Heme-FeIII Superoxide, Peroxide and Hydroperoxide Thermodynamic Relationships: FeIII-O2 a￠- Complex H-Atom Abstraction Reactivity

Article abstract of DOI:10.1021/jacs.9b12571

Establishing redox and thermodynamic relationships between metal-ion-bound O₂ and its reduced (and protonated) derivatives is critically important for a full understanding of (bio)chemical processes involving dioxygen processing. Here, a ferric

Full text of DOI:10.1021/jacs.9b12571

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Article
Heme-FeIII Superoxide, Peroxide and Hydroperoxide Thermodynamic
Relationships: FeIII-O2•– Complex H-Atom Abstraction Reactivity
Hyun Kim, Patrick J. Rogler, Savita K Sharma, Andrew
W. Schaefer, Edward I. Solomon, and Kenneth D. Karlin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b12571 • Publication Date (Web): 08 Jan 2020
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Heme-FeIII Superoxide, Peroxide and Hydroperoxide Thermo-
•–
dynamic Relationships: FeIII-O2 Complex H-Atom Abstraction
Reactivity
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Hyun Kim,† Patrick J. Rogler,† Savita K. Sharma,† Andrew W. Schaefer,‡ 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
ABSTRACT: Establishing redox and thermodynamic relationships between metal ion bound O2 and its reduced (and proto-
nated) derivatives is critically important for a full understanding of (bio)chemical processes involving dioxygen processing.
Here, a ferric heme peroxide complex, [(F8)FeIII-(O22–)]– (P) (F8 = tetrakis(2,6-difluorophenyl)porphyrinate), and superoxide
complex, [(F8)FeIII-(O2•–)] (S), are shown to be redox interconvertible. Using Cr(η-C6H6)2, an equilibrium state where S and P
are present was established in tetrahydrofuran (THF) at –80 ℃, allowing reduction potential determination of S as –1.17 V
vs. Fc+/0. P could be protonated with 2,6-lutidinium triflate, yielding the low-spin ferric hydroperoxide species, [(F8)FeIII-
(OOH)] (HP). Partial conversion of HP back to P using a derivatized phosphazene base gave a P/HP equilibrium mixture;
leading to the determination, pKa = 28.8 for HP (THF, –80 ℃). With the measured reduction potential and pKa, the O–H bond
dissociation free energy (BDFE) of hydroperoxide species HP was calculated to be 73.5 kcal/mol, employing the thermody-
namic square scheme and Bordwell relationship. This calculated O–H BDFE of HP in fact lines up with an experimental demon-
stration of the oxidizing ability of S, via hydrogen atom transfer (HAT) from TEMPO–H (2,2,6,6-tetramethylpiperdine-N-hy-
droxide, BDFE = 66.5 kcal/mol in THF), forming the hydroperoxide species HP and TEMPO radical. Kinetic studies carried out
with TEMPO–H(D) reveal second-order behavior, kH = 0.5, kD = 0.08 M–1s–1 (THF, –80 ℃), thus the hydrogen/deuterium kinetic
isotope effect (KIE) = 6, consistent with H-atom abstraction by S to be the rate-determining step. This appears to be the first
case where experimentally derived thermodynamics lead to a ferric heme hydroperoxide OO–H BDFE determination, that
FeIII-OOH species being formed via HAT reactivity of the partner ferric heme superoxide complex.
INTRODUCTION
porphyrinate. Note that in the resting state, the FeIII/FeII re-
duction potential ranges between –400 and –170 mV, and
thus is inactive to reduction.2 The ferrous heme binds diox-
ygen, giving rise to the initial ferric superoxide intermedi-
ate, often referred to as the oxy-heme or oxy ferrous inter-
mediate. The addition of a second electron converts super-
oxide to a ferric peroxide intermediate, and this step is over-
all rate-determining in the CYP450 catalytic cycle.17,22 Sub-
sequently, the peroxide is protonated to form a low-spin
ferric hydroperoxide intermediate, FeIII–OOH, which is
known as Compound 0. A second protonation on the distal
oxygen of hydroperoxide species causes heterolytic O–O
bond cleavage to produce a highly reactive high-valent fer-
ryl–oxo π-cation porphyrinate radical (Cmpd I), releasing a
molecule of water. Cmpd I is accepted as the active sub-
strate oxidant for the normal CYP450 cycle.9,23 This species
abstracts a hydrogen atom from the substrate to give a rad-
ical (R•) and the “OH” from the resulting iron(IV)-OH (Cmpd
II) undergoes ‘rebound’24 to the substrate radical, giving
products R–OH plus the original (in the cycle) ferric heme.
Cytochrome P450 monooxygenases (CYP450s) catalyze the
incorporation of one atom of molecular oxygen into biolog-
ical substrates, i.e., they hydroxylate nonactivated C–H
bonds in hydrocarbon and various other compounds.1–10
CYP450s are involved in critical biological processes, in-
cluding the biosynthesis of steroid hormones, degradation
of pharmaceutically derived drugs and detoxification of xe-
nobiotics including carcinogens.11–14 Many excellent review
articles concerning CYP450s exist, covering the relevant bi-
ochemistry, summarizing available protein X-ray struc-
tures15,16 and detailing aspects of the nature of intermedi-
ates and reaction mechanism. Theoretical/computational
investigations and analyses have been part of the research
efforts.1,3,5,10,17–21
The CYP450 catalytic mechanism as currently accepted is
depicted in Scheme 1. When the substrate (R–H) ap-
proaches the heme active-site, it displaces the distally
bound axial water molecule from the six-coordinate low-
spin ferric ion, converting it to a five-coordinate, high-spin
ferric-porphyrinate. The loss of the H2O causes an increase
of the redox potential of the heme iron by approximately
300 mV,4 which enables electrons transfer from the biolog-
Scheme 1. Catalytic Cycle of Cytochrome P450 Monoox-
ygenases (where R–H is the substrate).8
ical reductase producing
a
five-coordinate ferrous
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hydroperoxide complexes are relatively rare (and see dis-
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cussions below) even though researchers have suggested
that hydroperoxide species may serve as alternative oxi-
dant, since it is a common intermediate in the catalytic cy-
cles of monooxygenases.19,53–55
Reviews emphasizing synthesis and reactivity of Mn+-
•–
(O2•–) complexes are now available.56,57 Many Mn+-(O2
)
complexes of the first row transition metals, for example
Mn, Fe, Co, Ni, Cu and Zn have been synthesized via metal-
complex + O2 or metal-complex + superoxide reactions and
their substrate reactivity investigated. Iron-superoxide spe-
cies are considered to form in the first step of dioxygen ac-
tivation as the reactive intermediates in heme (vide supra)
and non-heme metalloenzymes (e.g., Myo-inositol oxygen-
ase (MIOX) and isopenicillin-N-synthase (IPNS)).57
In this report, we describe new synthetic analogues of
those intermediates early in the cycle, prior to O–O cleav-
age, namely the ferric heme superoxide, peroxide and hy-
droperoxide species (S, P and HP, respectively (Figure 1)).
We note that in CYP450, the superoxide, peroxide and hy-
droperoxide intermediates are thought to have an end-on
structure,9,10,58 only binding to iron with one oxygen atom
(the proximal O-atom), as depicted in Scheme 1. Utilizing
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As shown in Scheme 1, superoxide/peroxide/hydroper-
oxide intermediates are involved in catalytic cycle of
CYP450. These species also have been observed as interme-
diates in biological processes involving other O2 or H2O2 ac-
tivating heme enzymes.6 These include heme oxygenase
(HO; oxidatively breaking down hemes while also effecting
release of iron and CO (or other metabolite): superoxide
(FeII-O2) and hydroperoxide),25–27 nitric oxide synthase
(NOS; two-step reaction where hydroxylation of L-arginine
to N-hydroxy-L-arginine (L-NHA) is followed by oxidation of
L-NHA to citrulline and NO(g): superoxide, peroxide, and hy-
droperoxide),28,29 steroid hormone multifunctional CYPs
(hydroxylation and lyase activities: superoxide, peroxide,
and hydroperoxide),30–32 horseradish peroxidase (HRP;
H2O2 mediated substrate oxidation: hydroperoxide),6,9,33,34
chloroperoxidase (CPO; H2O2 mediated substrate halogena-
tion: hydroperoxide),9,35 aromatic peroxygenase (APO; oxy-
genation of aliphatic and aromatic hydrocarbons: hydrop-
eroxide),9,36 and tryptophan 2,3-dioxygenase (TDO; trans-
formation to formylkynurenine: superoxide).6,37 Any and all
aspects of CYP or other O2/H2O2 activating heme enzymes
(vide supra) reaction mechanisms have been or are being
investigated, including what key protonation events take
place and how, and what the structures of all intermediates
are such as atom-connectivity along with bonding (elec-
tronic structure). The initial oxy-heme, the O2-adduct with
ferrous ion (i.e., the ferric superoxide),38 the subsequently
reduced intermediate peroxide and protonated hydroper-
oxide (Cmpd 0) have not been detected in normal enzyme
turnover. However, comprehensive radiolytic cryoreduc-
tion studies25,39–42 have provided many details concerning
their structures and physical properties. Theoretical/com-
putational investigations have also provided insights into
the structures and thermodynamic inter-relationships for
intermediates in the CYP450 catalytic cycle (Scheme 1).5
However, over the years and continuing on, generation of
synthetic analogs of all the intermediates relevant to the
CYP450 catalytic cycle has been of great interest (and im-
portance). In this regard, there are many known heme FeIII-
superoxide synthetic compounds,8,43–52 O2-adducts of re-
duced ferrous hemes, including hemoglobin/myoglobin
mimics such as the “Picket-Fence” porphyrin O2-adduct.43
However, these do not possess thiolate axial ligation as in
CYP450s. Ferric heme peroxide and ferric heme
the F8 (F8
= tetrakis(2,6-difluorophenyl)porphyrinate)
heme framework, we have spectroscopically characterized
corresponding S, P and HP complexes, with UV-vis, electron
paramagnetic resonance (EPR) and resonance Raman (rR)
spectroscopies, and further chemically confirmed that P
and HP are peroxidic (vide infra).
More importantly, and the primary advance described in
this report, is that we have experimentally established the
thermodynamic relationships between these complexes:
(1) The reduction of ferric superoxide compound S is re-
versible, using chemical reagents, and a reduction potential
E°’ has been determined, (2) the ferric peroxo complex P,
possessing an intermediate spin and side-on bound perox-
ide dianion ligand, can be reversibly protonated to give the
low-spin ferric hydroperoxide HP. Thus, a pKa value for HP
has been established. Further, another finding is that the
ferric heme superoxide complex S can effect a hydrogen
atom abstraction reaction, and directly produce ferric heme
hydroperoxide complex HP (Figure 1). Only recently, Dey
and co-workers44 demonstrated for the first time that a fer-
ric heme superoxide complex could effect a HAT reaction;
removal of a hydrogen atom from a porphyrinate appended
catechol, thus an intra-molecular process, converted the su-
peroxide complex to the hydroperoxide analog. Such a
transformation represents the diagonal part of a thermody-
namic square scheme, relating S, P and HP complexes (Fig-
ure 1). Now, with our measured reduction potential (E°’)
and pKa values (as mentioned above), the O–H bond disso-
ciation free energy (BDFE) of ferric heme hydroperoxide
complex HP could be determined using the Bordwell rela-
tionship (eq. 1) where CG,solv represents the H+/H• standard
reduction potential in a particular solvent (also see be-
low).59
BDFE = 1.37(pKa) + 23.06Eo + CG,solv
(1)
Further, the calculated BDFE of complex HP, in fact is shown
to be in-line (consistent) with the actual experimental reac-
tivity of S toward a substrate with O–H bond (vide infra). To
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the best of our knowledge, the present study reports the
first example showing thermodynamics (reduction poten-
tial and pKa) involved and the generation of the ferric heme
hydroperoxide species by ferric heme superoxide complex
via HAT of an exogenously added substrate.
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Previously, our complex P was characterized by UV-vis,
EPR (g = 4.2 (S = 3/2); see Figure S1) and 1H NMR spectros-
copies.60 Further characterization of P was carried out and
2H NMR and rR spectroscopies are presented here. The pyr-
rolic proton in 2H NMR spectroscopy at –80 °C resonates at
δ 92.7 ppm (Figure S2), and this feature is consistent with
the previously reported 1H NMR spectroscopic signature
(90 ppm).60 Resonance Raman experiments confirmed per-
oxide formulation for P, with the observation of νO−O vibra-
tion (806 cm–1, ∆18O2 = –46 cm–1) and νFe−O stretch (466 cm–
1, ∆18O2 = –19 cm–1) as shown in Figure 2B. These observed
stretching frequency values match those calculated using
the harmonic oscillator model: ΔO−O,calc (16O2/18O2) = –47 cm–
1, ΔFe−O,calc (16O2/18O2) = –21 cm–1. The relatively low Fe–O
stretching frequency, which results from the symmetric
stretching vibration of a side-on bound peroxide ligand, is
similar to that in two other similarly structured heme
peroxo compounds (vide infra, Table S1)48 as well as non-
heme68–70 side-on peroxide complexes. It is notable that
νFe−O in these compounds is considerably reduced compared
to what is observed for oxy-heme (i.e., FeII-(O2)) species like
oxy-myoglobin (νFe−O = 570 cm–1),71 which we are referring
to as ferric superoxide complexes. When strong acid, triflic
acid (HOTf), is utilized to protonate peroxide species P, H2O2
is released as expected and a yield of 95.5% is obtained (see
Experimental Section and Figure S3). If no hydrogen perox-
ide had been released with the acidification, it would have
likely meant that reductive O–O cleavage (to give water)
had somehow occurred.72,73
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Figure 1. Sequential preparation of superoxide/perox-
ide/hydroperoxide heme analogs and thermodynamic
square scheme of reduction (E° as determined vs Fc+/0) and
protonation of a F8 ferric heme superoxide analog and O–H
BDFE of ferric heme hydroperoxide. (ArF = 2,6-difluoro-
phenyl group)
RESULTS AND DISCUSSION
Generation and Characterization of a Side-on Heme
Ferric Peroxide, [(F8)FeIII-(O22–)]– (P). As reported in pre-
vious work,60 addition of the outer sphere strong reductant
CoCp2 to the previously well-characterized ferric superox-
ide complex [(F8)FeIII-(O2•–)] (S) (λmax = 416 and 535 nm;
rRaman, νO−O, 1178 cm–1 (Δ18O2, –64 cm–1); νFe−O, 568 cm–1
(Δ18O2, –24 cm–1))61,62 at –80 ℃ in THF yields side-on ferric
heme-peroxide complex [(F8)FeIII-(O22–)]– (P) with UV-vis
spectral features at 434, 541 and 561 nm (Figure 2A). These
electronic absorption spectra are the same as those known
for other previously well-characterized side-on ferric por-
phyrin peroxide complexes which have low energy Soret
band and two peaks between 500 and 600 nm at α, β-re-
gion.11,48,63,64 Valentine and co-workers63 originally discov-
ered and characterized such species and synthesized them
by the reaction of potassium superoxide solubilized with
18-crown-6 ether, where the first superoxide anion reduced
a porphyrinate-FeIIICl (porphyrinate-H2 = TPP, OEP)65 to the
FeII form which then reacted with another equiv of superox-
ide anion to give the [(porphyrinate)FeIII(O22–)]– species
with side-on bound peroxide ligand (see diagram below).
These species do not possess an additional axial ligand, as
deduced previously,11,48,63,64 and supported by the X-ray
structure of a manganese analog [(TPP)Mn(η2-O22–)]–; here
the MnIII ion lies well above the porphyrinate plane toward
the peroxo ligand.66,67
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(HOTf) and applied the a quantitative horseradish peroxi-
dase assay (see experimental section and Figure S3).72,73
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Figure 3. UV-vis spectra of illustrating the conversion of
[(F8)FeIII-(O22–)]– (P) (blue) to form [(F8)FeIII-(OOH)] (HP)
(pink) by addition of [(LutH+)](OTf) in THF at –80 ℃.
The characterization of [(F8)FeIII-(OOH)] (HP) was fur-
ther corroborated by rR spectroscopy. The rR spectra of HP
are indicative of a clear change in the νFe−O vibrational
stretch mode on protonation, shifting from 466 cm-1 (∆18O2
= –19 cm–1) in side-on ferric peroxide species P to 576 cm-1
(∆18O2 = –25 cm–1) in hydroperoxide complex HP, whereas
the νO−O vibrational frequency remains the same, appearing
at 806 cm–1 (∆18O2 = –46 cm–1) as shown in Figure 4B. These
νFe−O and νO−O vibrational stretching frequency downshifts
when using 18O2 match well with values calculated using the
harmonic oscillator model: ΔFe−O,calc (16O2/18O2) = –26 cm–1,
ΔO−O,calc (16O2/18O2) = –47 cm–1. Also, these observed νFe−O and
νO−O vibrational values are quite similar to those obtained in
other low-spin end-on ferric heme hydroperoxide model
complexes (Table S1), those from the groups of Naruta48,49,51
and Dey80 and their co-workers. This also well matches data
obtained from cryogenically ferric hydroperoxide species
generated in CYP450’s,81,82 even though the latter have axial
cysteinate ligands which makes the coordination environ-
ment very different as compared to those for the synthetic
analogues (Table S1). The combined spectroscopic data
(UV-vis, EPR and rRaman) support the formulation of HP as
a low-spin end-on (η1) ferric hydroperoxide species. We
postulate that HP most likely possesses a solvent THF mol-
ecule (Figure 3), as we know this can confer low-spin char-
acter to six-coordinate ferric hemes with F8;61,62,83 there ex-
ists the possibility the axial ligand is 2,6-lutidine or DMF, in
exchange with THF.
Figure 2. (A) UV-vis spectra of [(F8)FeIII-(O2•–)] (S) (red) to
[(F8)FeIII-(O22–)]– (P) (blue) in THF at –80 ℃. (B) Resonance
Raman spectra of ferric peroxide complex P in frozen THF
obtained at 77 K with 413 nm excitation: Fe–O and O–O
stretching frequencies for the complex generated with 16O2
(black) or 18O2 (red). The 16O2–18O2 difference spectrum is
shown in blue. The bands at 900 and 1000 cm–1 are vibra-
tions within the porphyrin ring, which are generally ob-
served for hemes and minimally impacted by O2 isotope
substitution (i.e. the modes contain minimal O2 motion). For
full spectrum of P, see Figure S4.
Generation and Characterization of an End-on Hy-
droperoxide Species, [(F8)FeIII-(OOH)] (HP). The addi-
tion of one equiv 2,6-lutidinium triflate [(LutH+)](OTf) or
[(H)DMF](OTf)74 to the solution of [(F8)FeIII-(O22–)]– (P) at –
80 ℃ in THF leads to protonation of this side-on ferric heme
peroxide complex to form a corresponding ferric heme hy-
droperoxide species, [(F8)FeIII-(OOH)] (HP). UV-vis spectra
of HP exhibit absorptions at 418, 536 and 558 nm (Figure
3), while an EPR spectrum indicates distinctive changes
from the side-on peroxide g = 4.2 signal observed in P, to a
low-spin ferric hydroperoxide species characterized by g =
2.23, 2.14 and 1.96 signals (Figure 4A), very similar to pub-
lished values for other ferric end-on hydroperoxide syn-
thetically derived complexes,44,48,49,51,75–77 as well as for fer-
ric heme hydroperoxides generated within hemoglobin and
myoglobin.78,79 Note that the full EPR spectrum (Figure S5)
reveals that there is a small feature corresponding to lefto-
ver side-on ferric peroxide starting complex (at g = 4.2).
Consistent with our complex formulation as [(F8)FeIII-
(OOH)] (HP), a high yield (93.3%) of H2O2 was detected
when we acidified the complex solution with triflic acid
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protocols occurs with our case (vide supra); while Naruta
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and co-workers postulated that methanol was the proton
source converting peroxide to hydroperoxide complex, a
stronger acid ([(LutH+)](OTf) or [(H)DMF](OTf)) was re-
quired in our case, but a strong axial base ligand was not
necessary.
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Figure 5. Naruta group’s synthetic complexes; (A)
[(TMPIm)FeIII–(O22–)], (B) [(TMPIm)FeIII–(OOH)].48
Reduction Potential of the [(F8)FeIII-(O2•–)] (S)/
[(F8)FeIII-(O22–)]– (P) Redox Couple. When we set out this
study, one of the main goals was to see if we could deter-
mine a reduction potential for a superoxide [(F8)FeIII-(O2•–)]
(S) conversion to side-on peroxide complex [(F8)FeIII-(O2
2–
)]– (P). In order to do this accurately it needed to be shown
that reduction of S to P is a reversible reaction. In fact, we
find this to be the case; [(F8)FeIII-(O2•–)] (S) and peroxide
[(F8)FeIII-(O22–)]– (P) are interconvertible using redox rea-
gents. Addition of the strong oxidant, [(4-BrC6H4)3N](SbCl6)
(Tris(4-bromophenyl)ammoniumyl cation, known as Magic
Blue because of its intense royal blue color; E1/2 = 0.67 V vs.
Fc+/0 in MeCN)85 to the solution of P results in complete ox-
idative conversion of the peroxide to superoxide species
(Figure 6).
Figure 4. (A) Frozen THF solution EPR (10K) spectrum of
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HP. For full spectrum of HP, see Figure S5. (B) Resonance
Raman spectra of ferric hydroperoxide complex HP in fro-
zen THF obtained at 77 K with 413 nm excitation: Fe–O and
O–O stretching frequencies for the complex generated with
16O2 (black) or 18O2 (red). The 16O2–18O2 difference spectrum
is shown in pink. Note that complex P is present as an impu-
rity, observed as a set of 16O2/18O2 peaks at 466/447 cm–1
(marked with an asterisk). For full spectrum of HP, see Fig-
ure S4.
Comparison of This Work with Previously Reported
Hydroperoxide Ferric Hemes. It has been generally diffi-
cult to cleanly synthesize ferric heme hydroperoxide com-
pounds. Tajima and co-workers75,77,84 reported low-spin fer-
ric hydroperoxide complexes which are in accord with a
FeIII-heme(-OH)(-OOH) formulation, based on UV-vis and
EPR spectroscopies. Significant advances were made by
Naruta and co-workers48 who extensively characterized an
end-on ferric hydroperoxide complex, that shown in Figure
5B.14,20 It was generated by protonation of the seven-coor-
dinated side-on peroxide species [(TMPIm)FeIII-(O22–)] (Fig-
ure 5A) with ligated axial imidazole ligand from the cova-
lently attached tether, wherein a spin state change from S =
3/2 (νFe−O = 470 cm–1 (∆18O2 = –19 cm–1); νO−O = 809 cm–1
(∆18O2 = –45 cm–1)) to low-spin (νFe−O = 570 cm–1 (∆18O2 = –
26 cm–1); νO−O = 810 cm–1 (∆18O2 = –47 cm–1)) occurred (Ta-
ble S1).48 Therein, a covalently appended axial imidazole lig-
and was critical to give a stable 6-coordinate ferric heme hy-
droperoxide complex. In the absence of the imidazolyl axial
ligand (Figure 5), only decomposition occurred upon proto-
nation. In other case, Anxolabéhère-Mallart and co-work-
ers76 generated a ferric heme hydroperoxide complex in the
presence of 1-methylimidazole as sixth ligand by protona-
tion of its side-on ferric peroxide precursor which is formed
from the electrochemical reduction of an in situ formed fer-
ric superoxide species. A significant difference in synthetic
Figure 6. UV-vis spectra demonstrating the oxidation of
[(F8)FeIII-(O22–)]– (P) (blue) to form [(F8)FeIII-(O2•–)] (S)
(red) in THF at –80 ℃. A spectrum (gray lines) was recorded
every ~2 min; the reaction goes to completion in ~ 20 min.
Also, see the text.
Observations supporting this conclusion are that the
product solution containing [(F8)FeIII-(O2•–)] (S) is EPR si-
lent (10 K, THF) as expected; the low-spin FeIII ion is antifer-
romagnetically coupled with the unpaired electron of the
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directly coordinated superoxide radical anion. Interest- reported on the electrochemical generation of the side-on
peroxide species [FeIII(F20TPP)(O2)]− (EPR; g = 4.2) by ap-
plying a cathodic potential (Eapp = –0.60 V vs. SCE; –0.36 V
vs. SHE) to the corresponding superoxide precursor in di-
methylformamide at –30 °C. The electrochemical reduction
was found to be irreversible, nevertheless the finding of fer-
ric heme superoxide complex reduction at E°’ = –0.60 V vs.
SCE (–0.36 V vs. SHE) is most interesting since it lies very
close to our own result (vide supra). For Anxolabéhère-Mal-
lart, Fave and co-workers, the superoxide and peroxide
complexes employed the highly electron-withdrawing
heme (F20TPP) with 20 aryl fluorine substituents; no axial
base ligand was utilized. Roughly, the comparison of reduc-
tion potential of –0.36 V for the F20TPP containing ferric
heme superoxide, as well as the –0.39 V value for reversible
reduction of [(F8)FeIII-(O2•–)] with the far more negative po-
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ingly, the addition of the weaker reductant, Cr(η-C6H6)2 (E1/2
= –1.15 V vs. Fc+/0 in CH2Cl2),85 to the solution of S leads to
the formation of an equilibrium mixture of superoxide S and
peroxide P, allowing the determination of the reduction po-
tential for the superoxide (S)/peroxide (P) couple. A titra-
tion of superoxide S with varying amounts of Cr(η-C6H6)2 in
THF at –80 ℃ was monitored by UV-vis spectroscopy (Fig-
ure 7), allowing the determination of an equilibrium con-
stant value in each instance of added bis-benzene chro-
mium(0) titrant (Table S2); the UV-vis absorptions for S and
P gave direct determination of their concentrations, thus
defining the amount/concentration of Cr(η-C6H6)2 reacted
and of [Cr(η-C6H6)2]+ formed. From the collection of calcu-
lated equilibrium constants, corresponding reduction po-
tentials were determined by using the Nernst equation.
Thus, the reduction potential (E°’) for [(F8)FeIII-(O2•–)]
(S)/[(F8)FeIII-(O22–)]– (P) couple was calculated to be –1.17
± 0.01 V vs. Fc+/0 (for which E°’ = –0.39 V vs. SHE in THF).
See Table S2 for further details. A further demonstration of
the reversibility of the reduction and oxidation of complex
S/P was established; Peroxide species P generated by Cr(η-
C6H6)2 could be fully oxidized back to superoxide S using
Magic Blue and again, this resulting solution could be re-
duced to peroxide P with 5 equiv Cr(η-C6H6)2, to the extent
of ~85% (Figure S6).
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tentials (vide supra) observed for the various [(P)FeIII-(O2
)] complexes from Naruta and co-workers, possessing a
more electron-rich porphyrinate with axial imidazole
(TMPImOH,OEt; Figure S7), may make sense. (See Table S3 for
comparison of reduction potential values.)86 Interestingly,
computational chemists have estimated the reduction po-
tential for the ferric heme superoxide species in CYP450
(Scheme 1); depending on “chosen model, QM region, and
protonation state”, the values obtained range from –2.28 to
–1.04 V vs. SHE.5 Perhaps this is most reasonable, as the ax-
ial cysteinate ligand in CYP450’s is a strong anionic donor
known for its ability to “push”.9,23
Determination of the pKa Value of [(F8)FeIII-(OOH)]
(HP). As mentioned in the Introduction, for the thermody-
namic square scheme, the pKa value of hydroperoxide
[(F8)FeIII-(OOH)] (HP) has been determined in THF at –80
℃. It was evaluated by spectral titration using the derivat-
ized phosphazene base EtP2(dma) (pKa = 28.1 in THF, for
the conjugate acid at room temperature, see diagram be-
low).87
As shown by a titration followed by UV-vis spectroscopy
(Figure 8), the absorption band at 418 nm of HP decreased
with increasing concentration of added EtP2(dma), and the
absorption band shifted to 434 nm, with observation of an
isosbestic point at 426 nm. Such additions gave rise to an
equilibrium mixture of hydroperoxide HP and the side-on
peroxide complex [(F8)FeIII-(O22–)]– (P) (see Table S4). From
this data, an equilibrium constant for this reaction was cal-
culated in each instance and using the known pKa value of
EtP2(dma), a pKa value for deprotonation of HP was deter-
mined to be 28.8 ± 0.5 (THF, –80 °C). Following addition of
excess base EtP2(dma), only the deprotonated complex P re-
mained. As suggested by the data, the acid-base reaction in-
terconversion of HP and P is reversible. This was further
shown by adding [(LutH+)](OTf) to the solution generated
in this titration (Figure 8), this gives the fully regenerated
hydroperoxide complex HP (see Figure S8 for details). In or-
der to corroborate the [(F8)FeIII-(OOH)] (HP) pKa value
Figure 7. Conversion of [(F8)FeIII-(O2•–)] (S) (red) to
[(F8)FeIII-(O22–)]– (P) (blue) upon addition of Cr(η-C6H6)2 in
THF at –80 ℃, resulting in the generation of equilibrium
mixtures which allowed the determination of the reduction
potential (–1.17 V vs. Fc+/0) of the S/P redox couple. Inset:
monitoring the absorbance at 434 nm upon addition of var-
ious amounts of Cr(η-C6H6)2. See Table S2 for details.
Recently, Naruta and co-workers51 reported the reduc-
tion potential (–0.67 V and –1.1 V vs. SHE) of various ferric
heme superoxide synthetic compounds, not experimentally
but by employing density functional theory (DFT) calcula-
tions. Thus, these values are much more negative than what
2–
was found by us for the [(F8)FeIII-(O2•–)] (S)/[(F8)FeIII-(O2
)]– (P) redox couple, E°’ = –0.39 V vs. SHE in THF at –80 °C.
Anxolabéhère-Mallart, Fave and co-workers76 recently
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determined experimentally, a complementary experiment As mentioned, this is to our knowledge the first case
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was conducted employing the conjugate acid of EtP2(dma),
i.e., as expected, based on relative pKa values, the addition of
EtP2(dma)H+ (pKa = 28.1) to peroxide [(F8)FeIII-(O22–)]– (P)
results in the ready protonation and formation of HP (see
Figure S9).
where a BDFE value has been reported using experimen-
tally derived thermodynamics (reduction potential and pKa)
for a heme system, those involving O2 or its reduced deriva-
tives.90 Morokuma and co-workers91 computationally eval-
uated aspects of other heme-superoxide (as well as other
metal-superoxide) species, and their findings are also in ac-
cord with the present results, i.e., that for [(F8)FeIII-(OOH)],
BDFE = 73.5 kcal/mol. For heme ferric superoxide com-
plexes modeling protein active sites (e.g., hemoglobin,
CYP450) where a proximal ligand is either an imidazole or
an -SH group (the latter as a computational model for the
cysteinate proximal ligand in CYP450’s), i.e., (B)(porphy-
rinate)FeIII-O2•– (B = imidazole, SH) the ferric heme hydrop-
eroxide complex OO–H BDE’s are calculated to be in the
range 64–66 kcal/mol. Lai and Shaik18 also computationally
found that a CYP450 ferric heme superoxide is a “sluggish
oxidant”.
As discussed above, Naruta and co-workers51 syntheti-
cally generated several ferric heme hydroperoxide com-
plexes. They did address the question of pKa values, but only
computationally. For two ferric hydroperoxide complexes
they described, possessing modified superstructured syn-
thetic hemes (see Figure S7), their DFT determined pKa val-
ues were 25.1 or 32.3 in EtCN as a solvent. In reality, these
values are not so different from that of our own experimen-
tally determined pKa value (= 28.8, vide supra) for [(F8)FeIII-
(OOH)] (HP).
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Corresponding OO–H BDFE (or BDE) values for a few
product non-heme iron-hydroperoxide complexes are
known, but these tetramethylcyclam (TMC) ligand derived
complex results come only through computational evalua-
tion.91,92 These have recently been compared to experi-
mental results on cobalt and other heavier metal-superox-
ide complexes.93 Dicopper(II)-μ-hydroperoxide complex
OO–H BDFE’s have recently been determined.89,94 In one
case, the BDFE is 72 kcal/mol, while in another example, the
binucleating ligand utilized results in a hydroperoxide OO–
H BDFE of 81 kcal/mol. Thus, the corresponding dicop-
per(II)--superoxide complex is a quite strong oxidant for
HAT reactivity. Experimentation and compilation of O–H
bond thermodynamic parameters (i.e., BDE’s or BDFE’s) of
dioxygen-derived species (M-OH, M-OOH or M-oxo species
formed from O2-reduction/protonation) bound to metal
complexes, is of considerable general significance and im-
portance.95
Figure 8. UV-vis spectroscopic monitoring of the incremen-
tal addition of EtP2(dma) to a solution of [(F8)FeIII-(OOH)]
(HP) (pink) resulting in the formation of equilibrium mix-
tures of HP, EtP2(dma), [(F8)FeIII-(O22–)]– (P) (blue) and pro-
tonated base EtP2(dma)H+ which allowed the determina-
tion of the pKa value (28.8) of HP. Inset: monitoring of the
absorbance at 434 nm (blue).
Reactivity Studies of [(F8)FeIII-(O2•–)] (S) with O–H, C–
H and N–H substrates through HAT to Support for Our
Calculated BDFE. One of the utilities of BDFE data is to de-
termine or estimate the oxidative reactivity in this case of
the ferric heme superoxide complex. Hydrogen atom trans-
fer oxidation of substrates for [(F8)FeIII-(O2•–)] (S) would af-
ford the corresponding hydroperoxide analog, [(F8)FeIII-
(OOH)] (HP). With an HP BDFE experimentally determined
to be 73.5 kcal/mol, we can expect that S would only be ca-
pable of HAT reactions with substrates such as C–H, N–H or
O–H BDFE’s below or near this value.96 So, we have probed
these possibilities. Addition of an excess of higher BDFE
substrates than 73.5 kcal/mol (i.e., p-methoxyphenol, BDFE
= 83.1 kcal/mol in THF; fluorene, 76.8 kcal/mol in THF;
9,10-dihydroanthracene, 76 kcal/mol in DMSO; p-OMe-2,6-
di-tert-butylphenol, 75.8 kcal/mol in THF)59,89 to solutions
of [(F8)FeIII-(O2•–)] (S) at –80 ℃ in THF led to no reaction, as
expected. Also, no reaction occurs with similar BDFE sub-
strates for HP, such as 1,4-cyclohexadiene (72.9 kcal/mol in
MeCN) and xanthene (72.2 kcal/mol in THF).89 By contrast,
BNAH (1-benzyl-1,4-dihydronicotinamide, 70.7 kcal/mol in
DMSO),97 phenylhydrazine (70.4 kcal/mol in MeCN) and di-
phenylhydrazine (67.1 kcal/mol in DMSO)59 do react rap-
idly with complex S, but we were not able to determine if
the initial reaction is actually HAT; based on UV-vis spectro-
scopic monitoring, the expected hydroperoxide product,
Determination of the OO–H BDFE of [(F8)FeIII-(OOH)]
(HP). The bond dissociation free energy (BDFE) is the ther-
modynamic parameter used to compare the energy, i.e.,
bond strength, for homolytic bond cleavage of a two atom-
centered unit. In order to determine the BDFE, we utilized
the Bordwell equation (eq. 1)88 which uses Hess’ law with
the redox potential and acidity based on a thermodynamic
cycle. Thus, using the measured thermodynamic parame-
ters, E°’ (–1.17 V vs. Fc+/0, vide supra) and pKa (28.8, vide
supra), a thermodynamic square scheme is completed as
shown in Figure 1 and this leads to a BDFE of 73.5 ± 0.9
kcal/mol for the OO–H bond in hydroperoxide [(F8)FeIII-
(OOH)] (HP) according to eq. 2 where CG is 61 kcal/mol in
THF.89
BDFEO–H = 1.37(28.8) + 23.06(–1.17) + 61 = 73.5 kcal/mol
(2)
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[(F8)FeIII-(OOH)] (HP), was not observed at the end of the 0.63 ppm) in 2H NMR spectroscopy, indicative of a six-coor-
dinate ferric low-spin (S = 1/2) heme (Figure S2).98,99
For further insights into the nature of the hydrogen atom
abstraction from TEMPO–H by S, kinetic studies were car-
ried out in THF at –80 ℃ via UV-vis spectroscopy (Figure 9).
The rate of formation of hydroperoxide HP obeyed pseudo-
first-order kinetics (Figure S11) when the reaction was con-
ducted with excess TEMPO–H, and the observed pseudo-
first-order constants (kobs) are proportional to concentra-
tions of TEMPO–H as shown in Figure 10 with an effectively
zero intercept within error. According to this plot of kobs vs
[TEMPO–H], the second order rate constant k2 was obtained
to be 0.5 M–1s–1 in THF at –80 ℃. When deuterated sub-
strate, TEMPO–D, was treated to [(F8)FeIII-(O2•–)] (S), a sig-
nificant slowing of the reaction occurred; k2 = 0.08 M–1s–1 in
THF at –80 ℃ (Figure S12). The comparison of the rate con-
stants for H vs. D provides for a significant kinetic deuter-
ium isotope effect (KIE) of 6 in THF at –80 ℃ as shown in
Figure 10, strongly suggesting that H-atom abstraction by S
is the rate-determining step.
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reaction; probably HAT occurs, but side-reactions prevent
clean formation of HP. However, the addition of TEMPO–H
(66.5 kcal/mol in THF)89 to a solution of S in THF at –80 ℃
led to shifting of the UV-vis spectrum (Figure 9A), i.e., yield-
ing an absorption at 418 nm, corresponding to HP, suggest-
ing that substrate hydrogen atom abstraction reaction oc-
curred. Note that this HAT reaction from TEMPO–H to com-
plex S is thermodynamically downhill by ca. 7 kcal/mol,
thus predicted to be quite favorable.
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Figure 10. Plots of pseudo first order rate constants (kobs
)
plotted against various concentrations of TEMPO–H(D) to
obtain the second order rate constant, kH = 5.0 x 10–1 M–1s–1
(black circle) and kD = 0.8 x 10–1 M–1s–1 (blue circle) and KIE
in THF at –80 ℃.
Figure 9. (A) UV-vis spectroscopic monitoring the reaction
of [(F8)FeIII-(O2•–)] (S) (red) with TEMPO–H in THF at –80 ℃
to yield [(F8)FeIII-(OOH)] (HP) (pink). (B) 10 K EPR spec-
trum of the final products of TEMPO–H HAT by S in frozen
THF. The yield of hydroperoxide HP is 94.2 % based on the
absorbance and known absorptivity of HP at 418 nm. Spin
quantification finds that the EPR signal corresponds to 92%
of TEMPO radical.
There have been no prior reports of kinetic information
to compare the reactivity of [(F8)FeIII-(O2•–)] (S) with other
heme-superoxide complexes reacting with TEMPO–H.
{Note: There is a study from Mayer and co-workers,100
where a heme FeIII-OH complex reacts with TEMPO–H, k2 =
76 M−1s−1 at room temperature in toluene}. However, sec-
ond order rate constants (k2) for non-heme metal-superox-
ide (i.e., Mn+-(O2•–)) complexes (M = Cu, Mn, Co and Cr) in-
volved in HAT reactions with TEMPO–H have been reported
in a number of cases (see Table S5).93,94,101–104
As mentioned in the Introduction, recent review articles
detail metal-superoxide complexes of the first row transi-
tion metals.56,57 Prior computationally derived generaliza-
tions concerning metal superoxide reactivity have been
published;91,105 substrate reactivity depends on factors such
as electrophilicity, reaction driving force, and coupling be-
tween the metal center and superoxide ligand. A CYP450
ferric superoxide is a weak oxidant for HAT, possessing
large activation energy barriers.18 These results are
Complementary experiments were performed, confirm-
ing generation of hydroperoxide species from the reaction
between [(F8)FeIII-(O2•–)] (S) and TEMPO–H. For one thing,
addition of EtP2(dma) base to the product mixture led to the
formation of peroxide species P based on UV-vis spectros-
copy (Figure S10). Further, frozen solution EPR spectros-
copy was also performed and this revealed a signal at g = 2
attributed to the TEMPO radical, appearing ‘on top of’ the
signal expected for low-spin HP (Figure 9B), confirming H-
atom abstraction by S from TEMPO–H. In addition, the final
product exhibits upfield-shifted pyrrole resonance (δpyrr
–
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consistent with our findings where superoxide complex re-
synthesized according to previously published literature
procedures.110–112
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acts by HAT with an exogenous weak O–H (and perhaps N–
H and C–H, vide supra) substrate. Also, a few potentially bi-
ologically relevant non-heme iron superoxide complexes
are capable of abstracting hydrogen atom from weak X–H
substrates such as 2,4-di-tert-butyl phenol,106 2-hydroxy-2-
azaadamantane (AZADOL), phenylhydrazine (X–H < 72.6
kcal/mol)107 and 9,10-dihydroanthracene (DHA; BDE = 76
kcal/mol).108,109
The preparation and handling of air-sensitive compounds
were performed under a MBraun Labmaster 130 inert at-
mosphere (< 1 ppm O2 and < 1 ppm H2O) glovebox filled
with nitrogen. Dioxygen gas purchased from Airgas and
dried through Drierite. 18O2 gas was purchased from ICON,
Summit, NJ, and 16O2 gas was purchased from BOC gases,
Murray Hill, NJ.
All UV−vis measurements were carried out using a
Hewlett-Packard 8453 diode array spectrophotometer with
HP Chemstation software and a Unisoku thermostated cell
holder for low-temperature experiments. A 10 mm path
length quartz cell cuvette modified with an extended glass
neck with a female 14/19 joint, and stopcock was used to
perform all UV–vis experiments, as previously de-
scribed.99,113,114 1H and 2H NMR spectra were measured on a
Bruker 300-MHz NMR spectrometer at ambient or low tem-
peratures. Chemical shifts were reported as δ (ppm) values
relative to an internal standard (tetramethylsilane) and the
residual solvent proton peaks. Electron paramagnetic reso-
nance (EPR) spectra were recorded with a Bruker EMX
spectrometer equipped with a Bruker ER 041 × G micro-
wave bridge and a continuous flow liquid helium cryostat
(ESR900) coupled to an Oxford Instruments TC503 temper-
ature controller. Spectra were obtained at 10 K under non-
saturating microwave power conditions (ν = 9.428 GHz, mi-
crowave power = 0.201 mW, modulation amplitude = 10 G,
microwave frequency = 100 kHz, and receiver gain = 5.02 ×
103).
9
CONCLUSION
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We report here that the side-on ferric peroxide species
[(F8)FeIII-(O22–)]– (P) generated from [(F8)FeIII-(O2•–)] (S) by
one electron reduction, is protonated by acids such as
[(LutH+)](OTf) to form the end-on ferric hydroperoxide
complex [(F8)FeIII-(OOH)] (HP) which has been character-
ized by UV-vis, EPR and rR spectroscopies. Thus, the step-
wise generation of superoxide (S), peroxide (P) and hydrop-
eroxide (HP) analogs which are involved in catalytic cycle of
CYP450 (Scheme 1) or many other heme enzymes (see the
Introduction) are completed through reduction and proto-
nation processes as outlined in Figure 1. More importantly,
by direct experimentation, the reduction potential of the su-
peroxide species [(F8)FeIII-(O2•–)] (S), and the pKa value for
the hydroperoxide analog [(F8)FeIII-(OOH)] (HP) have been
established. With these parameters in hand, the O–H BDFE
of HP could be calculated (73.5 kcal/mol). This work de-
scribes the first example of experimentally determined
thermodynamics (reduction potential and pKa) interrelat-
ing S, P, and HP species while also demonstrating the gen-
eration of the ferric hydroperoxide complex (HP) using an
exogenous substrate (TEMPO–H) for HAT by the ferric
heme superoxide species (S). These results advance the lit-
erature concerning the thermodynamics of heme com-
plexes while further showing the utility of synthetic model
compounds to provide fundamental insights relevant to re-
action cycles of heme enzymes.
As mentioned in Introduction, the peroxide intermediate
in catalytic cycle of CYP450 (Scheme 1) is thought to have
an end-on (η1) geometry. Davydov, Sligar, Hoffman, and co-
workers42 detected this end-on bound peroxide intermedi-
ate by employing γ-irradiation at cryogenic temperature
with P450cam. Thus, one future goal will be research rele-
vant to thermodynamic analysis involving end-on ferric
heme peroxide chemistry. We wish to also further assess
whether heme or axial ligation modifications may lead to
enhancement of the oxidizing capability of a heme-FeII/O2
derived adduct.
The compounds (F8)FeII, and the pyrrole deuterated de-
rivative d8-(F8)FeII were synthesized as previously de-
scribed.61,115,116
UV-vis Spectroscopy.
Generation of [(F8)FeIII-(OOH)] (HP). After generating
complex [(F8)FeIII-(O22–)]– (P),60 1 equiv [(LutH+)](OTf) or
[(H)DMF](OTf) was added to the solution of P to form
[(F8)FeIII-(OOH)] (HP) in THF at –80 ℃. UV-vis: λmax = 418 (ԑ
= 157048 M–1 cm–1), 536 (ԑ = 9782.5 M–1 cm–1) and 558 (ԑ =
7322.5 M–1 cm–1) nm.
H2O2 Quantification by Horseradish Peroxidase
(HRP) Test. The spectrophotometric quantification of hy-
drogen peroxide was carried out by analyzing the intensity
of the diammonium 2,2′- azino- bis(3-ethylbenzothiazoline-
6-sulfonate)(AzBTS-(NH4)2) peaks (at different wave-
lengths to minimize error, Figure S3), which was oxidized
by horseradish peroxidase (HRP); this was adapted from
published procedures.72,73 Three stock solutions were pre-
pared: 300 mM sodium phosphate buffer pH 7.0 (solution
A), 1 mg/mL AzBTS-(NH4)2 (solution B) and 4 mg of HRP
(type II salt free (Sigma)) with 6.5 mg of sodium azide in 50
mL of water (solution C). 3.0 mL of the desired [(F8)FeIII-
(O22–)]– (P) or [(F8)FeIII-(OOH)] (HP) solution were gener-
ated in THF at –80 ℃, as previously described. The reaction
which is before and after being quenched by adding 100μL
of triflic acid (HOTf) solution (2.5 equiv) is subject to the
H2O2 analysis. Subsequently 100 μL of the cold THF sample
solution was removed via a syringe and quickly added to a
cuvette containing 1.3 mL of water, 500 μL of solution A,
100 μL of solution B, and 50 μL of solution C (all chilled in
an ice bath prior to use). After mixing for 15s, the samples
EXPERIMENTAL SECTION
Materials and Methods. All reagents and solvents pur-
chased and used were of commercially available quality ex-
cept as noted. Inhibitor-free Tetrahydrofuran (THF) was
distilled over Na/benzophenone under argon and deoxy-
genated with argon before use. Butyronitrile was distilled
over sodium carbonate and potassium permanganate and
deoxygenated with Ar before use. Cobaltocene was ob-
tained from Sigma Aldrich, sublimed at 75°C, and stored un-
der nitrogen in the glovebox freezer at –30 °C. The 2,6-
lutidinium Triflate [(Lu)(H)](OTf) and TEMPO–H(D) were
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were allowed to sit at room temperature for ~2 min until Electron Paramagnetic Resonance (EPR) Spectros-
copy. In a glovebox, 1 mM solutions of (F8)FeII in THF were
prepared and transferred to EPR tube and capped with
tightfitting septa. The sample tubes were placed in a cold
bath (dry ice/acetone) and oxygenated. The oxygenated
samples were set in a cold bath for 10 min and to the cold
THF solution of [(F8)FeIII-(O2•–)] (S) was added 1 equiv
CoCp2 for complex [(F8)FeIII-(O22–)]– (P). Subsequently, 1
equiv [(LutH+)](OTf) was added for complex [(F8)FeIII-
(OOH)] (HP). Then, the sample tubes were frozen in liquid
N2.
1
2
3
4
5
6
7
8
full formation of the 418 nm band was observed (Figure S3).
Determination of the reduction potential of [(F8)FeIII-
(O2•–)] (S). [(F8)FeIII-(O2•–)] (S) generated as previously
published61,62 was titrated with 0.25-2 equiv Cr(η-C6H6)2 in
THF at –80 ℃. For each equilibrium mixture, the concentra-
tion of each species in solution was measured using the ab-
sorption at 434 nm (Table S2). From these equilibrium con-
stants, corresponding reduction potentials were calculated
by using the Nernst equation.
9
2–
Reversibility of [(F8)FeIII-(O2•–)] (S) and [(F8)FeIII-(O2
10
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)]– (P). In a Schlenk cuvette, the addition of 1 equiv Magic
Blue to a solution of [(F8)FeIII-(O22–)]– (P) generated from
[(F8)FeIII-(O2•–)] (S) with 2 equiv Cr(η-C6H6)2 in THF at –80
℃ leads to the formation of [(F8)FeIII-(O2•–)] (S) and again,
For EPR data for the reaction of [(F8)FeIII-(O2•–)] (S) with
TEMPO–H, 10 equiv TEMPO–H was added to the 1mM solu-
tion of S generated as described above. The tube was left at
–80 ℃ for 1 hour 30 minutes and then frozen in liquid nitro-
gen.
2–
the addition of 5 equiv Cr(η-C6H6)2 gives back [(F8)FeIII-(O2
)]– (P) (Figure S6).
Determination of the pKa of [(F8)FeIII-(OOH)] (HP). In
a Schlenk cuvette, [(F8)FeIII-(OOH)] (HP) generated as de-
scribed above, was titrated by 0.75-3 equiv EtP2(dma) in
THF at –80 ℃. For each equilibrium mixture, the concentra-
tion of each species in solution was measured using the ab-
sorption at 434 nm (Table S4). From these equilibrium con-
stants, the pKa was calculated.
ASSOCIATED CONTENT
Supporting information
For [(F8)FeII], S, P and HP, associated spectroscopic data (2H-
NMR, EPR, rRaman and UV-vis), kinetic data for the reaction of
S with TEMPO–H(D), details concerning H2O2 quantification,
calculations relevant to reduction potential and pKa experi-
ments, control experimental details, and tabulated data from
the literature on (i) theoretically considered and computed
thermodynamics of ferric-heme superoxide reduction poten-
tial, ferric heme hydroperoxide pKa and OO–H BDFE, and (ii)
non-heme first-row transition metal ion superoxide complex
kinetics of reaction with TEMPO–H(D). This material is availa-
ble free of charge via the internet at http://pubs.acs.org
Reversibility of [(F8)FeIII-(O22–)]– (P) and [(F8)FeIII-
(OOH)] (HP). In a Schlenk cuvette, the addition of 3 equiv
EtP2(dma) to solution of [(F8)FeIII-(OOH)] (HP) generated as
2–
described above, results in the formation of [(F8)FeIII-(O2
)]– (P) in THF at –80 ℃ and again, the addition of 3 equiv
[(LutH+)](OTf) gives [(F8)FeIII-(OOH)] (HP) (Figure S8).
Reactivity study of [(F8)FeIII-(O2•–)] (S) with TEMPO–
H. Excess amount of TEMPO–H (0.4-3.6 mM) was added to
the solution of [(F8)FeIII-(O2•–)] (S) in THF at –80 ℃ in a
Schlenk cuvette.
AUTHOR INFORMATION
Corresponding Authors
Kinetic studies of [(F8)FeIII-(O2•–)] (S) with TEMPO–
H(D). The substrates TEMPO–H(D) (10, 30, 50, 70, 90
equiv) were added via syringe to a 0.04 mM solution of
[(F8)FeIII-(O2•–)] (S) and the following UV-vis spectra were
recorded (Figure 9). The hydroperoxide band at 418 nm ap-
peared with time and the pseudo-first-order rate plots were
observed. The values of kobs were calculated from plots of
log[(At-Af)/(Ai-Af)] vs time (s) (Figure S11, S12). According
to this plot of kobs vs [TEMPO–H(D)], the second order rate
constant k2 was obtained in THF at –80 ℃ (Figure 10).
Resonance Raman Spectroscopy. In the glovebox, 1 mM
solutions of (F8)FeII in THF were prepared and transferred
to rR tube and capped with tightfitting septa. The sample
tubes were placed in a cold bath (dry ice/acetone) and oxy-
genated using 16O2 or 18O2 gases. The oxygenated samples
were set in a cold bath for 10 min and to the cold THF solu-
tion of [(F8)FeIII-(O2•–)] (S) was added 1 equiv CoCp2 for
karlin@jhu.edu
edward.solomon@stanford.edu
Notes
The authors declare no competing financial interest.
ORCID
Hyun Kim: 0000-0001-9823-6457
Savita K. Sharma: 0000-0001-5489-8416
Andrew W. Schaefer: 0000-0001-6565-920X
Edward I. Solomon: 0000-0003-0291-3199
Kenneth D. Karlin: 0000-0002-5675-7040
ACKNOWLEDGMENT
This research was supported by the USA National Institutes of
Health (GM60353 to K.D.K. and GM040392 to E.I.S.).
complex [(F8)FeIII-(O22–)]– (P). Subsequently,
1 equiv
[(LutH+)](OTf) was added for complex [(F8)FeIII-(OOH)]
(HP). Then, the sample tubes were frozen in liquid N2 and
sealed by flame. Resonance Raman samples were excited at
413 nm, using a Coherent I90C-K Kr+ ion laser while the
sample was immersed in a liquid nitrogen cooled (77 K)
EPR finger Dewar (Wilmad). Power was ~2 mW at the sam-
ple, which was continuously rotated to minimize photo-
decomposition. The spectra were recorded using a Spex
1877 CP triple monochromator, and detected by an Andor
Newton CCD cooled to –80 °C.
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Low-Spin
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Complex―The Product of the Reaction of Nitrogen Monoxide (·NO(g)
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•–
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(90) For Naruta’s superstructured ferric heme superoxide, peroxide
and hydroperoxide complexes40 (with or without a H-bonding moiety
to interact with the O2-derived fragment) (Figure S7), theoretical
computations were carried out and superoxide-to-peroxide reduction
potentials and hydroperoxide/peroxide pKa values were provided;
however BDFE's for the ferric heme hydroperoxide complexes were
not evaluated.
(104) Wind, M.-L.; Hoof, S.; Braun-Cula, B.; Herwig, C.; Limberg, C.
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(95) Dhar and Tolman77a determined BDE = 90 kcal/mol for an O–H
bond of a CuIII-aqua complex, while Kieber-Emmons and coworkers77b
determined that a mono-hydroxo-bridged dicopper(II) complex pos-
sesses an O–H BDE of 77 kcal/mol; this species derives from HAT chem-
istry of the corresponding μ-oxo dicopper(II) complex. Mayer and
coworkers77c,d,e reported on several cases of coupled proton-electron
transfer (CPET) not involving O–H bond reactions; here, heme or non-
heme iron(III) compounds reacting with a hydrogen-atom donor sub-
strate react such that an electron transfers to the iron while the proton
transfers to a basic site on the ligand periphery. (a) Dhar, D.; Tolman,
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Hydroxide Complex. J. Am. Chem. Soc. 2015, 137, 1322-1329. (b) Ali, G.;
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ell, S.; Mayer, J. M. Intrinsic Barriers for Electron and Hydrogen Atom
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2000, 122, 5486-5498. (d) Mader, E. A.; Davidson, E. R.; Mayer, J. M.
Large Gournd-State Entropy Changes for Hydrogen Atom Transfer Re-
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at a Heme-Propionate in an Iron-Protoporphyrin-IX Model Compound.
J. Am. Chem. Soc. 2011, 133, 8544-8551.
(109) Kovacs and coworkers recently described a non-heme FeIII-su-
peroxide intermediate which abstracts hydrogen atoms from 1,4-cy-
clohexadiene (CHD) (BDE = 76 kcal/mol) but also THF solvent, that
possessing a strong C–H bond (BDE = 92 kcal/ mol); an FeIII-hydroper-
oxide complex is the product; Blakely, M. N.; Dedushko, M. A.; Poon, P.
C. Y.; Villar-Acevedo, G.; Kovacs, J. A. Formation of a Reactive, Alkyl Thi-
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Physical Properties of Early-Late Heterobimetallic Compounds
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(96) Perhaps Naruta and coworkers did not address the issue of
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(114) Wasser, I. M.; Huang, H.-w.; Moënne-Loccoz, P.; Karlin, K. D.
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Heme/Non-Heme Diiron(II) Complexes and O2 , CO , and NO Adducts as
Hodgson, K. O.; Hedman, B.; Karlin, K. D.; Solomon, E. I. Spectroscopic
Elucidation of New Heme/Copper Dioxygen Structure Type:
Implications for O⋯O Bond Rupture in Cytochrome c Oxidase. Angew.
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Reduced and Substrate-Bound Models for the Active Site of Bacterial
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a
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(116) Kieber-Emmons, M. T.; Qayyum, M. F.; Li, Y.; Halime, Z.;
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