II in various
enzymes, and determine the basicity of metal-oxo moieties.3
Many enzymatic and synthetic investigations remain con-
troversial,1,3 and significant effort is still necessary to inter-
rogate the properties of heme ferryl intermediates. Syn-
thetic high-valent model systems are useful in understand-
ing how ferryl intermediates dictate enzyme reactivity and
direct catalysis toward hydroxylation of unactivated C-H
bonds in P450s or electron transfer in peroxidases.7,12 Ef-
forts have also explored the use of Lewis acids, particularly
in non-heme or porphyrinoid systems, to examine the role
of Lewis-acidic ions in modulating chemistry, for instance
the H+ in cytochrome P450 or the Ca2+ ion of the oxygen
evolving Mn4O5 cluster of photosystem II. Reports have ex-
perimentally demonstrated the importance of Lewis acidic
ions (or secondary coordination sphere interactions) in en-
hancing reactivity.50–58 There have been no reports on syn-
thetic heme FeIV-OH species, which would be paramount in
understanding P450 chemistries and the conditions which
would allow for the generation of such species (and their
characterization) would be significant towards our under-
standing of such a key reactive intermediate.
Synthetic systems of these ferryl intermediates allow for
greater insight into the effects of various factors (e.g.,
nearby amino acid residues, proximal axial bases, or pKa val-
ues) that mitigate their formation, stability, efficiency, and
reactivity. Synthetic high-valent iron(IV) oxo heme systems
have been reported to be able to mimic the oxidative behav-
ior of analogous enzymatic reactive intermediates perform-
ing reactions such as alkene epoxidation, aliphatic and aro-
matic hydroxylation, N- or O-dealkylation, S-oxidation and
halogenation of alkanes.2,3,20–24,4,12,14–19 Synthetic model
studies have been found to give important mechanistic in-
sight into the behavior of cytochrome P450 oxygenases
(e.g., their roles in drug metabolism), however, the specifics
of key steps involving highly reactive intermediates remain
to be fully elucidated.3,25,26 More efficient and biomimetic
model systems of these enzymes are needed, especially of
cytochrome P450, which exhibits promising catalytic oxida-
tive properties for various biosynthetic pathways. This
would allow for the application of these enzymes beyond
their normal functions in selective catalytic oxidation of
strong aliphatic C-H bonds (e.g., hydroxylation of alkanes)
for renewable energy/fuel research;25,27 for example, heme
metalloenzymes have been employed as electrocatalysts to
develop an alternative energy source to replace the scarcity
of fossil fuels.28
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Herein, the full spectroscopic characterization of seven
synthetic high valent heme iron(IV) oxo (Compound II) spe-
cies is reported, wherein the physical properties of the
iron(IV)-oxo species were examined by addition of axial lig-
ands, Lewis acids, and both. A catalase Cmpd-II synthetic
model has been prepared, possessing a phenolate ligand,
although, as will be discussed below, its physical properties
turn out to be somewhat unexpected (vide infra). Ferryl de-
rivatives were prepared with the strong electron donating
exogenous bases 1,5-dicyclohexylimidazole (DCHIm), so-
dium 3,5-dimethoxyphenolate (hereafter referred to as
ArO–), and sodium imidazolate (Im–) to act as the axial lig-
and with tetrakis(2,6-difluorophenyl)porphyrinate (F8)
heme (Figure 2). A comparison was also made between a
tethered (PIm) and untethered (DCHIm) imidazole axial
base, (DCHIm)F8Cmpd-II vs. PImCmpd-II, to explore how the
tether addition affects the imidazole coordination to iron
and its effect on the bond length of the iron(IV) oxo moiety
(Figure 2). Interestingly, we report the first (that we know
of) Mössbauer and Extended X-ray Absorption Fine Struc-
ture (EXAFS) spectroscopic data on a synthetic imidazole
tethered heme Cmpd-II system. Further, the relative basic-
ity of the ligands and their character (anionic or neutral) on
the structural and electronic properties are assessed and
compared to known synthetic models and enzymatic sys-
tems. Results discussed in this work provide a significant
expansion on the heretofore limited axially-ligated heme
Cmpd-II synthetic model system literature and EXAFS data
comprised of a single report by Penner-Hahn et al.33
Balch and co-workers were the first to report on a syn-
thetic oxoiron(IV) porphyrin species18,29,30 and Groves and
co-workers were the first to prepare synthetic oxoiron(IV)
heme cation radical complexes.23,24,31–33 A number of syn-
thetic model studies focus on investigation of the nature/re-
activity of iron(IV)-oxo porphyrin -cation radicals,34–39
however reports on iron(IV)-oxo heme complexes are more
limited. Efforts have been predominantly directed towards
studying the protonation state of Cmpd-II as its basicity has
been shown to drive the reactivity of Cmpd-I (having impli-
cations for two-electron oxidations).1 Studies by Green and
coworkers have shown, employing rapid-mixing pH-jump
experiments coupled with spectroscopy, that the identity of
the axial ligand in heme enzymes plays a critical role in the
ferryl protonation of Cmpd-II.40–43 Notably, heme-thiolate
proteins such as chloroperoxidase (CPO) and P450s have
been shown to have a very basic Cmpd-II ferryl oxygen due
to the strong electron donation by the axial cysteine lig-
and.40 This “push” effect from the proximal ligand has been
reported to be key to ferryl protonation leading to Fe-O
elongation and shortening of the Fe-S bond.42–44 Work on
catalase and ascorbate peroxidase (APX) has demonstrated
that tyrosinate and histidine (with anionic character), re-
spectively, are electron-donating enough to maintain a
highly basic ferryl capable of generating an FeIV-OH spe-
cies.41,45 While most peroxidases contain a well-conserved
aspartate near the N of the proximal histidine (Figure 1),
horseradish peroxidase Cmpd-II is an authentic iron(IV)
oxo, 33,46–48 unlike APX Cmpd-II. In globins, where there is
not an aspartate near the proximal histidine (and thus, no
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support the formation of molecular Lewis acid ferryl ad-
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ducts (rather than full proton transfer occurring), wherein
rR and XAS data indicate the Lewis acid adducts possess a
slightly elongated iron (IV) oxo bond for F8Cmpd-II(LutH+).
Through the use of outer-sphere chemical reductants,
bracketed reduction potentials of (DCHIm)F8Cmpd-II and
F8Cmpd-II, with and without LutH+, have been obtained. It
was found that addition of the Lewis acid greatly increases
the oxidizing capabilities of these synthetic iron(IV) oxo
complexes; for example, the reduction potential of F8Cmpd-
II(LutH+) is greater than 890 mV more positive than that of
F8Cmpd-II. The results obtained provide leads toward a sys-
tematic approach to the development of additional biomi-
metic synthetic models to answer many of the controversial
questions concerning high-valent species in heme enzymes.
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Figure 2. Synthetic Cmpd-II complexes generated and studied
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as part of this report, including (1) the parent ferryl species,
and (2) derivatives formed by adding various exogenous
strongly electron donating axial ligands and (3) the tethered
derivative PImCmpd-II.
This study also includes two synthetic heme iron(IV) oxo
derivatives formed from addition of the protic Lewis acid
(LA) 2,6-lutidinium triflate (LutH+)), yielding two models to
study the interaction of a proton/acid-molecule with an
iron(IV) oxo species (Figure 3). This can give important in-
sights into the established protonated Compound II inter-
mediate complex found in key enzymes such as cytochrome
P450. The structure and electronic properties of these
seven various ferryl derivatives were characterized by UV-
vis, 2H-NMR, continuous wave (CW) X-band EPR, 54/57Fe la-
beled resonance Raman (rR), 57Fe-Mössbauer, Fe K-edge X-
ray Absorption (XAS) spectroscopies, and complementary
computational analysis experiments to obtain a spectro-
scopic handle on various axially ligated ferryl models and
Lewis acid Cmpd-II adducts; such data are lacking in the lit-
erature. XAS and 57Fe Mössbauer provide atom-specific in-
formation about the geometric and electronic structures
around Fe. The pre-edge region of the Fe K-edge XAS spec-
trum has intensity directly correlated to dipole character of
the 1s->3d transition – caused by 4p-3d mixing in non-cen-
trosymmetric systems.59 In our application. besides distin-
guishing 5 versus 6 coordination, the pre-edge intensity in-
creases as the Fe=O bond length decreases due to increased
4p mixing. 57Fe Mössbauer isomer shifts are connected to
the oxidation of the 57Fe atom – with lower values correlat-
ing to higher oxidation. Low spin Fe(IV) is distinguishable
from other oxidation and spin states providing a good han-
dle on sample purity. Quadrupole splitting parameters in
Mössbauer spectroscopy have less straightforward correla-
tions and these depend on the electric field gradient tensor.
An approximate trend related to our application here, is that
strong bonding along the molecular z-axis adds a negative
contribution to the quadrupole splitting – which in this case
would lower the magnitude of the splitting.60
Figure 3. Generation of high-valent ferryl Lewis acid adducts
by addition of 2,6-lutidinium triflate (LutH+) to F8Cmpd-II and
(DCHIm)F8Cmpd-II.
RESULTS AND DISCUSSION
Tethered/Exogenous Imidazole Axial Base: F8Cmpd-
II, (DCHIm)F8Cmpd-II, and PImCmpd-II. The UV-vis spec-
tra of F8Cmpd-II, (DCHIm)F8Cmpd-II, and PImCmpd-II were
previously reported,61–66 however, no further spectroscopic
evidence was provided and the conditions described there
were different than in the present work. The reduced iron
o
complex F8FeII or PImFeII was cooled to -90 C in a 1:9
MeTHF:toluene (MeTHF = 2-methyltetrahydrofuran) sol-
vent mixture and 1 equivalent (equiv.) of meta-chloroper-
benzoic acid (mCPBA) oxidant was added to form F8Cmpd-
II or PImCmpd-II, respectively. Subsequently, two equiva-
lents of the strong electron donating exogenous axial base
DCHIm was added to F8Cmpd-II, yielding (DCHIm)F8Cmpd-
II (Scheme 1).
Scheme 1. Addition of 1 equivalent of mCPBA to F8FeII
species in 1:9 MeTHF:toluene at -90 oC yielded F8Cmpd-
II. Further, treatment with an exogenous base or Lewis
acid resulted in the formation of an axial base ferryl de-
rivative and/or a Lewis acid iron(IV) oxo species.
Application of these spectroscopic methods leads to the
following, as described in this report: The purity of the
Lewis acid and axially ligated ferryl derivatives are con-
firmed (via Mössbauer spectroscopy) which also supported
that they are iron(IV) species (from the isomer shift) and
five or six-coordinate complexes based on the quadrupole
splitting. XAS, and 54/57Fe rR data correlate with slight elon-
gation of the iron-oxo bond with increasing donation from
the axial ligands. Mössbauer, rR, and XAS spectroscopic data
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Pre-
edg
e
Are
af
Soret,
Q-
bands
(nm)
(57Fe=O)
(57-54Fe)
(cm-1)
1
2
3
4
5
6
7

EQ
(mm/s) (mm/s)
833(5)
 
: 1574
F8Cmpd-
II
415,
544
54.3
(2.6)
0.11
2.06
8
9
811(5)
 
: 1573
(DCHIm
)F8Cmpd
-II
421,
552
27.6
(1.1)
0.11
0.11
1.07
1.23
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PImCmp
d-II c
422,
554
N/A
---d
(Im–)
F8Cmpd-
II e
828(6)
 
: 1571
420,
556
33.6
(0.7)
0.12
0.12
1.550
1.57
Initial Observations: Cmpd-II Complex Generation.
Preliminary synthetic experimentation revealed that pre-
paring F8Cmpd-II in coordinating solvents such as tetrahy-
drofuran, MeTHF, or n-butyronitrile (vs. non-coordinating
solvents as dichloromethane or toluene) resulted in a stable
and pure complex. This was supported by the lack of decom-
position and by the lack of ferric impurities observed by UV-
vis and EPR spectroscopies. However, F8Cmpd-II was less
reactive toward exogenous bases (e.g., phenolate, imidazo-
late, various imidazoles), substrates (i.e., phenols, C-H sub-
strates), or Lewis acids in coordinating solvents; thus, ex-
ploration of the use of different solvent systems was carried
out to optimize reactivity and stability to form stable syn-
thetic ferryl model complexes with interesting oxidative ca-
pabilities (Figures 2 & 3). Interestingly, it was found that
the mixture 1:9 MeTHF:toluene resulted in a solvent system
that allowed for the generation of F8Cmpd-II, in which it was
stable, pure, and reactive towards exogenous bases/Lewis
acids as observed by UV-vis, EPR, rR, EXAFS & Mössbauer
spectroscopies (vide infra). It was originally postulated that
F8Cmpd-II’s high stability and purity in coordinating sol-
vents was due to the solvent acting as a weakly bound axial
ligand to help stabilize the FeIV=O moiety. However, EXAFS
spectroscopy indicates that F8Cmpd-II is five-coordinate,
which was further corroborated by the pre-edge intensity
and Mössbauer spectroscopy (Table 1, vide infra). Thus, the
increased thermal stability imparted by the addition of a
small percentage of MeTHF in the solvent may be a result of
more favorable solvation of the iron(IV) oxo moiety.
(ArO–)
F8Cmpd-
II
829(6)
 
: 1572
420,
548
42.1
(0.9)
a All complexes are EPR silent as determined by using X-band
EPR spectroscopy at 10 K. The values for the major species
b
are reported in this table. See SI for minor Mössbauer species
fits and details. c The Mössbauer spectrum of PImCmpd-II is best
fit to three ferryl species due to the flexibility of the tethered
imidazole base; see the text for more details. d rR spectroscopic
data for PImCmpd-II was not collected. The Mössbauer spec-
e
trum of (Im–)F8Cmpd-II shows a 13% impurity of F8Cmpd-II.
f
Pre-edge area (standard deviations for the fits)
Addition of 1 equiv. of mCPBA to F8FeII resulted in a UV-
vis spectral shift from 422 to 415 nm in its Soret band and
from 542 to 544 nm in the Q-band, forming the parent
F8FeIV(=O) species (Scheme 1, Figures 2 & 4). The subse-
quent addition of DCHIm was characterized by a red shift in
the max of the Soret (415 to 421 nm) and of the Q-band (544
to 552 nm) generating (DCHIm)F8Cmpd-II. A similar red
shift was observed upon addition of 1 equiv. mCPBA to PIm-
FeII (419, 525 to 422, 554 nm) forming PImCmpd-II (Table 1
& Figures 2, 4 and S4) The complexes (DCHIm)F8Cmpd-II
and PImCmpd-II were stable in solution at –90 oC (>2 hours);
note that 2 equivalents of DCHIm were needed to ensure
full incorporation of the axial ligand (no F8Cmpd-II re-
mained). These spectral values are consistent with previous
reports (Table S1), wherein six coordinate or imidazole li-
gated synthetic heme iron(IV) oxo species exhibit red
shifted UV-vis absorption max values (vs. analogous 5-coor-
dinate species).
Table 1. Spectroscopic characterization of axially ligated F8
and PIm heme system (porphyrinate)FeIV oxo Cmpd-II com-
plexes.a
Fe
XAS
UV-vis
Mössbauerb
rR
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Chart 1. Proposed ferrous species based on Mössbauer param-
1
2
3
4
5
6
7
8
eters and similar assignments found in the literature. Two fer-
rous species were identified for F857FeII, with one or two
MeTHF solvent molecules bound. Three ferrous species were
detected for Pim57FeII with approximately 80% of the sample
favoring a 6-coordinate geometry. The six-coordinate com-
plexes may be ascribed to (1) tethered base and one solvent
molecule bound (with the tethered base possibly taking on two
conformations; i.e., coming out of the plane in one conformer)
or (2) two solvent molecules bound to the iron center.
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Figure 4. UV-vis monitoring of the formation of
(DCHIm)F8Cmpd-II at 0.01 mM, the Soret band (A) and 0.1 mM,
the Q-band (B) at -90 oC in 1:9 MeTHF:toluene.
Mössbauer Spectroscopic Characterization. To con-
firm the species’ purity, determine oxidation state (or elec-
tronic density at the metal center) via the isomer shift
[ (mm/s)], and the electric field gradient at the iron via the
quadrupole splitting [EQ (mm/s)], 57Fe Mössbauer spec-
troscopy was employed, in which all spectra were recorded
at 80 K in the absence of a magnetic field.67 The Mössbauer
spectrum of the ferrous precursor F857FeII in 1:9 MeTHF:tol-
uene was a mixture of two quadrupole doublets with pa-
rameters [major species (60%) EQ = 2.09 mm/s and
 =  mm/s and minor species (40%) EQ = 2.53 mm/s
and  =  mm/s] that are typical for high-spin ferrous
heme complexes (Table S2 & Figure S11).68–70 These param-
eters of the minor species were similar to the F857FeII spec-
trum previously reported by Karlin and co-workers71 in ac-
PIm57FeII was prepared for the first time in this work
(Note: See synthetic details in the Experimental Section).
The Mössbauer spectrum of PIm57FeII indicated three differ-
ent species (Chart 1), where there are two different six-co-
ordinate low-spin ferrous species (EQ = 1.68 mm/s
and  = 0.49 mm/s & EQ = 0.92 mm/s and  = 0.44 mm/s)
and one five-coordinate high-spin ferrous species (EQ =
2.48 mm/s and  = 0.96 mm/s) (Table S2 & Figure S13).
The high-spin parameters of PIm57FeII are in accordance with
the values of a typical high-spin 5-coordinate ferrous com-
pound,68–70 with either the tethered imidazole or solvent
binding, and with the parameters for the high-spin 5-coor-
dinate complex (MeTHF)F857FeII. It was previously estab-
lished that the tethered imidazole of the PIm system can eas-
ily coordinate to iron or persist free in solution. This was
demonstrated by the EPR of PImFeIIISbF6 or PImFeIIIOH
wherein two sets of signals were observed, that of a high-
o
etone at –80 C where they observed one species with pa-
rameters EQ = 2.66 mm/s  =  mm/s and suggested
that one solvent molecule is bound. Thus, F857FeII in 1:9
MeTHF:toluene was determined to be a mixture of the five-
coordinate (MeTHF)F857FeII (due to the similarity of its pa-
rameters to (acetone)F857FeII) and the six-coordinate
(MeTHF)2F857FeII species (due to its decreased quadrupole
splitting and its similarity to other high-spin ferrous spe-
cies68–70) (Chart 1).
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Page 6 of 23
The fits for these Mössbauer spectra indicated a single quadru-
pole doublet, implying high purity of the samples. Mössbauer
parameters are given in Table 1.
spin iron(III) species and that of a low-spin iron(III) com-
plex.63 Previous 1H-NMR data on PImFeII in various solvents
showed that it can exist in either a high-spin 5-coordinate
(in non-coordinating solvents, with the imidazole tether
bound axially) or low-spin 6-coordinate (in coordinating
solvent, with the tether and one solvent molecule bound ax-
ially) geometry.72 While the exact nature of the second low-
spin species observed in our Mössbauer (36%) cannot be
determined, we suggest that it could be (MeTHF)2PImFeII,
with the tether unbound or a species with one solvent mol-
ecule and the tether bound in a different orientation (Chart
1).
1
2
3
4
5
6
7
8
In previous reports, synthetic iron(IV) oxo species with
various porphyrins and with/without 1-methylimidazole
(1-MeIm) serving as an axial base ligand have been charac-
terized. Their Mössbauer spectroscopic parameters are
summarized in Table S1. Every synthetic ferryl system with
1-MeIm has an isomer shift around 0.1 mm/s and a quadru-
pole splitting around 1 mm/s in accordance with the values
reported here for (DCHIm)F8Cmpd-II (Tables 1, S1 & S2,
Figure 5). Consistent with the three PImFeII species observed
in the Mössbauer spectrum, there were three ferryl
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Samples for Mössbauer spectroscopy of F857FeIV=O,
(DCHIm)F857FeIV=O, and PIm57FeIV=O were all prepared in a
manner similarly to those utilized for UV-vis experiments in
PImCmpd-II species (EQ
=
1.06 mm/s
and
o
1:9 MeTHF:toluene at -90 C. The results confirm that all
three complexes possess the heme-iron in the FeIV oxida-
tion, based on the fitted isomer shifts found to be around 0.1
mm/s (Tables 1 and S2), a hallmark of other known syn-
thetic and enzymatic ferryl systems understood as lowered
3d electron shielding causing increased electron density at
the nucleus – which results in a decreased isomer shift rel-
ative to lower oxidations (Tables 1, S1, & S2 and Figures 5
and S13).11,42,44 F857FeIV=O (EQ = 2.06 mm/s  =  mm/s)
and (DCHIm)F857FeIV=O (EQ = 1.073 mm/s  =  mm/s)
were both fit with one quadrupole doublet and under the
aforementioned conditions were of high purity (Figure 5
and Tables 1 & S2). Addition of DCHIm to F8Cmpd-II re-
sulted in a decrease in the quadrupole splitting from 2.06 to
1.07 mm/s, which is consistent with the formation of a six-
coordinate species as the covalency of the axial base affects
the dz2 orbital due to an increased electron density upon
binding (Tables 1 & S2, Figure 5).
 =  mm/s; EQ = 1.22 mm/s and  =  mm/s; & EQ
= 2.06 mm/s and  =  mm/s) ascribed to the fluxional
nature of the tether (Chart 2, Figure S13, Tables 1 & S2).
Comparison of F8Cmpd-II and other 5-coordinate synthetic
heme ferryl systems (Tables 1 & S1) helped us to assign the
5-coordinate PImCmpd-II species with the tether free in so-
lution to the complex with the higher quadrupole splitting
around 2 mm/s. One might consider the possibility that the
tethered imidazole interacts with the oxo atom ligand (since
it is not coordinated to iron in the 5-coordinate ferryl spe-
cies) but this is most likely not occurring; the isomer shift
and quadrupole splitting parameters for F8Cmpd-II and the
5-coordinate PImCmpd-II are very similar and greater
changes might be expected if this interaction occurred. The
six-coordinate imidazole-tethered PImCmpd-II species had
values in agreement with (DCHIm)F8Cmpd-II (Chart 2 & Ta-
ble 1), so the imidazole being tethered or not does not seem
to have a major effect on the electron density or electric
field gradient at the iron center as observed by Mössbauer
spectroscopy. The second 6-coordinate species may arise
from a different conformation of the tethered axial base
when bound to the iron ion (e.g., the tether may be coming
out of the plane) or from solvent binding (see Chart 2). DFT
calculations support the conclusion that for PImCmpd-II the
major species in solution favors the tethered imidazole co-
ordinated to the iron metal center (vide infra).
Chart 2. Proposed PIm ferryl derivatives based on Mössbauer
parameters (Table S2 and Figure S13) and previous reports of
PIm based species.63,72,73 Approximately 76% of the sample fa-
vored being 6-coordinate, wherein we assign 58% of the sam-
ple to the tethered imidazole axial base coordinated to iron.
The second 6-coordinate species may be a different conformer
that the tethered base could adopt when bound to the iron ion
(bottom left, e.g., the tether could be coming out of the plane)
or a MeTHF solvent molecule bound to the iron ion (bottom
right).
Figure 5. 80 K zero applied magnetic field 57Fe Mössbauer
spectra (blue) with corresponding fits (red) at 2 mM in 1:9
MeTHF:toluene: Top. F8Cmpd-II, Bottom. (DCHIm)F8Cmpd-II.
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and toluene are 7.0 and 2.4 respectively, and protein interi-
1
2
3
4
ors are modeled with a dielectric of 4-10 – we don’t expect
our application of Badger’s rule to deviate significantly from
that of Green’s application to biological systems. Fe=O bond
distance determinations coming from XAS are discussed be-
low.
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Resonance Raman Spectroscopic Characterization of
Oxo-ferryl F8Cmpd-II and (DCHIm)F8Cmpd-II. Similar to
Bajdor and Nakamoto, who compared rR spectra of unla-
beled (56Fe) and 54Fe tetraphenylporphyrin iron(IV) oxo
complexes to identify Fe=O stretches,74 this study utilized
57/54Fe derivatives in synthetic heme iron(IV) oxo systems.
There are examples in the literature of labeling the oxidant
or adding labeled water to exchange iron(IV) oxo groups
(Table S1), but these approaches proved unsuccessful here.
Further, 57/54Fe resonance Raman spectroscopy was em-
ployed to aid the Mössbauer results and to examine the ef-
fects of the axial ligands on the iron(IV) oxo stretching vi-
bration (Table 1 and Figure 6). The heme complex,
F8FeIII(Cl), was prepared by metalating the F8 ligand with
either 57FeCl2 or 54FeCl2, followed by reduction to form the
previously published F857FeII and the newly synthesized (for
this report) heme complex F854FeII.71
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Figure 6. Resonance Raman spectra in the mid-frequency re-
gion for the F854/57FeIV=O complexes (57Fe spectra in red & 54Fe
spectra in black) without and with phenolate, imidazolate, and
DCHIm axial bases. (All spectra are normalized with respect to
the 1083 cm-1 band). The (Fe=O) for each derivative is labeled
and was observed to be in the expected 800 cm-1 region. See
the SI for addition details (e.g., rR spectra showing the oxida-
tion and spin state features).
Anionic Axial Ligands: (ArO–)F8Cmpd-II and (Im–
)F8Cmpd-II. Since various axial ligands, many of them hav-
ing an anionic nature, are present in heme-containing en-
zymes, attempts to synthesize F8Cmpd-II derivatives with
axially ligated substituted phenolate (ArO–) and unsubsti-
tuted imidazolate (Im–) donors were carried out. It is note-
worthy that phenolates without electron-donating moieties
are unreactive towards F8Cmpd-II in 1:9 MeTHF:toluene at
The rR data of F8Cmpd-II and (DCHIm)F8Cmpd-II con-
firmed their iron(IV) oxidation state by the oxidation state
marker band v4 around 1370 cm-1 (Table 1 and Figure S10).
The mid-frequency region also provides clear evidence for
the isotope sensitive (Fe=O) mode in the 800-850 cm-1 re-
gion (Figure 6 & Table 1). The iron(IV) oxo stretching fre-
quency of F8Cmpd-II shifted from 833 to 811 cm-1 (Δ = –22
cm-1) upon addition of DCHIm, consistent with electron do-
nation from the exogenous axial base causing slight elonga-
tion of the Fe=O bond (Figures 6, S7, and S8 & Table 1). This
is in good agreement with previous reports (Table S1),
wherein the addition of an axial base, either 1-methylimid-
azole or a strongly donating ligand, lowered the observed
(Fe=O). For instance the addition of 1-MeIm to
(TPP)FeIV(=O) or to (OEP)FeIV(=O) [TPP = tetraphenylpor-
phyrinate; OEP = octaethylporhyrinate], showed that the
(Fe=O) frequency decreased from 852 to 820 cm-1 (Δ –32
o
-90 C, failing to form a new species as determined by UV-
vis spectroscopy (no spectral change). However, when 2
equiv. of sodium 3,5- dimethoxyphenolate, in a mixture of
15-crown ether and butyronitrile, were added to F8Cmpd-II
o
in 1:9 MeTHF:toluene at -90 C there was a red shift in the
Soret and Q-band from (415, 544 nm to 420, 548 nm), the
latter values being similar to those observed for
(DCHIm)F8Cmpd-II (Figures 5 & S2 and Table 1). When 3,5-
dimethoxyphenol was added to F8Cmpd-II, there was an im-
mediate spectral change to a known ferric species (the re-
sulting UV-vis was similar to the authentic UV-vis signature
of F8FeIIIOH (Figure S35)) and further confirmed by ob-
servance of a high-spin ferric g = 6 signal via EPR spectros-
copy. This control experiment provided evidence that 3,5-
dimethoxyphenolate (not phenol) binds the iron center.
cm-1) (Table S1).74–77 Application of Badger’s rule with the
2/3
parameters determined by Green,78 re = 55.702/ve
+
1.003, indicates that based on the rR data the Fe=O bond is
slightly elongated, from 1.632 to 1.644 Å, upon addition of
DCHIm to F8Cmpd-II. As the dielectric constants for MeTHF
ACS Paragon Plus Environment

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The existence of the Cmpd-II system, (ArO–)F8Cmpd-II (Fig-
ure 2), and its characterization (here), may well facilitate fu-
ture efforts toward designing a protonated catalase Cmpd-
II model, to further elucidate compound structure, elec-
tronic-structure/bonding and reactivity, as pertains to the
findings uncovered by Green and coworkers, as mentioned
in the Introduction.
Page 8 of 23
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6
Addition of 2 equiv. of sodium imidazolate, in a mixture of
15-crown ether and butyronitrile, was also added to
F8Cmpd-II in 1:9 MeTHF:toluene at -90 oC, forming the (Im–
)F8Cmpd-II derivative (Figure 2), which showed the charac-
teristic red shift in the Soret and Q-band (as observed for
(DCHIm)F8Cmpd-II and (ArO–)F8Cmpd-II, Figures 4 and S2)
from 415, 544 nm to 420, 556 nm (Figure S3 and Table 1).
Although addition of imidazole (as a neutral unsubstituted
compound) to F8Cmpd-II resulted in a similar UV-vis signa-
ture as compared to the (Im–)F8Cmpd-II derivative, differ-
ent Mössbauer parameters were observed, supporting the
supposition that anionic imidazolate was bound in what we
formulate as (Im–)F8Cmpd-II (Figure S12 and Table S2).
The Mössbauer spectrum of (ArO–)F8Cmpd-II showed
one quadrupole doublet, supporting the compound’s purity
(EQ = 1.57 mm/s  =  mm/s), however, (Im–)F8Cmpd-
II (EQ = 1.55 mm/s  =  mm/s) had a minor (13%) fer-
ryl impurity of unreacted F8Cmpd-II (Figures 7 and Tables
1 & S2). Both derivatives were confirmed to be of the
iron(IV) oxidation state by the isomer shift around 0.1
mm/s and six-coordinate from the decreased quadrupole
splitting (~ 2.0 to 1.5 mm/s) upon adding imidazolate or
phenolate to F8Cmpd-II (Table 1). The isomer shift and
quadrupole splitting increased with imidazolate (vs. the
neutral imidazole, DCHIm), which is not consistent with the
expected increase in Fe-O elongation and shortening of Fe-
NIm bond imparted by a better electron donating axial lig-
and. These observations may be accounted for by consider-
ing the steric effects of the phenolate methoxy groups af-
fecting the angle at which the axial ligand interacts with the
iron center which thus may hinder electron donation from
the phenolate. It may also be due to the solvent mixture
(ArO–)F8Cmpd-II was prepared in, in that it is predomi-
nantly toluene (a non-polar solvent) which could well favor
ion pairing in solution. Thus, the phenolate may be interact-
ing with both the iron and the crowned sodium ion, which
may also be affecting the angle at which the phenolate coor-
dinates to the iron (this could also affect the donation ob-
served from (Im–)F8Cmpd-II). See also below, the discussion
of DFT calculations since computations for both (ArO–
)F8Cmpd-II and (Im–)F8Cmpd-II resulted in values closer to
the experimental spectroscopic data when a sodium ion was
included.
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Figure 7. 80 K zero applied magnetic field 57Fe Mössbauer
spectra (blue) with corresponding fits (red) at 2 mM in 1:9
MeTHF:toluene: Top. (Im–)F8Cmpd-II major (black) and minor
(purple) species (unreacted F8Cmpd-II) & Bottom. (ArO–
)F8Cmpd-II (one quadrupole doublet indicating high purity).
Changes in the absorption are due to differences in sample cell
path length. Major species parameters given in Table 1 and the
parameters for the minor species are given in Table S2.
The iron(IV) oxidation state and lack of strong electron
donation from the phenolate or imidazolate axial ligands
was also confirmed by rR spectroscopy employing 57/54Fe la-
beling, wherein DCHIm effected a greater change on the
Fe=O bond, which does not follow the expected trend (Fig-
ure 6 and Table 1). Phenolate coordination to F8Cmpd-II
slightly weakened the (Fe=O) from 833 to 829 and (Im–
)F8Cmpd-II showed a similar 5 cm-1 downshift from 833 to
828 cm-1. Based on Badger’s rule78 and the rR spectroscopic
data, (ArO–)F8Cmpd-II has an Fe=O bond length of 1.634 Å
and (Im–)F8Cmpd-II has an Fe=O bond length of 1.635 Å,
both of which are typical for ferryl-oxo systems as seen in
Table S1. As the results reported herein demonstrate, we
have established conditions to generate synthetic Com-
pound-II heme models with anionic axial ligands and future
studies with a more basic phenolate or different porphyrin
system may be able to better mimic catalase.
EXAFS/XANES Spectroscopy of the Axially Ligated
Ferryl Derivatives. EXAFS spectroscopic literature is ex-
tremely limited for synthetic heme iron(IV) oxo Cmpd-II
systems relative to Mössbauer or rR literature; there is only
one report, by Penner-Hahn and co-workers in 1986 for (1-
MeIm)(TTP)FeIV(=O) [TTP = meso-tetratolylporphyrin],
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Journal of the American Chemical Society
where the Fe=O bond distance was determined to be 1.64 – XAS. However, these features will be explored by high-en-
1.66 Å.33 Due to early difficulties modeling the significant
multiple scattering in hemes, there are few examples of syn-
thetic heme Fe=O EXAFS spectra; however, numerous
EXAFS spectroscopy reports on heme enzyme intermedi-
ates have been reported by Green and co-workers.1,3,40 The
present report will significantly add to the limited EXAFS
reports for synthetic heme iron(IV) oxo compounds.
ergy resolution fluorescence detected (HERFD) K-edge
spectroscopy.
1
2
3
4
5
6
7
8
The Fe EXAFS spectroscopy (k = 2-15.9 Å-1) fits for the
synthetic F8Cmpd-II models confirms the formation of Fe=O
units with an Fe=O distance of 1.66 Å for (DCHIm)F8Cmpd-
II and all other models at 1.65 Å, with the differences not
being significant (Figure S16 and Table S3). With the excep-
tion of the (ArO–)F8Cmpd-II, fits were unable to resolve the
axial ligand bond length from the pyrrole nitrogens in the
first coordination sphere. For the ArO– axial ligand system,
a marginally improved fit can be achieved by separating the
axial ligand scattering path from the pyrrole nitrogen path
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slightly above the experimental resolution (Raxial-Rpyrrole
=
0.12 Å; resolution = 0.11 Å). Only the F8Cmpd-II complex
had a best fit with an Fe-Np/N coordination number of 4
(Table S3), substantiated by the large pre-edge intensity
(Table 1 and see discussions below). The tethered PIm por-
phyrinate reveals a similar fit as the DCHImF8Cmpd-II, but
with a lower σ2 factor for the pyrrole/axial nitrogen paths.
While the EXAFS data are similar when comparing
F8Cmpd-II and its derivatives (due to the predominance of
the signals from the pyrrole structure), there are key differ-
ences in the intensities of the pre-edge region (X-ray Ab-
sorption Near-Edge Spectroscopy, XANES) for each model
(Figures 8 & S15A). The pre-edge quadrupole intensity is
governed by the 3d-hole metal character of the 1s->3d tran-
sition, and dipole intensity arises from 4p orbital mixing
with the 3d manifold for non-centrosymmetric molecules:59
in this case due to the strong axial distortion caused by the
short Fe(IV)=O bond.
Figure 8. XANES spectra of the tethered imidazole complex
(magenta) 5-coordinate F8Cmpd-II (black) and axially ligated
derivatives: DCHImF8Cmpd-II (blue), ImidazolateF8Cmpd-II
(red), and 3,5-dimethoxy PhenolateF8Cmpd-II (green). De-
creased pre-edge intensity was indicative of axial ligand
strength, which correlated well with the electronic influence of
the various axial ligands on the (Fe=O) obtained from rR.
Synthetic Heme Lewis Acid Adduct Ferryl Derivatives.
UV-vis spectroscopic monitoring of the addition of 1
equivalent of the Brønsted (and Lewis) acid 2,6-lutidinium
triflate (LutH+), dissolved in butyronitrile, to F8Cmpd-II or
(DCHIm)F8Cmpd-II resulted in the formation of the Cmpd-
II
Lewis
acid
adducts,
F8Cmpd-II(LutH+)
and
(DCHIm)F8Cmpd-II(LutH+), respectively (Figures 3, S5, & S6
and Table 2). The former adduct exhibited slightly shifted
Soret and Q-band absorptions (i.e., changes from 415, 544
nm to 413, 546 nm) while addition of LutH+ to
(DCHIm)F8Cmpd-II caused a blue shift in the Soret and Q-
band peaks (i.e., from 421, 552 to 418, 539 nm) (Figures S5
& S6 and Table 2). Interestingly, the interaction between
LutH+ and F8Cmpd-II or (DCHIm)F8Cmpd-II resulted in a de-
creased molar absorptivity in the Soret and Q-band (vs. the
increased molar absorptivity seen when an axial base was
added, vide supra).
Peak fitting of the pre-edge intensities reveals that the 5-
coordinate F8Cmpd-II has the largest pre-edge area (Table
1), due to a large dipole contribution caused by 4p mixing
into the 3d orbitals. Of the 6-coordinate models, the ArO–
derivative has the largest pre-edge area, followed by that for
the imidazolate, then the neutral imidazole (i.e., DCHIm) ax-
ially ligated species. The integrated pre-edge intensity area
positively correlates with the 57Fe=O stretching mode fre-
quency observed by rR which maps naturally onto a short-
ening of the Fe=O bond length, increased 4p-3d orbital mix-
ing, and concomitant increase in the dipole intensity of the
pre-edge (Table 1). It is noteworthy, that the difference in
pre-edge area between ArO– and Im– axial ligand systems is
not mirrored in the negligible difference in the Fe=O fre-
quency determined by rR spectroscopy. This may coincide
with the increased Fe-Oaxial bond length determined by
EXAFS spectroscopy (Table S3) relative to the unresolvable
Fe-Naxial distance in the Im– system. Attempts to fit the PIm
pre-edge resulted in a significantly different background
relative to the F8 complexes, likely related to the sample be-
ing a mixture of species (Table S2), thus we considered the
pre-edge unreliable for comparative purposes. Although
most obvious for the DCHIm complex (near 7117.8 eV), the
spectra featured an inflection of the rising edge region, but
could not be acceptably resolved by standard Fe K-edge
w
Table 2. Spectroscopic characterization of Lewis Acid F8Com-
pound-II derivatives.a
Fe
UV-vis
Mössbauer b
rR
XAS
Pre-
Soret,
Q-band
(nm)
(57Fe=O)
(57-54Fe)
(cm-1)

(mm/s
)
EQ
(mm/s
)
edge
Aread
819(4)
 
: 1573
413,
546
48.8
(1.5)
F8Cmpd-
II(LutH+)
0.08
2.23
(DCHIm)
F8Cmpd-
II(LutH+)
418,
539
25.0
(1.0)
0.07
1.30
---c
aAll these complexes are EPR silent via perpendicular mode
EPR spectroscopy at 10 K. bThe values for the major species are
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Journal of the American Chemical Society
Page 10 of 23
reported in this table. See SI for minor Mössbauer species fits
ferryl complex and the other to F8Cmpd-II. F8Cmpd-II(LutH+)
c
1
2
3
and details. Attempts to obtain the rR spectrum were unsuc-
cessful for DCHImF8Cmpd-II(LutH+). dPre-edge Area (standard
deviations for the fits)
was close to a 50/50 mixture with F8Cmpd-II, however,
(DCHIm)F8Cmpd-II(LutH+) was of high purity with only 10%
being F8Cmpd-II.
4
5
6
7
8
9
57Fe Mössbauer spectroscopy of (DCHIm)F8Cmpd-
II(LutH+) indicated the complex consisted of one species of
high purity as seen by the observation of one quadrupole
doublet (EQ = 1.30 mm/s and  =  mm/s) (Figure 9).
However, F8Cmpd-II(LutH+) (EQ = 2.23 mm/s and
 =  mm/s) had an impurity of unreacted F8Cmpd-II
starting material (Figure 9). The identity of this impurity
was later confirmed by 57/54Fe labeled rR spectroscopy (vide
infra, Figures S7 & S9, and Table 2). Both of the Lewis acid
ferryl derivatives were confirmed to be of the iron(IV) oxi-
dation state as reflected by the isomer shift around ~0.1
mm/s and for F8Cmpd-II(LutH+) this was further confirmed
by rR spectroscopy from the oxidation state marker band
being ~ 1370 cm-1 (Figure S10 and Table 2). Thus, addition
of LutH+ to both parent species resulted in a slight decrease
in the isomer shift and a slight increase in the quadrupole
splitting observed by Mössbauer spectroscopy. The in-
crease in quadrupole splitting in both cases is consistent
with a weakened Fe=O bond. Further, the decreased isomer
shift values are as expected due to the Lewis acid withdraw-
ing electron density away from the iron nucleus through its
interaction with the oxo ligand.
Resonance Raman spectroscopic data was only feasible to
be collected for F8Cmpd-II(LutH+) (Figure S9 and Table 2).
Unfortunately, attempts to obtain rR data for
(DCHIm)F8Cmpd-II(LutH+) were unsuccessful. Upon addi-
tion of LutH+ to F8Cmpd-II, the (Fe=O) decreased from 833
to 819 (Δ –14 cm-1) and required addition of 5 equiv. LutH+
to fully convert F8Cmpd-II to the F8Cmpd-II(LutH+) complex
(Figure S9). This (57Fe=O) downshift to 819 cm-1 indicates
that the Lewis acid caused a slight elongation of the Fe=O
bond of 0.007 Å (from 1.632 to 1.639 Å) according to
Badger’s rule.
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53
54
55
56
57
58
59
60
EXAFS spectroscopy is only accurate to ~0.02 Å and could
not detect this slight difference between F8Cmpd-II and
F8Cmpd-II(LutH+), however the pre-edge intensity de-
creases for the LutH+ adduct, indicating small elongation of
the Fe-O bond (Figure 10). EXAFS spectroscopy of F8Cmpd-
II(LutH+) and (DCHIm)F8Cmpd-II(LutH+) indicated that
both species were Lewis acid adducts and not fully proto-
nated FeIV–OH species, which is also inferred by the only
slight spectroscopic changes observed (Figure S17 and Ta-
ble S4). This is due to the presence of the very short Fe=O
bond (1.64 Å), while protonated Cmpd-II species have Fe–O
bond lengths greater than or equal to 1.8 Å.1 The interaction
of the Lewis acid with the oxo ligand causes the pre-edge
intensity to decrease from 54.3 to 48.8 units from F8Cmpd-
II to F8Cmpd-II(LutH+) and from 27.6 to 25.0 from
(DCHIm)F8Cmpd-II to (DCHIm)F8Cmpd-II(LutH+) indica-
tive of a small reduction in dipole character – caused by a
reduction in ligand field axial distortion (elongating Fe-O).
Although there is not full protonation, these may be prom-
ising systems for better understanding the interaction of
hydrogen bonding with ferryl complexes in enzymes, which
is known to contribute to the increased stability and reac-
tivity of analogous high-valent intermediates.
Figure 9. 80 K zero applied magnetic field 57Fe Mössbauer
spectra (blue) with corresponding fits (red) at 2mM in 1:9
Figure 10. X-ray absorption edge spectra of F8Cmpd-II (blue)
and (DCHIm)F8Cmpd-II (red) and as dashed lines F8Cmpd-
II(LutH+) (blue) and (DCHIm)F8Cmpd-II(LutH+) (red). Addi-
tion of LutH+ decreases pre-edge intensity, which is indicative
of slight elongation of the iron(IV)oxo bond length.
MeTHF:toluene: Top. F8Cmpd-II(LutH+)
&
Bottom.
(DCHIm)F8Cmpd-II(LutH+). Major species in (black) and minor
species in purple. Changes in absorption are due to differences
in sample cell path length. Parameters ascribed to the Lewis
acid species are given in Table 2 and parameters for the other
species seen are given in Table S2. Both species were fitted to
two quadrupole doublets, which was ascribed to the Lewis acid
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Journal of the American Chemical Society
Electronic Structure Calculations
(ArO-)
F8Cmpd-II
1.669
0.20
0.18
0.15
0.45
0.84
0.99
765.7
789.8
799.9
10.523
11.444
4.793
1
2
3
4
5
6
7
8
To obtain further insights into the geometric perturbations
caused by the presence of the various axial ligands, geome-
try optimizations with DFT were performed for each of the
F8-derived complexes (Table 3 and Figure S40). For each
complex, we calculated the Mössbauer parameters of the
geometry optimized structures and determined the p-or-
bital contributions to the metal-based frontier molecular
orbitals (FMOs) through Mulliken population analysis. In all
cases, the quadrupole splitting was calculated to be positive
agreeing with our assumption that strong axial bonding will
lower the observed magnitude of ΔEQ. Further, utilizing the
Badger’s rule parameters of Green, 78 we estimate the Fe=O
bond stretching vibrational frequency from the geometry
optimized bond length.
(Na+ ArO-)
F8Cmpd-II
1.655
1.649
PImCmpd-
II
Calculations including either of the ligands, ArO– or Im–,
result in longer Fe-O bonds (Table 3), coinciding with de-
creased Fe–O Badger’s rule stretch frequencies relative to
the DCHIm complex. This contrasts with the experimental
observations of higher stretching frequencies for the ani-
onic ligands than the six-coordinate DCHIm model. Like-
wise, the anionic computational models demonstrate poor
agreement with the experimental Mössbauer parameters.
Inclusion of a Na+ counterion near the anionic axial ligands
improves the correlation to the experimental Fe–O fre-
quency, with a concomitant shift in predicted Mössbauer
parameters towards the experimental values. These calcu-
lations suggest that the experimentally investigated anionic
ligand models are potentially forming ion-pair complexes
with the cation.
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59
60
The calculations reveal the 5-coordinate compound to have
the shortest Fe–O bond, which corresponds to the experi-
mentally observed highest rR stretching frequency and pre-
edge intensity relative to the other F8 models. Likewise, pre-
dicted isomer shift and quadrupole splitting values (0.09
and 2.23 mm/s, respectively) correspond well to the ob-
served experimental values for the major Mössbauer spe-
cies (Tables 1 and 3). The short Fe–O bond also coincides
with the greatest p-orbital metal-based hole character
which is reflected in the high experimental pre-edge inten-
sity. Addition of the DCHIm axial ligand results in an in-
crease of the calculated Fe–O bond length and decrease in
quadrupole splitting, reproducing the observed experi-
mental trends and coinciding with the addition of the ligand
along the z-axis. The calculations show addition of the axial
ligand also decreases the p-orbital character of the metal-
based FMOs by 42% relative to the 5-coordinate parent
complex which maps well to the analogous 49% decrease in
experimental pre-edge intensity.
The geometry optimized structure of the tethered imid-
azole complex, PIm, reveals a slight shortening of the Fe–O
bond, coupled with an increase in predicted quadrupole
splitting relative to the DCHIm-bound complex, mirroring
the experimental data for the major component of the PIm
Mössbauer sample. The Fe-Nax bond is longer for PIm and the
average Fe-Neq bonds are shorter with the net effect being a
higher ΔEQ despite having a slightly shorter Fe=O bond than
the DCHIm-bound model.
Inclusion of the LutH+ H-bonding to the oxo of the ferryl,
for the five-coordinate and DCHIm-ligated models, elon-
gates the Fe–O bond by ~0.02 Å (Chart 3). Addition of LutH+
also increases the predicted quadrupole splitting of both ad-
ducts relative to their respective non-LutH+ counterparts
and are in good agreement with the experimental Möss-
bauer parameters – consistent with an elongation of the
Fe=O bond. The Lewis acid adducts, relative to their non-
Lewis acid counterparts, have lower p-orbital character of
the metal based FMOs by 35 and 49 percent, respectively
(Table 3). This calculated reduction in p-orbital character is
not reflected by the change in pre-edge intensity for the six-
coordinate Lewis acid adduct. The five-coordinate Lewis
acid adduct XAS sample was a 50% mixture of the non-LA
species, therefore the DFT-derived p-orbital character can-
not be compared.
Table 3. DFT-derived Spectroscopic Parameters. See Figure
S40 for DFT structures.
Metal
FMOs
Fe=O
(Å)
Mössbauer
ν(Fe=O)
Cmpd-II
Derivative
ΔEQ
(mm
/s)
Badger’
s Rule
(cm-1)
σ
P-or-
bital %
(mm/s)
F8Cmpd-II 1.630
0.09
0.09
0.15
0.12
0.18
0.17
0.15
2.23
2.58
0.89
1.36
0.02
0.48
1.00
838.1
786.8
796.1
748.8
750.6
782.4
800.1
18.157
11.816
11.618
5.904
F8Cmpd-II
1.657
(LutH+)
(DCHIm)
1.652
F8Cmpd-II
Chart 3. DFT structures of F8Cmpd-II(LutH+) (A) and
(DCHIm)F8Cmpd-II(LutH+) (B). Hydrogen atoms (except for
the proton of LutH+) and the cyclohexyl groups of DCHIm
were omitted for clarity.
(DCHIm)
F8Cmpd-II 1.679
(LutH+)
(Im-)
1.677
12.208
12.424
10.766
F8Cmpd-II
(Na+ Im-)
1.659
F8Cmpd-II
(H+Im-)
1.649
F8Cmpd-II
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Journal of the American Chemical Society
Page 12 of 23
band at 420 and 530 nm, respectively. Further, EPR spec-
1
2
3
4
troscopy (X-band, run at 10 K) of the reaction mixtures after
5 equiv. Me10Fc or Me8Fc was added to (DCHIm)F8Cmpd-
II(LutH+) was silent, analogous to the EPR silent spectra of
(DCHIm)2F8FeII in 9:1 MeTHF:toluene at –90 oC.
5
6
7
8
9
Scheme 2. Addition of Me10Fc or Me8Fc to
(DCHIm)F8Cmpd-II(LutH+) results in a two-electron re-
duction to give (DCHIm)2F8FeII as the final product, as
supported by UV-vis and Mössbauer spectroscopic
measurements.
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59
60
Outer-sphere electron-transfer reduction of the
Lewis acid ferryl derivatives. Various outer-sphere re-
ductants (ferrocene derivatives, see SI for depicted struc-
tures) were added to F8Cmpd-II, (DCHIm)F8Cmpd-II,
F8Cmpd-II(LutH+), and (DCHIm)F8Cmpd-II(LutH+) to ap-
proximate the reduction potential of the iron(IV) oxo func-
tionality (Schemes 2 and S1-S12 and Figures 11 & S18-S29),
and to compare and contrast the effects of the presence of a
strongly donating axial ligand (i.e., DCHIm) and/or the
Lewis acid interacting with the oxo ligand. Reactivity stud-
ies, monitored by UV-vis spectroscopy, showed that the
Lewis acid derivative (DCHIm)F8Cmpd-II(LutH+) could re-
act with 10 equiv. of decamethylferrocene (Me10Fc, E1/2 = –
0.53 V vs. Fc+/0 in MeTHF)79 or 10 equiv. of octamethylfer-
rocene (Me8Fc, E1/2 = –0.43 V vs. Fc+/0 in MeTHF),79 but not
10 equiv. of dimethylferrocene (Me2Fc, E1/2 = –0.115 vs.
Fc+/0 in MeTHF)79 (Schemes 2, S2, and S3 & Figures 11, S19,
& S20). The reactions with Me10Fc and Me8Fc showed the
immediate spectral change from 418, 539 nm to 419, 530
nm, with two unique low energy bands in the 600-800 nm
region assignable to their respective ferrocenium products
(Schemes S2 & S3 and Figures 11, S19 & S20). However,
(DCHIm)F8Cmpd-II did not react with any of these reduct-
–
ants (Scheme S1 and Figure S18). The authentic B(C6F5)4
(BArF) salts of the oxidized forms of the reductants, decame-
thylferrocenium BArF (Me10Fc+) and octamethylferroce-
nium BArF (Me8Fc+) were prepared in order to quantify the
number of electrons transferred to (DCHIm)F8Cmpd-
II(LutH+) in these reactions. Quantification of the low en-
ergy ferrocenium peaks observed after completion of the
reduction reactions indicated that 2 equivalents of Me10Fc
or Me8Fc were consumed in the reaction with
(DCHIm)F8Cmpd-II(LutH+) (Figures S19 & S20), based on
standard curves produced from the authentic ferrocenium
salts (Figures S30 & S31 and Tables S5 & S7). To further
confirm this result, sequential addition of 0.5 equiv. of
Me10Fc to (DCHIm)F8Cmpd-II(LutH+), showed that no fur-
ther reaction occurred after adding 2 equivalents. Various
ferrous derivatives were prepared independently to iden-
tify the resulting heme reaction product. It was thought that
due to the 2 equivalents of DCHIm employed to form
(DCHIm)F8Cmpd-II that the final ferrous product might be
coordinated by DCHIm. (DCHIm)2F8FeII was prepared inde-
pendently by addition of two equivalents of DCHIm to F8FeII
in 9:1 MeTHF:toluene at -90 oC. The UV-vis absorption fea-
tures of (DCHIm)2F8FeII (Figures 11 (bottom) & S32) corre-
late directly with the spectrum observed for the final reac-
tion mixture of (DCHIm)F8Cmpd-II with Me10Fc or Me8Fc
(Figures 11 (top) and S19), all exhibiting a Soret and a Q-
Figure 11. UV-vis spectroscopy following the addition of 10
equiv. of Me10Fc to 0.1 mM (DCHIm)F8Cmpd-II(LutH+) in 1:9
MeTHF: toluene at -90 oC ; wherein there was an immediate
change from the LA ferryl adduct in orange to the resulting re-
action mixture in navy (Top). The products in the reaction mix-
ture were independently generated by adding 1 equiv. DCHIm
o
to 0.1 mM F8FeII in 1:9 MeTHF:toluene at -90 C (red) and by
adding a second equiv. of DCHIm to allow the favorable for-
mation of the major product (DCHIm)2F8FeII (blue) (Bottom),
a low-spin 6-coordinate d6 complex. The top reaction mixture
in navy and the bottom (DCHIm)2F8FeII complex in light blue
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Journal of the American Chemical Society
had comparable UV-vis features, Soret at 420 nm and Q-band
tom). The values in the resulting reaction mixture were com-
parable to (DCHIm)2F8FeII and thus (DCHIm)F8Cmpd-II(LutH+)
was reduced by 2 electrons forming the corresponding low-
spin ferrous species and water. Mössbauer parameters are
given in Table S2.
1
2
3
4
at 530 nm (Figures S19 & Figure S32). Quantification of the low
energy decamethylferrocenium peaks observed in the 700 –
800 nm region indicated two electrons were transferred at the
end of the reaction (Figure S19 & S30 and Table S5).
5
6
7
8
9
With all the data taken together, the reduction potential
of (DCHIm)F8Cmpd-II(LutH+) can be approximated. Unfor-
tunately, cyclic voltammetry was not able to be obtained for
the ferrocene derivatives in the 1:9 MeTHF:toluene reaction
mixture due to the insolubility of common electrolytes in
this solvent mixture. Thus, the reduction potential values of
the ferrocene derivatives in MeTHF79 were used to approx-
imate the reduction potential of the iron(IV) oxo species
(Table S9). The reduction potential of the iron(IV) oxo moi-
ety of (DCHIm)F8Cmpd-II(LutH+) can then be bracketed, –
0.43 < E1/2 < –0.115 V vs. Fc+/0 based on the reactivity stud-
ies described above (Table S9). Since (DCHIm)F8Cmpd-II
did not react with any of the reductants utilized (Scheme S1
and Figure S18), its reduction potential must be less than –
0.53 V vs. Fc+/0, at least 100 mV more negative than that of
(DCHIm)F8Cmpd-II(LutH+). This substantiates that
The Mössbauer spectrum of the reaction mixture of
(DCHIm)F8Cmpd-II(LutH+) with 10 equiv. Me10Fc showed
two quadrupole doublets with a major species of 90% [(EQ
= 1.01 mm/s and  =  mm/s)] and minor species con-
tributing 10% [(EQ = 2.45 mm/s and  =  mm/s)] (Fig-
ure 12, Table S2). Similar products were identified in the
Mössbauer spectrum of the reaction mixture of
(DCHIm)F8Cmpd-II(LutH+) with 10 equiv. Me8Fc (Figure
S14 and Table S2). In combination with the similar UV-vis
spectral features, comparable Mössbauer parameters of the
authentic standard of (DCHIm)2F8FeII (Table S2) deter-
mined the product mixture is predominantly composed
(90%) of the (DCHIm)2F8FeII species (Figure 12). It is postu-
lated that two equivalents of reductant and two protons,
one from LutH+ and one from 3-chlorobenzoic acid (the by-
product of mCPBA used to originally generate the oxo-ferryl
complexes) allows for the reduction of the iron(IV) species
to iron(II) (rather than iron(III)) due to the thermodynami-
cally favorable concomitant formation of the low-spin d6
(DCHIm)2F8FeII product, and, of course, the release of water
(Scheme 2).
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58
59
60
(DCHIm)F8Cmpd-II
is
a
worse
oxidant
than
(DCHIm)F8Cmpd-II(LutH+), as expected, and provides indi-
rect evidence for interaction of the Lewis acid with the oxo
ligand. It also shows that even though small structural
changes were observed (vide supra), large reactivity differ-
ences may be seen. Thus, (DCHIm)F8Cmpd-II(LutH+) can be
used to examine the interaction of a proton with the oxo lig-
and of a ferryl species to give insights into the protonated
Cmpd-II intermediate in cytochrome P450 monooxygenase
and/or Lewis acid adducts of other enzyme or synthetic
high-valent heme iron(IV)-oxo species. Also, due to hydro-
gen bonding being a prevalent factor in metalloenzymes to
stabilize the formation of high-valent species, and/or mod-
ulate their reactivity, these or related compounds can be
used as good models to support how hydrogen bonded in-
teractions help dictate an enzyme’s reactivity.
Analogous reactivity studies were carried out for
F8Cmpd-II and F8Cmpd-II(LutH+). In contrast to
(DCHIm)F8Cmpd-II, these studies showed that F8Cmpd-II
can be reduced by Me10Fc, but not Me8Fc (Schemes S4 & S5
and Figures S21 & S22). Addition of 10 equiv. of Me10Fc to
F8Cmpd-II, monitored by UV-vis spectroscopy (Scheme S4
and Figure S21), resulted in the formation of
(MeTHF)F8FeIII(X), with X = solvent or 3-chlorobenzoate.
Quantification of the Me10Fc+ peaks was not possible due to
the overlapping intense low energy ferric band, however,
the ferric state was confirmed by low-temperature 2H-NMR
spectroscopy employing the deuterated pyrrole d8-F8 ana-
log (Figure S37). Though the reaction stopped at iron(III)
and
exhibited
a
similar
2H-NMR
shift
as
(MeTHF)F8FeIII(OH), the final product was found to not be
the iron(III)-hydroxide complex due to differences in UV-vis
absorption features (Schemes S4, S13 & S14 and Figures
S21, S33 and S34).
Figure 12. 80 K zero applied magnetic field 57Fe Mössbauer
spectra (blue) with total fits (red): Addition of 5 equiv. Me10Fc
to 2 mM (DCHIm)F8Cmpd-II(LutH+) in 1:9 MeTHF:toluene at -
Interestingly, especially based on the small structural
changes observed for F8Cmpd-II(LutH+) compared to
F8Cmpd-II, the addition of LutH+ greatly increased this spe-
cies’ reduction potential. Previous reports,80–84 on non-
heme iron(IV) or manganese(IV) oxo complexes have
o
90 C (Top). (DCHIm)2F8FeII was prepared by adding 2 equiv.
of DCHIm to 2 mM F8FeII in 1:9 MeTHF:toluene at -90 oC (Bot-
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Journal of the American Chemical Society
shown that addition of Lewis acids such as Sc3+ or HClO4 in-
creased the reduction potential of these species by 0.5 – 0.9
mV (however, the greater difference in magnitude, as ob-
served herein, were usually only detected when excess
amounts of the Lewis acid were added). Thus, the unusually
large increase in reduction potential observed here, with
the addition of 1 equiv. LutH+ (as a molecular adduct and
not just a proton transferred, vide supra), is notable. In fact,
F8Cmpd-II(LutH+) was found to react with Me10Fc, Me8Fc,
Page 14 of 23
1
2
3
4
5
6
7
8
Me2Fc, ferrocene (Fc), acetylferrocene (AcetylFc, E1/2
=
9
+0.235 V vs. Fc+/0 in MeTHF79), and diacetylferrocene (Diac-
etylFc, E1/2 = +0.460 V vs. Fc+/0 in MeTHF, Table S9)
(Schemes 3, S6-S9 & S11 and Figures S23-S26 & S28). How-
ever, F8Cmpd-II(LutH+) did not react with tris(4-bromo-
phenyl)amine (E1/2 = +0.695 V vs. Fc+/0 in MeTHF, Table S9)
(Scheme S11 and Figure S28). These reactions with
F8Cmpd-II(LutH+) resulted in the formation of more compli-
cated reaction mixtures. The stronger reductants Me10Fc
and Me8Fc were able to predominately reduce the iron(IV)
oxo Lewis acid adduct by two electrons, forming (X)2F8FeII,
with X= MeTHF or 3-chlorobenzoic acid (Scheme 3 Pathway
3 and Schemes S6, & S7 and Figures S23 & S24), whose as-
signment is based on similar absorption features (max of
428 and 530 nm) as (DCHIm)2F8FeII (max of 420 and 530
nm). The low-energy ferrocenium peaks were quantified
and supported the conclusion that ~2 equiv. of reductant
were consumed (Tables S6 & S8); a minor ferric impurity
could be observed in the Soret peak region at 411 nm and
slightly in the Q-band (Figures S23 and S24). Also, EPR spec-
troscopy (X-band, run at 10 K) of the reaction mixtures after
5 equiv. Me10Fc or Me8Fc was added to F8Cmpd-II(LutH+)
was silent, analogous to the EPR silent spectrum of
(DCHIm)2F8FeII in 9:1 MeTHF:toluene at -90 oC. Thus, a six-
coordinate ferrous complex was the product when employ-
ing these stronger reductants.
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48
49
50
51
52
53
54
55
56
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58
59
60
However, addition of weaker reductants such as Me2Fc,
Fc, AcetylFc, and DiacetylFc did not result in two-electron
reduction of the iron(IV) oxo to iron(II); predominately a
ferric species was observed via UV-vis spectroscopy
(Scheme 3 Pathway 2A, Figures S25, S26, & S28 & Schemes
S8, S9 & S11). To determine the final product from the reac-
tions with the weaker reductants, 3-chlorobenzoic acid was
added to F8FeIIIOH, supporting that (MeTHF)F8FeIII(OH2) is
the resulting species based on the similar Soret of 411 nm
and highly characteristic multiple peak Q-band signature
between the authentically generated species and the reac-
tion mixture (Scheme S10 & Figures S27). However, due to
the relatively intense, broad low energy band of the ferric
species spanning ~650 – 1100 nm, the ferrocenium prod-
ucts for the weaker reductants were not able to be quanti-
fied due to the overlap of the peaks. The ferric nature of the
Scheme 3. F8Cmpd-II(LutH+) can be reduced by various fer-
rocene derivatives. When stronger reductants (Me10Fc or
Me8Fc) are used, the iron(IV) is reduced by two electrons
to (X)2F8FeII (Pathway 3). However, when weaker reduct-
ants (Fc, AcetylFc, or DiacetylFc) are used, one-electron re-
duction occurs, giving (MeTHF)F8FeIII(OH2) (Pathway 2
(A)), as determined by comparison with the independently
generated (MeTHF)F8FeIII(OH2) prepared by addition of 3-
chlorobenzoic acid to F8FeIII(OH) (Pathway 1 (A)). Further,
the assignment of (MeTHF)F8FeIII(OH2) is supported due to
its ability to be reduced by Me10Fc or Me8Fc to give
(X)2F8FeII, which similarly occurs when those reductants
are added to the Fc, AcetylFc, or DiacetylFc reaction mix-
ture (B). All reactions were carried out in 1:9 MeTHF:tolu-
2
reaction product was supported by H-NMR spectroscopy,
which showed a paramagnetic shift around 124 ppm (Fig-
ure S38). Also, addition of 5 equiv. AcetylFc and DiacetylFc
to the EPR silent F8Cmpd-II(LutH+) complex resulted in a
high-spin ferric signal (g = 6 and 2) (Figure S28). To further
confirm the iron(III) oxidation state, 10 equiv. of Me10Fc
was added to the Fc and AcetylFc reaction mixtures, result-
ing in an immediate spectral change to 530 nm, similar to
2
the UV-vis signature and H-NMR shift of the reaction mix-
tures when Me10Fc or Me8Fc were used (Scheme 3 Pathway
2B, Figure S38). Further evidence for the reduction of the
ferric species to ferrous was the quantification of the deca-
methylferrocenium peaks, which showed a one-electron re-
duction (as observed by the low-energy bands, Schemes S10
and S12 & Figures S27 and S29). The independently gener-
ated (MeTHF)F8FeIII(OH2) was also able to be reduced by
Me10Fc (Scheme 3 Pathway 1B), forming a similar 530 nm
UV-vis signature and the low energy Me10Fc+ peaks were
quantified to indicate a 1-electron reduction occurred
(Scheme S14 & Figure S34). The comparable UV-vis (Fig-
o
ene at –90 C. See Supporting Information for further de-
tails.
2
ures S25, S26, S28 & S33) and H-NMR signatures (Figure
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Journal of the American Chemical Society
(DCHIm)F8Cmpd-
II(LutH+)
-430 < E1/2 < -
S38
&
S39) of the independently generated
Me8Fc
-430
-530
(MeTHF)F8FeIII(OH2) and the Me2Fc, Fc, AcetylFc reaction
mixtures supports that that the ferric aqua complex was
formed; additional support is that both can be further re-
duced by one electron (Figures S27, S29, S34, S38, & S39).
1
2
3
4
5
6
7
8
115
Me10Fc
F8Cmpd-II
-530 < E1/2 < -430
(DCHIm)F8Cmpd-II
< -530
These reactivity studies allow us to bracket the reduction
Values collected in MeTHF,79 see SI for further details.
(N(ArBr)3 = tris(4-bromophenyl)amine)
a
potential of F8Cmpd-II, –0.53 < E1/2 < –0.43 V vs. Fc+/0
,
demonstrating that F8Cmpd-II is a stronger oxidant than
(DCHIm)F8Cmpd-II (Tables 4 and S9). However, the reduc-
tion potential of F8Cmpd-II(LutH+) is bracketed to be be-
tween +0.460 and +0.695 V vs. Fc+/0, since DiacetylFc was ca-
pable of reducing this species, but no reaction occurred with
tris(4-bromophenyl)amine (Scheme S11 and Figure S28C).
Thus, addition of LutH+ to F8Cmpd-II increases the reduc-
tion potential of the complex by greater than 0.890 V, indi-
cating that F8Cmpd-II(LutH+) is a strong oxidant (Tables 4
and S9). The reversibility of these reduction reactions was
not examined because it is unlikely that these reactions are
reversible due to the formation of stable reduced products
that would not be easily oxidized. For example, FeIII(OH2) →
FeIV(OH2) is thermodynamically unfavorable due to the
likely instability of a protonated iron(IV) oxo complex while
(DCHIm)2F8FeII → (DCHIm)F8Cmpd-II(LutH+) would be un-
favorable because the former is a highly stable low-spin d6
complex. These synthetic heme iron(IV) oxo Lewis acid de-
rivatives may pave the way for the preparation of a fully
protonated FeIV–OH model.
Conclusions
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High-valent ferryl intermediates with various axial lig-
ands catalyze numerous oxidative processes in metalloen-
zymes. Investigations utilizing synthetic systems may allow
for a greater understanding of structures and bonding (i.e.,
electronic structure), plus mechanistic insights into enzy-
matic pathways. This aids in the understanding of the na-
ture of these highly oxidizing intermediates which can be
harnessed for numerous practical applications (drug devel-
opment, catalysis, synthesis, fuel cells, etc.). However, there
have been limited reports on synthetic heme Cmpd-II mod-
els with varying axial ligation, with no synthetic model sys-
tems studied for catalase. Herein, the full characterization
of synthetic Cmpd-II models with and without various axial
bases was achieved and the electronic properties examined
using a wide array of spectroscopic techniques including
UV-vis, EPR, 2H-NMR, Mössbauer, 57/54Fe labeled resonance
Raman, and EXAFS.
Addition of axial bases such as imidazole, imidazolate, or
phenolate cause a slight elongation of the Fe=O bond. This
is the first report of a synthetic heme Cmpd-II model con-
taining phenolate axial ligation as a model for the enzyme
catalase. This synthetic model complex could also poten-
tially be used in future electrocatalytic studies to under-
stand how catalase (and analogous enzymes) dispropor-
tionate reactive oxygen species (e.g., H2O2) minimizing
harmful ROS reactions.28 The first Mössbauer and EXAFS
data of a synthetic heme axially ligated phenolate or teth-
ered imidazole iron(IV) oxo species is also reported. The
work reported herein offers significantly expanded EXAFS
characterization of synthetic heme iron(IV) oxo complexes
than is currently reported in the literature. Further, the
iron(IV) oxo vibrational frequency was identified by rR via
employing 54Fe and 57Fe derivatives (vs. needing to synthe-
size 18-O labeled oxidant (e.g., mCPBA) or using 18-O la-
beled water for an exchange reaction, which leads to ad-
verse reactions).
Table 4. Approximation of the reduction potential of various
ferryl derivatives by reacting them with ferrocene derivatives.
This work reports on the first hydrogen bonded synthetic
heme Lewis acid iron(IV) oxo adducts with and without an
axial base. Addition of the protic Lewis acid, 2,6-lutidinium
triflate, to F8Cmpd-II or (DCHIm)F8Cmpd-II resulted in a
slight elongation of the Fe=O bond, which was supported by
rR and/or XAS spectroscopic analysis. These novel ferryl
Eo vs
Oxidant, paired
with strongest re-
acting reductant
Reduct-
ant
Fc+/0
(mV
Bracketed E1/2
of FeIV=O (mV)
a
)
N(ArBr)3
695
460
235
0
Lewis
acid
adducts,
F8Cmpd-II(LutH+)
and
(DCHIm)F8Cmpd-II(LutH+), showed increased (more posi-
tive) reduction potentials compared to their counterparts
without LutH+; in fact, the reduction potential of F8Cmpd-II
increased by almost a volt upon addition of LutH+. Thus, ad-
dition of LutH+ causes a direct interaction with the oxo lig-
and and makes these synthetic Cmpd-II compounds much
more oxidizing, further supporting the importance of the
Diace-
tylFc
+460 < E1/2 <
F8Cmpd-II(LutH+)
+695
AcetylFc
Fc
Me2Fc
-115
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Page 16 of 23
protonation or hydrogen bonding state of Cmpd-II in enzy- a slight (<0.1eV) change in the edge – the EXAFS analysis
matic systems.
1
presented here utilized both scans per spot, but no signifi-
cant differences were observed by fitting the first scans
only. The pre-edge area analysis for the 6-coordinate
lutidinium complex utilized only the first measured scans.
Data were background subtracted, splined, and normalized
by the PYSPLINE software.86 Theoretical EXAFS scattering
amplitudes were calculated by FEFF (version 7.0).87 These
amplitudes were then used in the fitting performed through
the EXAFSPAK suite for the parameters: Fe-X path distance
(R), the path mean square deviation σ2, and threshold en-
ergy E0. Two multiple scattering paths (Fe-N-Cα/β) were in-
cluded with σ2 fixed to that of the pertinent Fe-Cα/β paths
consistent with previous heme EXAFS studies.88 For the first
coordination sphere, alternative fits were attempted to iso-
late the axial ligand path from the equatorial pyrrole nitro-
gens path – but resulted in unreasonable σ2 values and/or
distances not resolvable by the experimental parameters,
except for the ArO-axial ligand as discussed in the text. Pre-
edge areas were determined by fitting one or two pre-edge
peaks, two background peaks to model the rising-edge, and
one peak for the edge jump utilizing least squares fitting in
the Igor Pro version 6.37 with Pearson VII functions and in-
tegrals were taken using the Igor program “areaxy” trape-
zoidal integration function. The averages and standard de-
viations were taken from 11 best fits for each spectrum (in
the range 7105-7122 eV) where the width of the edge jump
peak was fixed from the first fit and adjusted by alternating
steps of ±0.1 eV then all other parameters were refit again
to a maximum of 5 fits in each direction.
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8
9
Experimental
General
All reagents and solvents purchased and used were of
commercially available quality except as noted. Inhibitor-
free 2-methyltetrahydrofuran (MeTHF) and tetrahydrofu-
ran (THF) were distilled over Na/benzophenone under Ar
and deoxygenated with Ar before use. Toluene was distilled
over calcium hydride and deoxygenated with Ar before use.
Butyronitrile was distilled over sodium carbonate and po-
tassium permanganate and deoxygenated with Ar before
use. Solvent deoxygenation was achieved by bubbling Ar
through the desired solvent for ≥ 45 minutes via an addition
funnel connected to a receiving Schlenk flask. All solvents
were stored in amber bottles under 4 Å sieves. The oxidant,
3-chloroperbenzoic acid (mCPBA) was purified according
to published procedures.85 Air-free manipulations were
performed in a Vac atmosphere OMNI-LAB drybox or under
argon atmosphere using standard Schlenk techniques. Low
temperature UV-vis spectroscopy experiments were car-
ried out by using a Cary-50 Bio spectrophotometer
equipped with an Unisoku USP-203A cryostat using with a
modified Schlenk cuvette with a 1 cm path quartz cell. The
spectrometer was equipped with Cary WinUV Scanning Ki-
netics software. All NMR spectra were recorded in 9 inch, 5
mm o.d. NMR tubes on a Bruker 300 MHz NMR instrument
equipped with a tunable deuterium probe to enhance deu-
terium detection. The 2H chemical shifts were calibrated to
natural abundance deuterium solvent peaks. EPR spectra
were collected with an ER 073 magnet equipped with a
Bruker ER041 X-Band microwave bridge and a Bruker EMX
081 power supply: microwave frequency = 9.42 GHz, micro-
wave power = 0.201 mW, attenuation = 30 db, modulation
amplitude = 10 G, modulation frequency = 100 kHz, temper-
ature = 10 K. Mössbauer spectra were recorded on a spec-
trometer from SEE Co. (Edina, MN) operating in the con-
stant acceleration mode in a transmission geometry. The
sample was kept in an SVT - 400 cryostat from Janis (Wil-
mington, MA), using liquid N2 as a cryogen for 80 K meas-
urements. Isomer shifts were determined relative to the
centroid of the spectrum of a metallic foil of α–Fe collected
at room temperature. Data analysis was performed using
version F of the program WMOSS (www.wmoss.org) and
quadrupole doublets were fit to Lorentzian lineshape. XAS
data were collected at beam lines 7-3 and 9-3 at the Stan-
ford Synchrotron Radiation Lightsource (SSRL) at the SLAC
National Accelerator Laboratory under ring operating con-
ditions of 500 mA over an energy range of 6785 to 8100 eV,
with the samples maintained at 10K. To mitigate X-ray in-
duced sample damage, we parsed each sample into 6-8
spots and collected 1-2 scans per spot. However consistent
in our experience with many synthetic model complexes
frozen in dry organic solvents, we observed no significant
scan-to-scan changes in the pre-edge nor edge positions at
the Fe K-edge energies and we were able to utilize the first
and second scans for nearly all the models. The exception
was the 6-coordinate lutidinium complex which had a ~4%
scan-to-scan reduction in maximum pre-edge intensity and
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Density Functional Theory geometry optimizations were
performed in vacuum with the program ORCA version 4.089
using the BP8690 functional and Ahlrich’s triple-zeta basis
set Def2-TZVPP91 for all atoms at the grid6 integration grid
level and the CP(PPP) basis set for Fe at the grid7 integra-
tion grid level. 57Fe isomer shift calibration coefficients used
were those calculated previously.92 Inclusion of dispersion
effects (D3)93 for the calculations caused no significant de-
viation from observables of the non-corrected calculations
(Table S10) presented in the main text, except in the case of
the Lewis-acid adducts where addition of dispersion correc-
tion caused a larger tilt in the Lewis-acid relative to the Fe-
O axis. The difference does not significantly change the Fe-
O bond length, but changes the calculated quadrupole split-
ting for the 6-coordinate complex by −0.32 mm/s. For (ArO–
)F8Cmpd-II and (Im–)F8Cmpd-II, calculations were per-
formed with and without the presence of a sodium ion. Cal-
culations with a sodium ion present yielded results closer to
the experimentally observed parameters. See Supporting
information for further details.
Synthesis
The complexes F8FeII, F857FeII, PImFeII and d8-F8FeII were
synthesized as previously described.63,71,72,94 54FeIICl2 andthe
compounds (F8TPP)54FeIII-Cl and (F8TPP)54FeII were synthe-
sized using the same literature methods by Karlin and
coworkers to make the 57Fe derivative,71 with the only dif-
ference being that 54FeIICl2 was utilized in the metalation of
F8.
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Journal of the American Chemical Society
were synthesized as previously described.62–65,72 High-va-
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6
7
8
lent iron(IV) oxo complexes were generated by preparing a
3.0 mL 1:9 MeTHF:toluene solution (0.01 mM or 0.1 mM) of
F8FeII or PImFeII in a 10 mm pathlength quartz Schlenk cu-
vette, which was then sealed with a rubber septum in the
glovebox. The cuvette was then cooled in the cryostat cham-
(F8TPP)54FeIII-Cl: UV-vis (CH2Cl2): 411, 503, 639, 772. 1H-
NMR (CDCl3): δ 80.1 (s, br, 8 H), 13.8 (s, 4 H), 12.5 (s, 4 H),
7.6 (s, 4 H). Calculated for C44H20ClF8FeN4: C, 62.32; H, 2.38;
N, 6.61. Found: C, 62.44; H, 2.51; N 6.58.
(F8TPP)54FeII: UV-vis (1:9 MeTHF:toluene): 422, 542. 1H-
NMR (THF-d8): δ 56.1 (s, 8 H), 8.4 (s, 4H), 7.2 (s, 8H). Calcu-
lated for C44H20F8FeN4: C, 65.04; H, 2.48; N, 6.90. Found: C,
64.56; H, 2.83; N 6.87.
o
ber to –90 C, and the solution was then allowed to ther-
mally equilibrate (~10 minutes) before various reagents
were added. The reduced iron(II) precursors were sub-
jected to 1 equivalent of mCPBA dissolved in toluene at -90
oC, forming the F8Cmpd-II or PImCmpd-II species after five
minutes (denoted by a characteristic increase in the extinc-
tion coefficient of the Q-band). Subsequently, 2 equivalents
of 1,5-dicyclohexylimidazole (DCHIm) dissolved in toluene,
2 equivalents sodium 3,5-dimethoxyphenolate (ArO–), or 2
equivalents of sodium imidazolate (Im–) was added to
F8Cmpd-II, resulting in a red shift in the Soret and Q-band
yielding (DCHIm)F8Cmpd-II, (ArO–)F8Cmpd-II, or (Im–
)F8Cmpd-II, respectively. Due to solubility issues, sodium
3,5-dimethoxyphenolate and sodium imidazolate were
added in a 4:1 15-crown-ether: butyronitrile solution in-
stead of in toluene.
9
The compound PImFeIII_Cl was synthesized using the same
procedure Karlin and coworkers used to prepare the previ-
ously reported PImFeIIIOH.72 However, the only difference
was that the solution was stirred in HCl and then neutral-
ized to pH 7 with sodium bicarbonate, instead of NaOH at
the end of metalation procedure. Likewise, PIm57FeIII–Cl was
made using the same procedure, except starting with
57FeIICl2 instead of 56FeIICl2. The newly synthesized PIm57FeII
was formed using the same procedure72 Karlin and cowork-
ers used to prepare the previously reported PIm56FeII.
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(PIm
61.36; H, 3.30; N, 8.95. Found: C, 61.45; H, 3.28; N 9.18.
(PIm 57FeII •2H2O: Calculated for C55H35N7F6FeO3: C, 65.29;
)
57FeIIICl•CH2Cl2: Calculated for C56H33N7Cl3F6FeO: C,
1 equiv. of 2,6-lutidinium triflate, prepared as previously
described by Tilley and coworkers,95 was added in bu-
tyronitrile to F8Cmpd-II or (DCHIm)F8Cmpd-II, generating
F8Cmpd-II(LutH+) or (DCHIm)F8Cmpd-II(LutH+), respec-
tively. Various ferrocene derivatives (Me10Fc, Me8Fc, Me2Fc,
Fc, AcetylFc, DiacetylFc) or tris(4-bromophenyl)amine
were added to F8Cmpd-II, (DCHIm)F8Cmpd-II, F8Cmpd-
II(LutH+), and (DCHIm)F8Cmpd-II(LutH+), wherein the reac-
)
H, 3.49; N, 9.69. Found: C, 64.65; H, 3.54; N 9.45.
Sodium 3,5-dimethoxyphenolate: In the glovebox, a vial
of 3,5-dimethoxyphenol (0.510 g, 3.31 mmol) was dissolved
in 2 ml of THF. 1 equivalent of sodium hydride (0.079 g, 3.31
mmol) was added and the reaction was stirred for 20
minutes. Over the course of the reaction, bubbles were ob-
served indicating hydrogen being released and the vial got
slightly warm. Subsequently, the reaction was concentrated
2
tions were monitored by UV-vis, H-NMR, or Mossbauer
spectroscopies. The absorbance at 784 nm was monitored
for various concentrations of decamethylferrocenium BArF
(0.025 – 0.29 mM) and at 779 nm for octamethylferroce-
nium BArF (0.025- 0.20 mM), forming standard curves (Fig-
ures S30 & S31) to quantify the number of electrons trans-
ferred at the end of the reaction. See Supporting Infor-
mation for further details.
1
to give a white solid. H-NMR (DMSO): δ 5.3 (d, 2.12 Hz, 2
H), 5.1 (t, 2.16 Hz, 1 H), 3.5 (s, 6 H).
Decamethylferrocenium BArF: To a suspension of 350
mg (1.07 mmol) decamethylferrocene in 125 mL acetoni-
trile 173.1 mg (1.02 mmol) of solid AgNO3 and 731.8 mg
(1.02 mmol) of KBC24F20 was added, causing an immediate
color change. The solution was allowed to react for 30 min
at which time the acetonitrile solution was extracted with
(2 x 75 mL) of hexane. The acetonitrile solution was saved
and solvent removed. The solid was suspended in ~120 ml
tetrahydrofuran and filtered through a pad of celite. The tet-
rahydrofuran was removed yielding a green solid which
was crystalized from chilling a hot methanol:ethanol:ace-
tonitrile:water solution which after filtration and drying
yielded 825 mg (76% yield) of Me10FcBArF. Calculated for
C44H30BF20Fe: C, 52.57; H, 3.01. Found: 52.37; H, 3.49.
X-band EPR. Compound-II derivatives were generated
by adding 1 equivalent of mCPBA and, depending on the de-
rivative, 1 equivalent of axial ligand to a 1:9 MeTHF:toluene
solution containing 1 mM of F8FeII or PImFeII (total volume
= 0.6 mL) at –90 ºC (acetone/liquid nitrogen cold bath).
Upon addition of reagent(s), the solution was bubbled with
Ar for 20 seconds to ensure thorough mixing. In each case,
upon adding mCPBA the solution was allowed to react for 5
minutes (F8Cmpd-II and PImCmpd-II were frozen after 5
minutes), following the addition of 2 equiv. of axial ligand to
form (DCHIm)F8Cmpd-II, (ArO–)F8Cmpd-II, and (Im–
)F8Cmpd-II. 1 equiv. of 2,6-lutidinium triflate was added in
butyronitrile to F8Cmpd-II or (DCHIm)F8Cmpd-II, generat-
ing F8Cmpd-II(LutH+) and (DCHIm)F8Cmpd-II(LutH+). After
the complexes were generated, tubes were frozen in N2(liq)
,
Octamethylferrocenium BArF: Synthesized by the same
procedure as Me10FcBArF above, using octamethylferrocene
in place of decamethylferrocene. Calculated for
C42H26BF20Fe: C, 51.62; H, 2.68. Found: 51.67; H, 2.67.
and all spectra were recorded at 10 K. All complexes were
EPR silent (X-band) consistent with an integer spin elec-
tronic spin value and the typical EPR signature of an S=1
Compound-II species.
54/57Fe labeled rR. The oxoiron(IV) complexes were gen-
erated in the same fashion as the EPR spectroscopy sam-
ples, however samples were made at 2 mM and employed
the 57Fe and 54Fe derivatives with a total volume of 0.3 mL
Spectroscopic Sample Preparations
UV-vis. F8Cmpd-II, (DCHIm)F8Cmpd-II, and PImCmpd-II
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Journal of the American Chemical Society
Page 18 of 23
at –90 °C (acetone/liquid nitrogen bath) in a 7-inch, 5 mm,
ORCID
1
2
3
4
5
6
7
8
rubber septum-capped, NMR tube. Resonance Raman spec-
tra were obtained with a 407 nm laser excitation (4 mW) on
samples maintained at 110 K and with constant spinning as
described previously.94
Melanie A. Ehudin: 0000-0002-4324-4721
Leland B. Gee: 0000-0002-5817-3997
Sinan Sabuncu: 0000-0001-8346-8175
Pierre Moënne-Loccoz: 0000-0002-7684-7617
Augustin Braun: 0000-0002-9487-5769
Edward I. Solomon: 0000-0003-0291-3199
Kenneth D. Karlin: 0000-0002-5675-7040
2H-NMR. The oxoiron(IV) complexes were generated in
the same fashion as the EPR spectroscopy samples, how-
ever they were prepared using the pyrrole-deuterated ana-
log, d8-F8FeII, at 5.0 mM concentrations with a total volume
of 0.6 mL at -90 oC (acetone/liq. N2) in a 9-inch, 5mm, rubber
septum-capped, NMR tube. Upon addition of reagent(s), the
solution was bubbled with Ar for 20 seconds to ensure thor-
Author Contributions
¶These authors contributed equally to this work.
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Funding Sources
2
ough mixing. H NMR spectra were acquired on a Bruker
AVANCE 300 MHz NMR spectrometer at 46.072 MHz. Ex-
periments were carried out at –90°C using a recycle delay of
0.01s and a total of 5120 scans were collected. The peaks
were referenced to the one of toluene solvent peaks at 7.1
ppm (the -CH group of toluene, which is the most upfield).
No competing financial interests have been declared.
ACKNOWLEDGMENT
The research support of the U.S. National Institutes of Health
(GM60353 to K.D.K.) is gratefully acknowledged. This research
was supported by the National Institute of General Medical Sci-
ences under award numbers R01GM040392 (E.I.S.),
R01GM074785 (P.M.-L.), and F32GM122194 (L.B.G.). The SSRL
Structural Molecular Biology Program is supported by the DOE
Office of Biological and Environmental Research and by Na-
tional Institutes of Health, National Institute of General Medical
Sciences Grant P41GM103393 (to K.O.H. and B.H.). Some com-
putational work was performed under the XSIM project on the
CORI computing system at NERSC, a U.S. Department of Energy
Office of Science User Facility operated under Contract No. DE-
AC02-05CH11231. We also gratefully acknowledge Dr. David P.
Goldberg for the use of his Mössbauer instrument and Jesse B.
Gordon for assistance in running the samples. M.A.E. would
also like to especially thank Dr. David A. Quist for helpful dis-
cussions and for aiding in sample preparation. We
acknowledge the help of Drs. Matthew Latimer and Erik Nelson
for assistance at SSRL beam lines 9-3 and 7-3. L.B.G. also
acknowledges Dr. Thomas Kroll for assistance in pre-edge fit-
ting.
57Fe Mössbauer. Samples were prepared similarly to
how they were generated for UV-vis spectroscopy. How-
ever, the reduced F857FeII or PIm57FeII complexes were em-
ployed. The iron starting material was prepared as a 2.0 mL
1:9 MeTHF:toluene solution at 2 mM under Ar in a 10 ml
Schlenk flask in the glovebox, which was then subsequently
cooled to -90 oC for ten minutes. Reagents were then added
accordingly to form desired complexes as described in the
UV-vis section. See Supporting Information for further de-
tails. Samples were prepared by cooling a pipette in liquid
nitrogen and then quickly filling a pre-chilled Delrin Möss-
bauer cup. All Mössbauer spectra were taken at 80 K in the
absence of a magnetic field using liquid nitrogen as a cryo-
gen.
XAS. Samples were prepared in a similar fashion to the
Mössbauer samples as above. All samples except the DCHIm
ligated sample were prepared in 10% MeTHF and 90% tol-
uene with a concentration at 2.0 mM employing the F857FeII
analog. Mössbauer spectroscopy was run on all the XAS
samples to confirm the purity of that particular batch and
the reported Mössbauer purities reflect the purity of the
corresponding XAS spectra. The DCHIm samples were the
only exception; the samples were prepared in 100% toluene
and were 90% pure by Mössbauer with the impurity being
the ferrous precursor. Samples were prepared by cooling a
pipette in liquid nitrogen and then quickly filling pre-chilled
Delrin cell, and immediately frozen in liquid nitrogen. Every
compound was generated twice and measured for XAS.
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