Electrocatalytic O2-reduction by synthetic cytochrome c oxidase mimics: Identification of a bridging peroxo intermediate involved in facile 4e-/4H+ O2-reduction

Article abstract of DOI:10.1021/jacs.5b06513

A synthetic heme-Cu CcO model complex shows selective and highly efficient electrocatalytic 4e^-/4H⁺ O₂-reduction to H₂O with a large catalytic rate (>10⁵ M^-1 s^-1). While the heme-Cu model (FeCu) shows almost exclusive 4e^-/4H⁺ reduction of O₂ to H₂O (detected using ring disk electrochemistry and rotating ring disk electrochemistry), when imidazole is bound to the heme (Fe(Im)Cu), this same selective O₂-reduction to water occurs only under slow electron fluxes. Surface enhanced resonance Raman spectroscopy coupled to dynamic electrochemistry data suggests the formation of a bridging peroxide intermediate during O₂-reduction by both complexes under steady state reaction conditions, indicating that O-O bond heterolysis is likely to be the rate-determining step (RDS) at the mass transfer limited region. The O-O vibrational frequencies at 819 cm^-1 in ¹⁶O₂ (759 cm^-1 in ¹⁸O₂) for the FeCu complex and at 847 cm^-1 (786 cm^-1) for the Fe(Im)Cu complex, indicate the formation of side-on and end-on bridging Fe-peroxo-Cu intermediates, respectively, during O₂-reduction in an aqueous environment. These data suggest that side-on bridging peroxide intermediates are involved in fast and selective O₂-reduction in these synthetic complexes. The greater amount of H₂O₂ production by the imidazole bound complex under fast electron transfer is due to 1e^-/1H⁺ O₂-reduction by the distal Cu where O₂ binding to the water bound low spin Fe^II complex is inhibited.

Full text of DOI:10.1021/jacs.5b06513

Article
pubs.acs.org/JACS
Electrocatalytic O ‑Reduction by Synthetic Cytochrome c Oxidase
2
Mimics: Identification of a “Bridging Peroxo” Intermediate Involved
−
+
in Facile 4e /4H O ‑Reduction
2
†
†
‡
,‡
,†
Sudipta Chatterjee, Kushal Sengupta, Shabnam Hematian, Kenneth D. Karlin,* and Abhishek Dey*
†Department of Inorganic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
‡Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, United States
*
S Supporting Information
ABSTRACT: A synthetic heme−Cu CcO model complex shows
−
+
selective and highly efficient electrocatalytic 4e /4H O -reduction
2
5
−1 −1
to H O with a large catalytic rate (>10 M s ). While the heme-
2
−
+
Cu model (FeCu) shows almost exclusive 4e /4H reduction of O
2
to H O (detected using ring disk electrochemistry and rotating ring
2
disk electrochemistry), when imidazole is bound to the heme
(
Fe(Im)Cu), this same selective O -reduction to water occurs only
2
under slow electron fluxes. Surface enhanced resonance Raman
spectroscopy coupled to dynamic electrochemistry data suggests the
formation of a bridging peroxide intermediate during O -reduction
2
by both complexes under steady state reaction conditions, indicating
that O−O bond heterolysis is likely to be the rate-determining step
(
RDS) at the mass transfer limited region. The O−O vibrational
−1 16 −1 18
−1
−1
frequencies at 819 cm in O (759 cm in O ) for the FeCu complex and at 847 cm (786 cm ) for the Fe(Im)Cu
2
2
complex, indicate the formation of side-on and end-on bridging Fe-peroxo-Cu intermediates, respectively, during O -reduction in
2
an aqueous environment. These data suggest that side-on bridging peroxide intermediates are involved in fast and selective O2-
reduction in these synthetic complexes. The greater amount of H O production by the imidazole bound complex under fast
2
2
−
+
II
electron transfer is due to 1e /1H O -reduction by the distal Cu where O binding to the water bound low spin Fe complex is
2
2
inhibited.
−
1
INTRODUCTION
s ) which is then readily transferred to heme-a (at an overall
3
■
5
−1
rate ∼10 s ) forming an initial Fe−O adduct analogous to
oxy-hemoglobin/myoglobin.
(O ) -Cu (either side-on or end-on) intermediate preceding
PM has been characterized spectroscopically and structurally in
some CcO active sites; their relevance in catalytic O reduction,
while invoked in a few investigations, is not clear (Figure
2
Cytochrome c oxidase (CcO), a heme-copper enzyme, catalyzes
2
,14,17
III
−
+
A bridging peroxide Fe -
the 4e /4H reduction of O to H O in the terminal step of the
2
2
2−
II
1
−4
2
electron transport chain during respiration.
tions of oxygen reduction by synthetic heme-Cu models are also
related to fuel cell cathode catalysts as they provide fundamental
The investiga-
2
insight into the factors that lead to efficient O reduction
2
18−24
1
).
materials. CcO has attracted paramount interest of synthetic
inorganic and bioinorganic chemists due to its great
fundamental and practical importance in understanding
dioxygen activation/reduction, and proton translocation,
The investigation of synthetic heme-Cu systems has greatly
assisted our understanding of the reaction mechanism of
dioxygen reduction and factors that lead to selective 4e /4H
reduction of O avoiding formation of detrimental partially
reduced oxygen species (PROS).
workers have reported the initial formation of an Fe−O adduct
followed by P -like intermediate formation with their functional
CcO model under rate limiting conditions,
have stressed on the formation of a bridging peroxide as a
possible intermediate during O -reduction under single turnover
−
+
5
−10
2
etc.
The X-ray structures of CcO reveal the active site
19,26−30
While Collman and co-
responsible for O binding and reduction, to be a binuclear site
2
comprising of heme-a , with a proximal histidine (His) along
2
3
3
,11,12
with a distal side histidine bound copper (CuB).
these three ligating His residues is covalently cross-linked to a
nearby tyrosine (Tyr) residue which is said to act as a source of
One of
M
27,26
Karlin’s group
electron and proton in the reduction of O and proton
2
2
19,31−33
1
3,14
conditions.
These bridging peroxo intermediates can also
translocation.
This chemistry stems from the character-
IV
II
be interconverted, in some cases, from a side-on to an end-on
ization of an intermediate P (ferryl Fe O and Cu −OH/
M
H O), formed after the O−O bond cleavage in CcO, which
2
1
5,16
suggests the presence of a Tyr radical.
Recent literature
Received: June 23, 2015
8
suggests that O initially binds to Cu (at an overall rate ∼10
2
B
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b06513
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Journal of the American Chemical Society
Article
We find that 1 and 2 selectively reduce O to H O under fast
2
2
electron-transfer (ET) flux, while 3 is less selective in doing so
under similar conditions. In fact, the production of PROS
decreases with a decrease in ET rate for complex 3 in contrast to
complexes 1, 2 and all other reported Fe−Cu sys-
6
,26,28,38−40
tems.
The SERRS and SERRS-RDE techniques
employed here not only allow identification of intermediates
and probable ORR mechanism, but also help to deduce rate-
determining steps, supplementing the information obtained
from electrochemical data alone.
RESULTS AND ANALYSIS
Cyclic voltammetry (CV) of complex 1, which has been
physiabsorbed on an edge plane graphite (EPG) electrode in
deoxygenated pH 7 phosphate buffer, shows a reversible Fe
■
Figure 1. Binuclear active site structures of CcO showing (A) side-on
1
2
1
2
25
(μ−η :η ) (η at Fe center and η at Cu center) (PDB ID: 3ABM)
and (B) end-on (μ-1, 2) (PDB ID: 3S8F) bridging peroxo complexes.
III/II
couple at −210 mV (Figure 3A, blue). Complex 2 shows two
34,35
18,29,32
product in synthetic myoglobin
or CcO models
upon
incorporation of an external strong field ligand like imidazole or
its analogues. In spite of obtaining significant knowledge and
insight into the O reduction mechanism under single turnover
2
conditions in organic solvents, the mechanistic details in an
aqueous environment remains unresolved due to the lack of
direct spectroscopic evidence, which motivates our present
study.
Examples of electrocatalytic O -reduction reactions (ORR)
2
employing synthetic CcO model complexes containing Fe-only
and Fe−Cu centers have been reported at physiological
2
9,30,36
pH.
Although the presence of Cu ion was initially
−
thought to be essential for selective 4e reduction of O , it has
2
now been established that the presence of Cu is only necessary
26,28
when the electron transfer (ET) from the electrode is slow.
The presence of a strong sixth ligand coordinated to the heme
center may influence and alter the ORR reactivity. Recently, an
experimental setup has been developed where dynamic
electrochemistry (rotating disk electrochemistry; RDE) is
coupled to a powerful spectroscopic tool, surface enhanced
resonance Raman spectroscopy (SERRS) which allows direct in
situ investigations of the reactive intermediates formed under
steady state conditions and thus aids the elucidation of the
37
reaction mechanism for electrocatalytic ORR.
Figure 3. (A) CV data of complexes 1 (blue), 2 (red), and 3 (green)
physiabsorbed on an EPG electrode in deoxygenated pH 7 buffer under
an argon atmosphere at a scan rate of 50 mV/s using Ag/AgCl as
reference and Pt wire as counter electrodes. SERRS data of 1 (B), 2
(
C), and 3 (D), physiabsorbed on a C SH modified Ag disk, under
8
oxidizing (red) and reducing (green) conditions in pH 7 buffer under
anaerobic conditions at a constant rotation rate of 200 rpm. (E) and
(
F) are the ν , ν , and ν bands of the spectra obtained at reducing
4 3 2
conditions of complex 2 and 3, respectively, along with their Lorentzian
fits showing different components.
6
6
6
Figure 2. L-Fe (1), L-FeCu (2), and L-Fe(Im)Cu (3) are the
synthetic model complexes used for this study.
In this study, the electrocatalytic O -reductions of a heme-
copper L-FeCu complex (2, Figure 2) and its imidazole adduct
L-Fe(Im)Cu (3, Figure 2) have been investigated at
physiological pH under steady state conditions, in order to (i)
evaluate the efficiencies of this group of complexes under
aqueous conditions and (ii) detect intermediates formed during
reversible CV waves at −155 and 173 mV, which may be
2
6
III/II
II/I
ascribed to the E1 for the Fe
and Cu
processes,
couple of
/2
6
III/II
respectively (Figure 3A, red). The E1 for the Fe
/2
−
1, which has an OH as the axial ligand, appears at a more
negative potential than 2. Moreover, the presence of distal Cu
III/II
site in complex 2 is responsible for slight positive shift in Fe
O -reduction. Results from study of the Fe-only complex (1,
Figure 2) serve as a control to evaluate the role of the copper.
potential compared to complex 1. The CV of 2 obtained here on
EPG under aqueous condition is different from that in other
2
B
DOI: 10.1021/jacs.5b06513
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Journal of the American Chemical Society
Article
CcO models obtained in aqueous or organic media, where both
Chart 1
III/II
II/I
19,28
II/I
the Fe
and Cu CV waves appear at same potentials.
III/II
The CV of 3 indicates that the E1/2 for the Fe
and Cu
processes, though very close, are somewhat distinguishable in
the cathodic process but overlap completely in the anodic
reaction (Figure 3A, green). Therefore, it is quite ambiguous to
extract the respective E1/2 values from the CV data. The E1/2
values for the two processes appear at more negative potentials
41
compared to complex 2. This is likely due to the binding of
imidazole as the sixth ligand to the Fe center (when one
equivalent of imidazole is added externally to complex 2, it binds
to the Fe site only as observed from the distinct resonance
Raman signal in homogeneous medium; see Figure S1,
Supporting Information and the Experimental Details section)
which weakens trans Fe−O bond, strengthening the Cu−O
bond in this complex, which lowers the E values of both the
1
/2
Fe and Cu centers compared to those in complex 2. Note that in
CcO as well as in its synthetic mimics (where imidazole is
covalently bound to heme center), the heme and Cu ion
28,26
potentials are almost the same.
Integration of the charge
In the case of complex 2, the resting oxidized state is observed
to be LS when immobilized on the electrode (Figure 3C) unlike
yields the surface coverage, i.e., the number of the electroactive
species present on the electrodes. Complexes 1 and 2 show
33
in an organic solvent where it is high spin. The change in spin
−
11
−11
state in 2 is likely due to the fact that complex 2 binds water
from bulk solvent) as an axial ligand (Chart 1). However, for
coverages of (2.36 ± 0.33) × 10 and (1.3 ± 0.05) × 10
2
III/II
(
mol/cm integrating the Fe
redox process, respectively.
6
complex 3 the sixth position has already been occupied by the
external imidazole ligand before immobilization and it is LS in
both organic solvent and aqueous medium (Chart 1, Figure 3D
Analogously, the coverage of Cu in the L-FeCu is estimated to
−11
2
be (0.97 ± 0.06) × 10 mol/cm implying that the Fe:Cu ratio
in complex 2 is close to 1:1, consistent with the formulations of
the complex. Note that the individual coverage of Fe and Cu in
complex 3 cannot be determined due to overlapping of the
II
and Figure S1A, Supporting Information). Upon reduction, Fe
II
HS and Fe LS species have been obtained as the major
III/II
II/I
components for complexes 2 and 3, respectively. Hence, it may
be proposed that these two complexes exist as 5C water bound
Fe
and Cu redox processes (Figure 3A, green). However,
−11
2
the total integrated charge of (2.08 ± 0.12) × 10 mol/cm for
complex 3 is two times greater than the individual coverage of
Fe and Cu obtained for complex 2 consistent with the presence
of two electroactive redox centers in 3.
II
II
Fe HS and Im-water bound 6C Fe LS, respectively (Chart 1,
right panel) when reduced. A similar distribution of components
along with their assignments has been observed previously in
38,40
SERRS experiments with similar Fe-porphyrin complexes.
Note that imidazole and water bound LS Fe species, although
proposed in some synthetic systems, are yet to be structurally
SERRS was performed on the catalysts 1, 2 and 3 immobilized
II
on C SH self-assembled monolayer (SAM) modified roughened
8
Ag surfaces. SERRS data were obtained, both when the catalysts
were in their respective resting oxidized state and also under
reducing conditions in aqueous pH 7 buffer. SERRS data for 1
47,48
characterized.
The electrocatalytic O reduction using complexes 1, 2 and 3
2
has been investigated using the rotating disk electrochemistry
RDE) technique in air-saturated pH 7 buffer. Linear sweep
voltammetry (LSV) of the complexes at different rotation rates
under resting conditions exhibits oxidation state (ν ),
4
(
coordination number (ν ) and spin state (ν ) marker bands at
3
2
−
1
1
362, 1439, and 1556 cm , respectively, indicating the presence
shows a substrate diffusion limited catalytic O -reduction
III
2
of a five-coordinated (5C) Fe high-spin (HS) species on the
current below −100 mV vs Ag/AgCl (Figure 4). This current
42,43
surface (Figure 3B, red).
Upon reduction, the formation of a
increases with increasing rotation rates following the Koutecky−
II
HS Fe species, having the ν and ν bands at 1347 and 1543
cm , respectively, is observed (Figure 3B, green and Figure S2,
Supporting Information).
oxidizing potentials (0 V vs Ag/AgCl), i.e., under resting
conditions has the ν , ν , and ν bands at 1365, 1451, and 1566
−1
−1
−1
4
2
Levich (K−L) equation, I = i (E) + i , where i (E) is the
−
1
K
L
K
potential dependent kinetic current and i is the Levich current
where i is expressed by 0.62nFA[O ](DO ) ω v
4
0,42,44,45
L
The SERRS spectrum of 2 at
2/3 1/2 −1/6
and n is
L
2
2
the number of electrons transferred to the substrate, A is the
2
4
3
2
macroscopic area of the disk (0.096 cm ), [O ] is the
2
−1
cm , respectively, which correspond to a six-coordinated (6C)
low-spin (LS) Fe species (Chart 1 and Figure 3C, red). Upon
concentration of O in an air saturated buffer (0.26 mM) at
2
III
−5
2
2
5 °C, DO is the diffusion coefficient of O (1.8 × 10 cm
2 2
1
−
reduction, new ν , ν , and ν bands at 1347, 1436, and 1544
4
3
2
s ) at 25 °C, ω is the angular velocity of the disk and v is the
2
49,50
−1
II
−1
cm are observed which correspond to a HS Fe species (Chart
, Figure 3C, green, and Figure 3E). SERRS data of 3 at
kinematic viscosity of the solution (0.009 cm s ) at 25 °C.
46
−1
1
From the slope of a plot of I versus the inverse square root of
the angular rotation rate (ω− ), the number of electrons
delivered to the substrate during electrocatalytic ORR can be
evaluated. The slopes obtained from the experimental data for
catalysts 1 and 2 are almost identical to the theoretical slope
1/2
oxidizing potential show the ν , ν , and ν bands at 1367, 1453,
4
3
2
−
1
and 1568 cm , respectively, indicating the presence of a 6C
Fe LS species (Chart 1 and Figure 3D, red). This complex 3,
when reduced, mainly leads to the formation of a LS Fe species
with the ν , ν , and ν bands at 1358, 1447, and 1559 cm ,
III
II
−1
predicted for a 4e− O reduction process (Figure 4B, D).
4
3
2
2
−
respectively (Chart 1 and Figure 3D, green). However, a
However, for complex 3, the slope corresponds to a 3.4e
Lorentzian fit of this reduced spectrum shows the presence of
some HS Fe component as well (Figure 3F).
process (Figure 4F). The values of n obtained indicate that
under conditions of very fast electron transfer (as occurs on
II
C
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Journal of the American Chemical Society
Article
Figure 5. Percentage of PROS formed by the catalysts 1 (red), 2
(yellow), and 3 (green) in air saturated pH 7 buffer under fast (EPG),
slow (C SH SAM on Au), and very slow (C SH SAM on Au) electron
8
16
fluxes measured by a RRDE experiment using Ag/AgCl reference and
Pt wire counter electrodes. Rotation speed = 300 rpm, scan rate = 10
mV/s.
to 1, and the difference in PROS production is more prominent
under slow electron flux (Figure 5 and Figure S4, Supporting
28
Information); the estimated PROS generated by 2 are 6.3 ±
.1%, 7 ± 0.1%, and 13.5 ± 1% when physiabsorbed on EPG,
C SH SAM, and C SH SAM, respectively. As observed for
0
8
16
26,38−40
other CcO mimics and porphyrin complexes,
1 and 2
also show the same general trend of increasing PROS generation
with a decrease in the rate of electron transfer from the
electrode. However, complex 3 shows the opposite trend, where
on EPG the amount of PROS produced is 25 ± 2%, i.e., only
Figure 4. LSV of 1 (A), 2 (C), and 3 (E) physiabsorbed on EPG in air
saturated pH 7 buffer at a scan rate of 50 mV/s at multiple rotations
using Ag/AgCl as reference and Pt wire as counter electrodes. The K−
L plots of the respective catalysts at various potentials are given in (B),
−
+
7
5% of the O was fully reduced by a 4e /4H process to give
2
H O (Figure 5). In fact, this agrees well with the observed
2
finding of n = 3.4 (n = 3.5 theoretically if 25% PROS is
produced), that obtained from the K−L analyses (vide supra).
−
−
−
(
D), and (F). The theoretical plots for 2e , 3.4e , and 4e processes are
also indicated in the figures.
On C SH modified Au electrode, the magnitude of PROS is 10
8
±
1% which further decreased to 4 ± 1% on further slowing
−
EPG electrodes), O undergoes almost complete 4e reduction
down the electron transfer by employing a C SH SAM
2
16
to H O when catalyzed by 1 and 2 but not by 3 (vide infra). The
modified surface (Figure 5, and Figure S5, Supporting
2
−
+
second order rate of catalysis (k ) for ORR can also be
Information). The diminished amount of 2e /2H reduction
product (i.e., H O ) with a decrease in ET rate is unprecedented
cat
obtained from the intercept of this K−L plot using the formula:
i = nFAk Γ [O ]. The k values are obtained to be (3.65 ±
−1 −1
2
2
for synthetic heme-Cu systems. To better understand this,
SERRS-RDE experiments under steady state conditions have
been performed for these catalysts.
K
cat cat
2
cat
6
6
6
1
.1) × 10 , (4.49 ± 0.9) × 10 , and (1.28 ± 0.11) × 10 M s
for complexes 1, 2, and 3, respectively. These facts indicate that
−
+
the selectivity and efficiency for 4e /4H O -reduction are
SERRS-RDE data, collected on C SH SAM modified Ag
2
8
better for the FeCu complex as compared to its imidazole
adduct. It should be noted that the second order rate constant of
disks, of the iron only catalyst 1 during steady state O2
reduction, i.e., catalytic turnover (the electrode is held at −0.5
V vs Ag/AgCl) (Figure 6A, blue) shows the presence of four
−
+
4
e /4H ORR by complex 2 is 1 order of magnitude greater
6,28
than FeCu complexes reported earlier. The slower reactivity
of those synthetic heme-Cu systems may be due to a slower rate
species in the high frequency region. The major species has ν
4
−1
and ν at 1362 and 1556 cm , respectively, corresponding to
2
28,47,51
III
of O binding to the 6C LS reduced iron center.
HS Fe species (Figure 6D, red). There are two additional
2
The selectivity of this electrocatalytic O reduction by the
catalysts 1, 2 and 3 and its dependence on the ET rate from the
minor components having the ν and ν bands at 1350 and 1545
cm (Figure 6D, green) and at 1375 and 1572 cm (Figure
2
4
2
−1
−1
electrodes has been further evaluated by rotating ring disk
6D, black), respectively. The first minor species correspond to a
2
6,28,38,52
II
IV
electrochemistry (RRDE).
Physiabsorption of the
HS Fe species and the other may correspond to a Fe O
3
7,42,54
catalysts on EPG, C SH modified Au and C SH modified Au
species as observed for other Fe−porphyrin complexes.
8
16
5
−1
3
−1
III
electrodes allows fast (kET > 10 s ), moderate (k ∼ 10 s )
Note that a very weak Fe LS component in the ν band could
also be noticed at 1565 cm . Under similar steady state
ET
2
−
1
−1
and slow (k
respectively.
∼ 4−6 s ) electron transfer fluxes,
ET
2
6,53
RRDE measures the amount of PROS
turnover, the FeCu catalyst 2 also gives rise to a mixture of
species as observed in this high frequency region (Figure 6B).
Lorentzian fits of the ν and ν bands show the presence of three
produced (in situ), if any, due to incomplete O reduction.
2
The amount of PROS produced at pH 7 by 1 under fast,
moderate, and slow electron fluxes are 6 ± 1%, 9 ± 0.5%, and 23
4
2
III
components (Figure 6E). The major species is a LS Fe
complex with ν and ν bands at 1366 and 1567 cm .
4 2
The other components correspond to a HS Fe species having
ν and ν bands at 1349 and 1542 cm , respectively, and a HS
−1
55,56
±
3%, respectively (Figure 5 and Figure S3, Supporting
II
Information). We find that with the “extra” redox center, the
copper ion in complex 2, PROS quantities decrease, compared
−1
4
2
D
DOI: 10.1021/jacs.5b06513
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Journal of the American Chemical Society
Article
Figure 7. SERRS-RDE data in the low frequency region under steady
state conditions in the presence of air and 18O saturated pH 7 buffer
2
for complex 2 (A) and complex 3 (B) on C SH modified Ag surfaces.
8
The difference spectra are shown in dotted line (black).
52
RRDE setup as for Fe-porphyrin complexes. Here, for 2, the
lack of significant current at the Pt ring (held at a negative
III
−3
potential where [Fe (CN) ]
can be reduced to
6
II
−4
IV
[
Fe (CN) ] ) suggests that no Fe O is accumulated during
6
O reduction consistent with the lack of Raman signal (Figure
2
S6A, Supporting Information). When the SERRS-RDE experi-
ment is performed in air saturated pD 7 buffer, the O−O peak
appears at 819 cm− as observed in case of air saturated pH 7
buffer, which further suggests that the Fe-peroxo species formed,
1
2
2
−
is a species with a peroxo bridge (i.e., Fe−O −Cu), and not an
III
iron hydroperoxide (Fe −OOH) complex (Figure S7,
Supporting Information). Unfortunately, the low frequency
region of the spectrum does not reveal corresponding Fe−O/
Cu−O vibrations for the bridged-peroxo complex 2 (Figure
5
7
S8A, Supporting Information). However, the ν
absolute
O−O
value and its observed 60 cm−
1 16/18
O shift (vide supra) are
Figure 6. SERRS-RDE data for complexes 1 (A), 2 (B) and 3 (C) in
the high frequency region at oxidized (red) and steady state conditions
consistent with the O−O bond stretching frequency of Fe-
peroxo species, excluding the possible formation of any high-
(
blue) in air saturated pH 7 buffer under aerobic conditions at a
IV
−1
valent Fe O species where a 25−30 cm downshift is
constant rotation rate of 200 rpm. The difference spectra are shown in
18
IV
generally observed on O substitution (Fe O is also
green. The ν and ν bands of the spectra obtained during steady state
4
2
excluded based on the ferrocyanide experiment discussed
O reduction of complex 1, 2, and 3 along with their Lorentzian fits
2
58
showing different components are given in (D), (E), and (F),
respectively.
above).
For complex 3 with imidazole “base”, a prominent peak at 847
−1
cm appears, under steady state conditions in an air saturated
pH 7 buffer, in the SERRS-RDE data which is shown to shift to
FeIII species (very low population in steady state) with the ν
−1
−1 16/18
18
4
786 cm (61 cm
O downshift) in O (Figure 7B).
2
2
−1
16/18
and ν bands at 1359 and 1555 cm , respectively (Figure 6E).
Note that the magnitudes of the
O isotope shifts (>60
2
−1
SERRS-RDE data for 3 during steady state O reduction shows
cm ) for both the complexes are larger than the calculated
2
III
II
II
the presence of LS Fe , LS Fe , and HS Fe species (Figure 6C,
blue). The major component is the LS Fe species with the ν
and ν bands at 1368 and 1569 cm (Figure 6E). Fits to the ν4
values for the harmonic O−O diatomic oscillators which is likely
III
4
due to the mixing or mode-coupling of the fundamental O−O
−1
16/18
2
stretching mode with porphyrin vibrations. A weak
O
2
and ν region clearly show the presence of bands at 1345 and
sensitive ν
band could be identified for complex 3 at 545
2
Fe−O
−1
II
−1
−1
−1
18
1
544 cm corresponding to a HS Fe species, whereas the
bands at 1357 and 1559 cm correspond to a LS Fe species
Figure 6E). Note that a larger intensity of the LS Fe species
cm , which shifts to 524 cm (21 cm downshift) upon O-
substitution (Figure S8B, Supporting Information). Although
another weak signal at 532 cm is observed for the same
−1
II
4
2
II
−1
(
16
18
is observed relative to the HS component that can be due to the
higher resonance enhancement and/or a greater population, the
latter being more likely.
complex 3 in O , the shift in O is not detectable. Here too,
2 2
IV
no high-valent Fe O intermediates could be detected when
II
carrying out the same K [Fe (CN) ] assay (Figure S6B,
4
6
SERRS-RDE data for complex 2 in the low frequency region
shows the formation of an iron-peroxo intermediate (either
bridged or terminal), which gives rise to an O−O stretching
Supporting Information). This relatively high energy of νO−O
in complex 3 compared to complex 2 is likely due to the
formation of an end-on (μ-1,2) bridged peroxo complex. This is
because of the presence of a stronger σ-donor imidazole ligand
in complex 3 which facilitates the formation of an end-on
bridging peroxo moiety and not a side-on coordination, resulting
in lower νO−O stretching vibrations, as also known from previous
studies on similar CcO model complexes in organic
−1
16
−1
vibration (ν ) at 819 cm in O , shifting to 759 cm in
O−O
2
1
8
O (Figure 7A). Unlike for simple Fe-porphyrin systems, a
2
IV
IV
Fe O vibration was not observed. High-valent Fe O
systems produced during O reduction have been shown to be
2
II
able to oxidize K [Fe (CN) ] present in solution and the
4
6
III
−3
32,59,60
[
Fe (CN) ] produced can be detected in the Pt ring in a
media.
6
E
DOI: 10.1021/jacs.5b06513
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
DISCUSSION
Scheme 1. Plausible Mechanistic Pathways during Steady
■
State O -Reduction by Complex 2
2
A combination of electrochemical and spectroscopic techniques
has been used to investigate the oxygen reactivity of CcO model
complexes (i.e., a heme-Cu system with and without imidazole)
physiabsorbed on electrode surfaces under physiological
conditions. The parent heme-Cu L-FeCu complex (2)
selectively reduces O by a 4e /4H process under fast ET
6
−
+
2
conditions (only 6% PROS) with a high overall k for the ORR
cat
6
−1 −1
(
4.49 × 10 M s ), that being 1 order of magnitude higher
6,28
than any other synthetic heme-Cu system. The SERRS-RDE
data obtained on this complex under steady state conditions
II
III
III
reveal the presence of HS Fe , HS Fe and LS Fe species. The
II
fact that the Fe center is in its HS state in the reduced complex
and was established to be in its LS Fe state in previously
II
2
reported synthetic mimics of CcO likely explains the faster ORR
49
kinetics observed in the former relative to the latter. The
−1
additional vibration observed at 819 cm which shifts to 759
−1
18
cm in O but does not shift on deuteration indicates that (a)
2
a bridging peroxide is involved in the mechanism and (b) the
O−O bond cleavage of this peroxy species is likely to be a slow
61
step in the mass transfer limited region (vide infra). By
contrast, the imidazole bound analogue of the parent heme-Cu
6
6
L-FeCu complex, L-Fe(Im)Cu (imidazole bound trans to the
Scheme 2. Plausible Mechanistic Pathways during Steady
bridging hydroxo ligand in the resting oxidized state), produces
State O -Reduction by Complex 3
2
significant amounts of PROS (25%) during O reduction on an
2
EPG electrode. SERRS-RDE data on this complex shows the
1
6/18
−1
presence of a
O sensitive O−O vibration at 847 cm (786
−
1
−1
−1
cm ) and Fe−O vibration at 545 cm (524 cm ). Thus, a
bridging peroxide ligand is an intermediate in the O reduction
2
cycle even with an imidazole axial ligand and the heterolytic O−
O bond cleavage is a slow step as well. Note that the presence of
end-on peroxide species has been demonstrated in X-ray
20,21,62
structures of CcO
and their involvement in O reduction
2
has been proposed for “mixed valence” CcO (i.e., where just the
13
FeCu active site is reduced).
In the SERRS-RDE experiments, species for which the rate of
formation is faster than its rate of decay will accumulate and can
be detected. Analyses of marker bands indicate the accumulation
II
II
III
of Fe HS, Fe LS and Fe LS in the case of Fe−Cu complexes.
SERRS-RDE is performed at a potential where the catalytic
current is mass transfer limited, i.e., no change in the catalytic
current occurs with an increase in the driving force and the
current only depends on the supply of species from the aqueous
phase. This implies that no species whose decay involves an
electron transfer can be rate determining and accumulate during
steady state at these potentials. This automatically excludes the
possibility that an electron transfer step is the rate-determining
(
from bulk) with subsequent O−O bond cleavage leading to the
formation of high-valent compound I (d, Schemes 1 and 2).
However, compound I cannot be isolated as an intermediate
during steady state, perhaps because of the immediate reduction
of this high valent species by electron transfer from the electrode
which is held at −0.5 V vs Ag/AgCl during SERRS-RDE
experiments i.e. its decay is faster than its formation precluding
its accumulation during steady state. Thus, the data indicate that
both O binding and heterolytic cleavage of O−O bond are
slow. However, from the greater intensity LS Fe component in
the ν and ν region relative Fe component, we propose that
decay of the bridging peroxide species via protonation is slower
step. The two components of the reaction (reduction of O to
2
H O) that diffuse from bulk solution to electrode surface are
2
II
oxygen and protons. Consistently, it is observed that Fe species
(
both HS and LS), which decay by binding oxygen (derived
from bulk solvent), accumulate in the SERRS-RDE data. This is
2
II
III
because of the formation of Fe (a, Schemes 1 and 2) through
III
II
reduction of Fe (via ET from electrode) is faster at these
4
2
potentials than O binding. While O binding in the distal
2
2
II
pocket (i.e., containing the Cu) will lead to O−O cleavage and
than decay of Fe resulting from O binding.
2
subsequent O reduction, O binding to the proximal site,
The relative values of the νO−O suggest that the peroxo
2
2
6
replacing the axial water ligand, will likely result in the
intermediate formed under steady state conditions in the L-
production of PROS (c, Scheme 1). Similarly, we believe the
Fe LS species observed is a bridging peroxide (b, Scheme 1
FeCu complex has a side-on coordination, whereas the one
formed in the L-Fe(Im)Cu analogue is bound in an end-on
III
6
and c, Scheme 2) (the metal ligand region in the SERRS-RDE
spectra also reflects the same) because its decay involves proton
geometry. These assignments are in good agreement with the
previously reported homogeneous data where similar shifts are
F
DOI: 10.1021/jacs.5b06513
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
3
1,32,59
observed upon switching a side-on to end-on intermediate.
Instruments (model CHI710D Electrochemical Analyzer). A Bio-
potentiostat, reference electrodes and a Teflon plate material evaluating
cell (ALS Japan) were purchased from CH Instruments. The rotating
ring disk electrochemical (RRDE) setup from Pine Research
Instrumentation (E6 series ChangeDisk tips with AFE6M rotor) was
used to obtain the RRDE data. Surface Enhanced Resonance Raman
data were collected with a Trivista 555 spectrograph (Princeton
6
Considering the fact that the peroxo adduct of the L-FeCu
complex is 6C LS (based on the ν and ν values), the binding
motif of the bridging side-on peroxo to the Fe and Cu centers is
4
2
1
2
2
1
likely μ−η :η (η at Cu and η at Fe; b, Scheme 1) in an
2
1
aqueous environment and not μ−η :η as observed in organic
3
1,32,59
solvents.
The ν
observed in the SERRS-RDE data,
+
O−O
Instruments) using 413.1 nm excitation from a Kr laser (Coherent,
−1
here, in an aqueous environment, is ∼30 cm higher than the
Sabre Innova SBRC-DBW-K). Mass spectra were recorded by a QTOF
Micro YA263 instrument.
values observed in structurally characterized high-spin Fe−
2
1
6
(
μ−η :η -peroxo)−Cu model compounds in organic solu-
Synthesis. The heme-(μ-hydroxo)-copper complex L-FeCu (2)
3
1,33
6
tion.
These differences may arise from differences in
and its copper free heme analogue L-Fe (1) were synthesized as
6
7,68
6
6
reported in the literature.
The imidazole adduct of L-FeCu, L-
polarity of the medium or hydrogen bonding from water, or
due to the change from high-spin to low-spin, or of course due
to proposed change to being side-on to Cu (Scheme 1).
Complex 3 shows selective 4e /4H O reduction under slow
ET flux (<4% PROS when physiabsorbed on C SH modified
SAM on Au) whereas under fast ET, where complex 2 and all
known heme-Cu complexes show 4e /4H O reduction, this
complex with an axial imidazole ligand shows significant 2e /
H reduction of O . SERRS-RDE data on this complex reveals
accumulation of LS Fe species; presumably with a water
molecule as the sixth axial ligand (a, Scheme 2). Ferrous
porphyrins in the LS ground state are known to have sluggish
It is likely that under fast ET on EPG
i.e., when kET > kO2 for the LS Fe center), the distal Cu
reduces O to produce PROS (b, Scheme 2). As the ET rate is
slowed and becomes comparable to or even slower than the O2
binding rate to LS Fe (i.e., kET ≈ k or kET < k ), the O2
slowly) binds to iron and the 4e /4H ORR proceeds via a
Fe(Im)Cu (3), was prepared by adding imidazole dissolved in CHCl3
to a 1 mM solution of 2 in CHCl . The formation of the complex was
3
monitored by UV−vis (Figure S1B, Supporting Information) and TOF
MS ES+ (Figure S1C) and was also confirmed by rR spectroscopy
(Figure S1A). Note that since imidazole has been added to the complex
−
+
2
1
6
2
(as synthesized) in a noncoordinating organic medium, it binds to the
−
+
2
open face of the heme as a sixth ligand (i.e., trans to the bridging
hydroxo moiety, see Figure 2).
−
+
2
Construction of Electrodes. Formation of Self-Assembled
Monolayer (SAM). Au wafers and disks were cleaned electrochemically
by sweeping several times between 1.5 V to −0.3 V (vs. Ag/AgCl) in
2
II
0
.5 M H SO . Ag disks were cleaned in alumina (size: 1, 0.3 and 0.05
2 4
μm) and then roughened in 0.1 M KCl solution as described in
literature. SAM solutions were prepared in ethanol using 0.4 mM
concentration of the corresponding alkanethiols. Freshly cleaned Au
wafers, disks and freshly roughened Ag disks were initially rinsed with
40,47
rates of O binding.
2
II
(
2
tripled distilled water, ethanol, purged with N gas and then immersed
2
II
in the depositing solution of C SH or C SH in ethanol for 4 and 20 h,
O2
O2
8
16
−
+
(
respectively.
Physiabsorption of Catalysts on to EPG and SAM. EPG disks were
cleaned on silicon carbide (SiC) grit paper followed by sonication in
bridging peroxo intermediate (c, Scheme 2). The ferrous center
in its LS state may also reduce O via outer sphere O reduction
2
2
−
51,63,64
ethanol and dried under N gas. The disks were then mounted on a
2
by 1e when the ET rate is fast.
Thus, the product, i.e.,
platinum ring disk assembly (Pine Instruments). The solutions of the
catalysts were prepared in chloroform followed by drop-casting on the
EPG electrode for 30 min for complete loading. The surfaces were then
thoroughly rinsed with chloroform followed by ethanol and triply
distilled deionized water and dried before CV, RDE and RRDE
experiments. In case of modified Au and roughened Ag disks, the disks
were taken out of the depositing solutions, rinsed with ethanol and
chloroform and then mounted on the RRDE setup. The catalysts were
loaded as described for EPG. After each respective loading, the surfaces
were thoroughly rinsed with chloroform, ethanol and triply distilled
water before carrying out electrochemical or SERRS/SERRS-RDE
experiments.
water, can bind the heme iron in its reduced state and act as a
47,65,66
competitive inhibitor of the substrate, O2.
For facile O2
reduction, it is important that the water formed is released from
the active site; this effect may also be operative in the native CcO
3
,65
enzyme active site through proper water channelling.
CONCLUSION
■
In summary, bridging peroxo intermediates are shown to be
involved during electrocatalytic O -reduction in aqueous media
2
by heme-Cu complexes which are otherwise known to form
2−
such Fe−O2 −Cu species in organic solvents. The kinetics of
Electrochemical Experiments. All electrochemical experiments
were carried out in pH 7 buffer (unless otherwise mentioned)
containing 100 mM Na HPO .2H O and 100 mM KPF (supporting
O -reduction compare fairly well with those of the heme−Cu
2
complexes that are proposed not to involve a bridging peroxide
type intermediates. Thus, pathways involving bridging peroxide
2
4
2
6
electrolyte) using Pt wire as the counter electrode and Ag/AgCl as the
reference electrode.
−
+
intermediates can lead to facile and selective 4e /4H O -
2
Cyclic Voltammetry. The CVs of the catalysts were obtained by
physiabsorbing the complexes on EPG electrodes under anaerobic
conditions in deoxygenated buffer at a scan rate of 50 mV/s.
Coverage Calculation. The coverage for a particular redox couple
is estimated by taking the average of the integrated area under the
corresponding oxidation and reduction currents of the respective
species obtained from their reversible voltammogram. The experiments
were repeated three times and an average value with standard deviation
has been presented.
Ring Disk Electrochemistry (RDE). The RDE measurements were
performed on a CHI 700D bipotentiostat with a Pine Instruments
Modulated speed rotor fitted with an E6 series Changedisk tip. The
complexes were physiabsorbed on the disks as described earlier. The
Koutecky−Levich (K−L) experiments were performed for all the
complexes at the following rotations: 100, 200, 300, 400, 500, and 600
reduction.
EXPERIMENTAL DETAILS
Materials. All reagents were of the highest grade commercially
available and were used without further purification. Octanethiol
■
(
C SH), hexadecanethiol (C SH), potassium hexafluorophosphate
8
1
6
(
KPF ), chloroform (CHCl ) and all buffers were purchased from
6
3
Sigma-Aldrich. Disodium hydrogen phosphate dihydrate (Na HPO ·
2
4
H O), potassium chloride (KCl), imidazole (Im), conc. sulfuric acid
2 4 2
2
wafers were obtained from Platypus Technologies (1000 Å of Au on 50
Å of Ti adhesion layer on top of a Si(III) surface). Edge Plane Graphite
(
EPG), Au disks for the RRDE experiments and Ag disks for SERRS
experiments were purchased from Pine Instruments.
Instrumentation. UV−vis absorption data were taken in an Agilent
technologies spectrophotometer model 8453 fitted with a diode-array
detector. All electrochemical experiments were performed using a CH
rpm. The second order rate of catalysis (k ) can be evaluated from the
cat
intercept of the K−L plot (see Figure 4) using the equation i
=
K
nFA[O ]k Γ
where i is expressed as the inverse of the intercept
K
2
cat catalyst
G
DOI: 10.1021/jacs.5b06513
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
−
1
obtained upon plotting I at different rotation rates with respect to
ACKNOWLEDGMENTS
−1/2
■
ω
and Γ
is the surface coverage of the catalyst obtained from
catalyst
The authors sincerely thank SB/S1/IC-25/2013 (A.D.),
Department of Science and Technology, Govt. of India for
funding this research. S.C. and K.S. acknowledge CSIR, India,
for Senior Research Fellowships. S.H. and K.D.K. acknowledge
the National Institutes of Health (USA) for financial support.
the integrated current of the CV taken under anaerobic conditions.
Rotating Ring Disk Electrochemistry (RRDE): Partially
Reduced Oxygen Species (PROS) Detection and Calculation.
The platinum ring and the Au disk were both polished using alumina
powder (grit sizes: 1, 0.5, and 0.03 μm) and electrochemically cleaned
and inserted into the RRDE tip which was then mounted on the rotor
and immersed into a cylindrical glass cell equipped with Ag/AgCl
reference and Pt counter electrodes (Figure 8A). In this technique, the
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b06513.
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between the Cu and both oxygen atoms of the bridging peroxides are
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show Figure 1A depicted with a side-on geometry.
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(
AUTHOR INFORMATION
Corresponding Authors
icad@iacs.res.in
karlin@jhu.edu
Notes
■
(
*
(
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J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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3
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(
II
the presence of minor LS Fe component as well having ν , ν , and ν
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(
58) However, the use of scrambled isotope would provide a definite
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I
DOI: 10.1021/jacs.5b06513
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX