Catalytic formation of hydrogen peroxide from coenzyme NADH and dioxygen with a water-soluble iridium complex and a ubiquinone coenzyme analogue

Article abstract of DOI:10.1021/acs.inorgchem.6b01220

A ubiquinone coenzyme analogue (Q₀: 2,3-dimethoxy-5-methyl-1,4-benzoquinone) was reduced by coenzyme NADH to yield the corresponding reduced form of Q₀ (Q₀H₂) in the presence of a catalytic amount of a [C,N] cyclometalated organoiridium complex (1: [Ir^III(Cp?)(4-(1H-pyrazol-1-yl-κN²)benzoic acid-κC³)(H₂O)]₂SO₄) in water at ambient temperature as observed in the respiratory chain complex I (Complex I). In the catalytic cycle, the reduction of 1 by NADH produces the corresponding iridium hydride complex that in turn reduces Q₀ to produce Q₀H₂. Q₀H₂ reduced dioxygen to yield hydrogen peroxide (H₂O₂) under slightly basic conditions. Catalytic generation of H₂O₂ was made possible in the reaction of O₂ with NADH as the functional expression of NADH oxidase in white blood cells utilizing the redox cycle of Q₀ as well as 1 for the first time in a nonenzymatic homogeneous reaction system.

Full text of DOI:10.1021/acs.inorgchem.6b01220

Article
pubs.acs.org/IC
Catalytic Formation of Hydrogen Peroxide from Coenzyme NADH
and Dioxygen with a Water-Soluble Iridium Complex and a
Ubiquinone Coenzyme Analogue
,
†
†
,†,‡,§
Tomoyoshi Suenobu,* Satoshi Shibata, and Shunichi Fukuzumi*
†Department of Material and Life Science, Graduate School of Engineering, Osaka University, ALCA and SENTAN, Japan Science
and Technology, Suita, Osaka 565-0871, Japan
‡
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea
§
Faculty of Science and Engineering, Meijo University, ALCA and SENTAN, Japan Science and Technology Agency, Nagoya, Aichi
468-0073, Japan
*
S Supporting Information
ABSTRACT: A ubiquinone coenzyme analogue (Q : 2,3-
0
dimethoxy-5-methyl-1,4-benzoquinone) was reduced by coen-
zyme NADH to yield the corresponding reduced form of Q0
(
Q H ) in the presence of a catalytic amount of a [C,N]
0 2
III
cyclometalated organoiridium complex (1: [Ir (Cp*)(4-(1H-
pyrazol-1-yl-κN )benzoic acid-κC )(H O)] SO ) in water at
2
3
2
2
4
ambient temperature as observed in the respiratory chain
complex I (Complex I). In the catalytic cycle, the reduction of
1
by NADH produces the corresponding iridium hydride
complex that in turn reduces Q to produce Q H . Q H
2
0
0
2
0
reduced dioxygen to yield hydrogen peroxide (H O ) under slightly basic conditions. Catalytic generation of H O was made
2
2
2
2
possible in the reaction of O with NADH as the functional expression of NADH oxidase in white blood cells utilizing the redox
2
cycle of Q as well as 1 for the first time in a nonenzymatic homogeneous reaction system.
0
INTRODUCTION
use of various spectroscopic methods in solution to provide
valuable mechanistic insight.
Complex I is responsible for the catalytic reaction expressed
■
NADH-coenzyme Q oxidoreductase so-called respiratory chain
Complex I” at inner mitochondria membrane of eukaryotes as
well as cell membrane of bacteria (prokaryotes) accepts
electrons from 1,4-dihydronicotinamide adenine dinucleotide
NADH) and passes electrons to ubiquinone coenzyme Q
“
by eq 1, where NADH is oxidized with ubiquinone (UQ) to
+
+
NADH + H + UQ + 4H
in
(
(
1
,2
+
+
UQ) to generate ubiquinol (UQH2). UQH is known to
carry the electrons to the next complex (Complex III,
→ NAD + UQH + 4H
2
out
(1)
2
cytochrome bc complex) through the supercomplex formation
between Complex I and Complex III. UQH2 is also
produce the corresponding oxidized form of NADH, that is,
1
3
NAD+ and the reduced form of UQ, that is, UQH
10,11
2
transported from Complex II (succinate-coenzyme Q reduc-
tase) that catalyzes the oxidation of succinate to fumarate with
(ubiquinol).
The transportation of two electrons from
NADH to UQ is known to proceed via intervening Fe−S
4
,5
the reduction of UQ to UQH2. Inside the mitochondria
membrane, UQH2 exists together with UQ in so-called
clusters and cytochromes with being associated with trans-
portation of four protons from inside (H in) to outside (H+out
+
)
12,13
6
across the membrane.
Besides four protons transported
through membrane, another one proton (H ) would be
ubiquinone pool (Q-pool) being relatively mobile. UQ is
+
also synthesized in the endoplasmic reticulum and Golgi
membrane system. In these nonmitochondrial membranes,
consumed to form UQH in the overall stoichiometric reaction
2
in eq 1, indicating that the reaction represented by eq 1 might
be acid-promoted as suggested by the electrochemical studies
on the proton-coupled electron-transfer reduction of a
UQH functions as an antioxidant, which is essential for the
2
7
defensive system against oxidative stress in tissues. Actually,
UQ was found to be an effective antioxidant for the medical
14
8
ubiquinone coenzyme analogue. In this context, Complex I-
dependent reduction of UQ has so far been examined in terms
of the effect of the surrounding amino acid residues on the
treatment of neurodegenerative diseases. In this context,
mechanistic insight into oxidation of UQH and its analogues
2
with molecular oxygen has been studied with respect to
9
autoxidation. However, the antioxidant effect of UQ on
autoxidation of UQH has yet to be revealed in a homogeneous
Received: May 19, 2016
2
reaction system, where reaction kinetics can be analyzed with
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.6b01220
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Inorganic Chemistry
Article
redox cofactors in various enzymatic aqueous reaction
systems.
otherwise noted. UV−vis absorption spectra were recorded on a
Hewlett−Packard 8453 diode array spectrophotometer with a quartz
cuvette (light-path length = 1 cm) at 298 K. The mixed gas was
controlled by using a gas mixer (Kofloc GB-3C, KOJIMA Instrument
Inc.), which can mix two or more gases at a defined partial pressure
1
5
However, in nonenzymatic reaction systems mimicking the
function of Complex I, reduction of various substituted 1,4-
benzoquinones as a model of UQ with NADH analogues has so
far been investigated with regard to the proton-coupled
and mass flow rate. A defined concentration of O in an aqueous
2
solution was prepared by a mixed gas flow of O and N controlled by
2
2
16−18
electron transfer in both aqueous and organic solvents.
using a gas flow meter (KOJIMA Instrument Inc.) appropriate for each
−
1
1
However, there has so far been no report on the reaction
between an NADH analogue and a UQ analogue bearing both
dimethoxy and methyl substituents, since the strong electron-
donating effect of these substituents has prohibited the
reduction of UQ with NADH even under the acidic conditions.
Furthermore, NADH is known to be unstable in an acidic
medium at room temperature. Thus, the appropriate range of
gas under normal pressure (1.0 × 10 MPa). The H NMR spectra
were recorded on JEOL JNM-AL300 spectrometer and Varian UNITY
INOVA600. The pH values were determined by a pH meter (TOA,
HM-20J) equipped with a pH combination electrode (TOA, GST-
5
725C). The pH of the solution was adjusted by using 1.00−10.0 M
NaOH/H O without buffer unless otherwise noted.
2
Chemicals. Chemicals were purchased from commercial source
and used without purification, unless otherwise noted. A water-soluble
iridium complex 1 was synthesized and characterized as reported
19
pH for the use of NADH is limited at pH > 7. The extremely
2
9,31
III
low aqueous solubility of UQ as well as UQH has also
previously.
An aqueous solution (50 mL) of [Ir (Cp*)(H O) ]-
2
2 3
precluded further exploration of their redox reactivity in a
(SO ) (0.20 g, 0.423 mmol) and 4-(1H-pyrazol-1-yl)benzoic acid
4
homogeneous reaction system irrespective of the number (n =
(0.085 g, 0.454 mmol) was stirred under reflux for 12 h. The reaction
solution was filtered with a membrane filter (Toyo Roshi Kaisha, Ltd.,
H100A025A; pore diameter, 1 μm). The filtrate was evaporated under
reduced pressure to yield a yellow powder of 1 and was dried in vacuo.
6−10) of isoprenoid subunits in their side chain (Chart 1).
Chart 1. Ir Aqua Complex 1, the Hydride Complex 2,
NADH, UQ, and 2,3-Dimethoxy-5-methyl-1,4-benzoquinone
2
,3-Dimethoxy-5-methyl-1,4-benzoquinone (>97.0%) was purchased
from Wako Chemical, Ltd. 2,3-Dimethoxy-5-methylhydroquinone
(>97.0%) was purchased from Astatech, Inc. β-Nicotinamide adenine
dinucleotide disodium salt hydrate, reduced form, was purchased from
Tokyo Chemical Industry Co., Ltd. Oxo[5,10,15,20-tetra(4-pyridyl)-
porphinato]titanium(IV) ([TiO(tpyp)]) was supplied from Tokyo
(
Q0)
Chemical Industry Co., Ltd. (TCI). H (99.99%; Japan Air Gases Co.)
2
and O (99.5%; Sumitomo Seika Chemicals Co., Ltd.) gases were used
2
without further purification. Purification of water (18.2 MΩ cm) was
performed with a Milli-Q system (Millipore; Direct-Q 3 UV).
Detection of Hydrogen Peroxide. The amount of produced
hydrogen peroxide was determined by spectroscopic titration with an
4+
32
acidic solution of [TiO(tpypH )] complex (Ti-TPyP reagent). The
4
Ti-TPyP reagent was prepared by dissolving 34.03 mg of the
[
TiO(tpyp)] complex in 1000 mL of 50 mM hydrochloric acid. A
small portion (100 mL) of the reaction solution was sampled and
diluted with water. To 0.25 mL of the diluted sample, 0.25 mL of 4.8
M perchloric acid and 0.25 mL of the Ti-TPyP reagent were added.
The mixed solution was then allowed to stand for 5 min at room
temperature. This sample solution was diluted to 2.5 mL with water
Complex I by itself independent of other respiratory chain
complexes is also responsible for the reduction of O by NADH
2
•
−
to form reactive oxygen species such as superoxide (O2 ) and
hydrogen peroxide (H O ) as an expression of NADH oxidase
2
2
and used for the spectroscopic measurement. The absorbance (A ) at
20,21
S
function of white blood cells.
However, there has so far
λ = 434 nm was measured by using a Hewlett-Packard 8453 diode
array spectrophotometer. A blank solution was prepared in a similar
manner by adding distilled water instead of the sample solution in the
been no report on the reaction of O with NADH catalyzed by
2
a ubiquinone coenzyme analogue (UQ) to selectively form
H O , although O was selectively reduced by 1,4-hydro-
same volume with its absorbance designated as A . The difference in
2
2
2
B
quinones via the autocatalytic production of H O in
absorbance was determined as follows: ΔA434 = A
B
− A . On the basis
S
2
2
22
of ΔA and the volume of the solution, the amount of hydrogen
nonenzymatic reaction system. H O has merited increasing
434
2
2
3
2
peroxide was determined according to the literature.
attention as an attractive clean energy and is expected to be
23−28
pH Adjustment. The pH values of the solutions were determined
by a pH meter (TOA, HM-20J) equipped with a pH combination
electrode (TOA, GST-5725C). The pH of solution was adjusted by
utilized in H O fuel cell.
2
2
We report herein the successful reduction of 2,3-dimethoxy-
5
-methyl-1,4-benzoquinone (Q ) as a ubiquinone analogue
0
using 1.00−10.0 M NaOH/H O without buffer unless otherwise
2
with NADH to produce the corresponding ubiquinol (Q H )
0
2
noted.
in water by using a water-soluble iridium aqua complex
III
2
3
[
(
Ir (Cp*)(4-(1H-pyrazol-1-yl-κN )benzoic acid-κC )-
RESULTS AND DISCUSSION
■
H O)] SO [1] ·SO , which can react with NADH to produce
2
2
4
2
4
29
an Ir-hydride complex (2). The coenzyme analogue Q is
The synthesis and characterization of 1 were performed
0
soluble up to ∼4.0 mM in water at pH 7 in contrast to water-
according to the literature and are briefly described in the
30
29,31
insoluble ubiquinone coenzyme Q . Moreover, the catalytic
Experimental Section.
The carboxylic acid group in 1 is
1
0
+
reduction of dioxygen by NADH was made possible to
selectively generate hydrogen peroxide in the presence of
deprotonated to give the carboxylate form 1-H as shown in eq
2 at pH > 7.0, since pK of 1 was determined to be 4.0 (eq
a
2
9,31
[
1] ·SO and a ubiquinone coenzyme analogue, Q .
2).
2
4
0
The characteristic absorption band of the UV−vis absorption
EXPERIMENTAL SECTION
General Methods. All experiments were performed under an Ar
or N2 atmosphere by using standard Schlenk techniques unless
spectrum of NADH at λ = 260 and 340 nm and that of 2,3-
■
max
dimethoxy-5-methyl-1,4-benzoquinone (Q ) at λmax = 269 nm
0
gradually decreased, and finally the new absorption band at λmax
B
DOI: 10.1021/acs.inorgchem.6b01220
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Inorganic Chemistry
Article
+
=
260 nm assignable to NAD appeared in the presence of a
33
+
catalytic amount of 1-H at pH 7.0, as shown in Figure 1. In
1
Figure 2. H NMR spectra in the reaction of Q (11.8 mM) with
0
NADH (12.3 mM) catalyzed by 1 (0.10 mM) for 10 min in a
Figure 1. Changes in the UV−vis absorption spectra observed at t = 0
black line) and 10 min (red line) in the reaction of Q (25 μM) with
NADH (25 μM) in the presence of 1 (2.5 μM) in an aqueous
phosphate buffer (2.0 mL, pH 7.0) with stirring under argon at 298 K
after addition of an aliquot of the solution of 1 (50 μL, 0.10 mM)
injected by a syringe to the solution at t = 0.
deaerated aqueous phosphate buffer solution (pH 8.0) at 298 K. (a)
H NMR spectral of the authentic sample of NADH (12.3 mM) and
reaction solution after 10 min; consumption of NADH. (b) The
1
(
0
conversion from Q (δ = 2.01 ppm) to Q H (δ = 2.08 ppm). (c) The
0 0 2
+
formation of NAD (δ ≈ 8.0−9.5 ppm).
3
5,36
conversion from Q0 to Q H (Figure 2a,b).
The
0
2
+
simultaneous formation of NAD clearly yields the character-
contrast, there was no change in the UV−vis absorption spectra
in the absence of 1 under otherwise the same experimental
conditions (Figure S1 in Supporting Information). No
29,37
istic peaks at δ ≈ 8.0−9.5 ppm in Figure 2c.
+
1
-H was converted to the iridium(III) hydride complex 2 by
formation of semiquinone radical anion (Q0• ) at λ = 320
−
the reduction with NADH in water at pH 8.8 as reported
max
29
3
4
previously (eq 4). The pK value of the protonated 2 may be
and 450 nm was confirmed throughout the reaction. These
results indicate that 1-H catalyzes the reduction of Q with
a
+
0
NADH to form 2,3-dimethoxy-5-methylhydroquinone (Q H )
0
2
+
and NAD as expressed by eq 3 and Scheme 1.
similar to that of 1 (4.0) because the pK value is similar to that
a
38
of benzoic acid (4.19). The formation of 2 was detected by
H NMR and electrospray ionization mass spectrometry. The
hydride complex 2 was also produced in the reaction of 1-H
with hydrogen (H ) as reported previously (eq 5).
1
The progress of the reaction was also monitored by H NMR
as shown in Figure 2. The disappearance of the peak at δ = 6.91
ppm was due to the consumption of NADH, and the new peak
appeared at δ = 2.08 ppm that corresponds to a methyl group
1
29
+
29,31
2
(
−CH ) of Q H at 5 position indicating the stoichiometric
3
0
2
Scheme 1. Catalytic Cycle for the Reduction of Q by NADH
0
with 1
The resulting 2 reduced Q (λ = 269 and 412 nm) to
0
max
produce 2,3-dimethoxy-5-methylhydroquinone (Q H : λ =
0
2
max
2
85 nm) in water (pH 7.0) at room temperature as expressed
1
by eq 6 that was confirmed by UV−vis and H NMR. Thus, Q
0
C
DOI: 10.1021/acs.inorgchem.6b01220
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Inorganic Chemistry
Article
was catalytically reduced with H to yield Q H in the presence
2
0
2
+
of 1-H as shown by Figure 3. The characteristic absorption
Figure 3. Changes in the UV−vis absorption spectrum during the
reduction of Q (50 μM) with H (bubbling for 5 min) by using 1 (25
0
2
μM) in water (pH 7.0) at 298 K. An argon-saturated reaction solution
−
1
of 1 and Q (black line) was bubbled with H (1.0 × 10 MPa) for 5
0
2
min thus resulting in the formation of Q H (red line).
0
2
band due to Q at λ = 269 nm gradually disappears, and the
0
max
new absorption bands appear at λmax = 285 and 254 nm
assignable to QH and 2, respectively. The stoichiometry of the
2
reaction is determined as given by eq 7 based on the extinction
4
−1
−1
coefficients of Q at λ = 269 nm (ε = 1.5 × 10 M cm )
0
max
3
−1
−1 39
and Q H at λ = 285 nm (ε = 4.1 × 10 M cm ).
0
2
max
Figure 4. (a) UV−vis spectral changes of Q H by the addition of
0
2
NaOH in deaerated H O at pH 7.0 (black line), pH 10.8 (blue line),
2
and pH 12.4 (red line) at 298 K. (b) Change in absorbance at λ = 311
nm by the addition of 0.10−5.0 M NaOH in deaerated H O at 298 K.
2
When the pH of a Q H solution was increased from pH 7.0
0
2
to 10.8 by adding an aliquot of 0.1−5.0 M NaOH solution
several times, a new absorption band at λ = 307 nm appeared
max
with an isosbestic point at λ = 289 nm as shown in Figure 4a,
−
indicating the formation of deprotonated anion (Q H ) in eq
0
8. Further addition of NaOH to the solution resulted in the rise
of new absorption bands due to the corresponding dianion
2
−
(
Q0 ; eq 8), that is, doubly deprotonated Q H , with the shift
0 2
of the absorption maxima from λmax = 307 to 311 nm. From the
UV−vis absorption spectral titration in Figure 4b, the pK
Figure 5. Changes in the UV−vis absorption spectra during the
oxidation of Q H (30 μM) by O in an O -saturated aqueous borate
a
0
2
2
2
values of Q H were determined to be pK = 9.90 and pK =
buffer solution (pH 8.0) at 298 K at t = 0−10 min detected every 50 s.
0
2
a1
a1
1
1.4, respectively. These pK values are similar to those of
a
40
A 25 μL aliquot of a solution of Q H (2.0 mM) was injected into 2.0
0
2
hydroquinone.
mL of an O -saturated aqueous borate buffer solution (pH 8.0) at 298
2
K.
Although Q H is stable in neutral water under air for a few
0
2
minutes, Q H is gradually oxidized by O (eq 9) in water at
0
2
2
became much faster with increasing pH as observed in Figure 6
and Figure S2 in Supporting Information. With the progress of
the reaction, the characteristic UV−vis absorption band of
Q H at λ = 285 nm changes gradually to that of Q at λ =
0
2
max
0
max
2
69 nm, exhibiting the isosbestic points at λ = 232 and 289 nm
(
Figure 5). The amount of H O produced in this reaction
2
2
various pH as observed by UV−vis absorption spectral change
determined by the spectral titration with use of the oxo-
[5,10,15,20-tetra(4-pyridyl)porphyrinato]titanium(IV) com-
in Figure 5. The rate of the reaction to generate Q and H O
2
0
2
D
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Inorganic Chemistry
Article
solution at various pH (8.0, 9.0, and 10.4) is shown in Figure 8.
At pH 8.0, H O was generated efficiently. When 1 (1.0 μM),
2
2
Figure 6. Time course of formation of Q in the reaction of Q H
2
0
0
(
100 μM) with O (1.4 mM) in aqueous phosphate buffer at 298 K at
2
various pH values (pH 6.0, 7.0, and 8.0; black ●, blue ▲, and red ■,
respectively).
Figure 8. Time course of H O production by the reaction of NADH
2
2
3
2
plex in water (see Experimental Section) agrees well with that
of Q H consumed according to eq 9 based on the extinction
(
0.20 mM) with O (1.4 mM) catalyzed by 1 (5.0 μM) and Q (50
2
0
0
2
μM) in aqueous borate buffer at 298 K at various pH values (pH 8.0,
3
−1
−1
9.0, and 10.4; red ●, green ▲, and blue ■
, respectively).
coefficients for Q H (ε = 1.9 × 10 M cm ) and Q (ε = 1.5
0
2
0
4
−1
−1
×
10
M
cm ). A linear relationship between the
concentrations of initial Q H and resulting H O was obtained
with a slope of 1.0 as shown in Figure 7. This indicates the
validity of the stoichiometry of the reaction in eq 9.
Q
0
(50 μM), and NADH (0.20 mM) were used, the turnover
0
2
2
2
number (TON) of H production with respect to 1 reached
O
2 2
+
54 at 40 min. Under more basic conditions, 1-H complex
released a proton from the aqua ligand to form the
corresponding hydroxo complex 3, leading to substitution
inertness at the Ir metal center of the iridium complex 1. The
29
pK value was determined to be 9.5 (eq 10). The reactivity of
a
Q H for reduction of O increased with increasing pH (Figure
0
2
2
6
), whereas the iridium complex is deactivated by the
deprotonation from the H O ligand (eq 10). In such a case
2
Figure 7. Plot of the concentration of H O produced by the catalytic
pH 8.0 was found to be suitable for the catalytic H O
formation from NADH and O by using 1 and Q .
2 0
2
2
2
2
−1
reduction of O (1.0 × 10 MPa) with Q H (50 μM−0.50 mM) at
2
0
2
298 K in an aqueous phosphate buffer solution (pH 8.0).
To determine the rate-determining step in the catalytic cycle,
dependence of r on concentrations of substrates were examined
in the catalytic generation of H O from NADH and O in the
2
2
2
By combining eqs 4, 6, and 9, the overall catalytic cycle for
presence of 1 and Q in an aqueous borate buffer (pH 8.0).
0
the selective H O generation in the reaction of NADH with O
2
2
2
When the concentrations of 1 and NADH were varied, the
by using 1 and Q as cocatalysts is shown in Scheme 2, where
0
+
rates of H O2 generation (r) were independent of the
2
1
-H reacts with NADH to produce the hydride 2, which
concentrations of 1 and NADH as shown in Figures 9 and
10, respectively (the time courses are shown in Figures S3 and
S4 in Supporting Information, respectively). These results
indicate that 1 and NADH are not involved in the rate-
determining step in the catalytic generation of H O .
reduces Q to Q H followed by reduction of O with Q H to
0
0
2
2
0
2
+
produce H O , accompanied by regeneration of 1-H and Q .
2
2
0
The generation of H O in the reaction of NADH (0.20
2
2
mM) with O (1.4 mM) in the presence of catalytic amount of
2
2
2
1
(5.0 μM) and Q (50 μM) in an aqueous borate buffer
0
In contrast, when the concentrations of Q and O were
0
2
varied, the rates of H O generation (r) are proportional to
2
2
Scheme 2. Overall Catalytic Cycle for the Selective H O
2
2
concentrations of Q and O as shown in Figures 11 and 12,
0
2
Generation from NADH and O with 1 and Q
2
0
respectively (the time courses are shown in Figures S5 and S6
in Supporting Information, respectively). Thus, the reaction
rate is expressed by eq 11, where
r = k[Q ][O ]
0
2
(11)
k is the second-order rate constant. The slope of the linear plot
of r versus the concentration of Q as shown in Figure 11
0
E
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Figure 12. Plot of the initial rate of H O2 production (r) vs
Figure 9. Plot of the initial rate of H O2 production (r) vs
2
2
concentration of O (0.35, 0.70, 1.05, and 1.40 mM) in the production
concentration of 1 in the production of H O from NADH (1.0
2
2
2
of H O from NADH (1.0 mM) and O catalyzed by 1 (5.0 μM) and
mM) and O catalyzed by 1 (5.0, 30, and 50 μM) and Q (50 μM) in
2
2
2
2
0
Q (50 μM) in an aqueous phosphate buffer (pH 8.0) at 298 K.
0
an O -saturated aqueous phosphate buffer (pH 8.0) at 298 K.
2
From kobs and fixed [Q ], k was determined to be 0.28 M−
1
0
−1
s . These two second-order rate constants obtained from
different plots agree well with each other, suggesting that the
rate-determining step in the overall catalytic H O production
2
2
is the reduction of O by Q H . The rate constant of formation
2
0
2
+
−1 −1
of 2 from 1-H with NADH was reported to be 44 M
s
at
98 K, which is much larger than the k value mentioned
above. Thus, the reaction kinetics of oxidation of Q H by O
2
29
2
0
2
(
eq 9) was examined in the absence of NADH (vide infra).
The spectral change during the oxidation of Q H by O was
0
2
2
shown in Figure 5. The time course of the reaction was
monitored by the increase in absorbance at λ = 269 nm due to
Q as shown in Figure 13, which exhibits a sigmoidal curve with
0
Figure 10. Plot of the initial rate of H O2 production (r) vs
2
concentration of NADH (0.30, 0.50, 1.00, and 1.66 mM) in the
production of H O from NADH and O catalyzed by 1 (5.0 μM) and
2
2
2
Q (50 μM) in an O -saturated aqueous phosphate buffer (pH 8.0) at
0
2
298 K.
Figure 13. Time course of change in absorbance at λ = 269 nm for the
formation of Q in the reduction of O (1.4 mM) with Q H (25 μM)
0
2
0
2
in the absence and presence of a catalytic and stoichiometric amount
of Q (0, 5.0, 10, and 25 μM; black, green, blue, and red, respectively)
0
in an aqueous borate buffer solution (pH 8.0) at 298 K. In this figure,
the amount of Q that is added into the solution was withheld.
an induction period. Interestingly, when a catalytic and
Figure 11. Plot of the initial rate of H O2 production (r) vs
2
stoichiometric amount of the product (Q ) was added to a
0
concentration of Q in the production of H O from NADH (1.0
0
2
2
reaction solution, the induction period disappeared, and the
rate of formation of Q0 was accelerated with increasing
mM) and O catalyzed by 1 (5.0 μM) and Q (25, 50, and 100 μM) in
2
0
an O -saturated aqueous phosphate buffer (pH 8.0) at 298 K.
2
concentration of Q (Figure 13 and Figure S7 in Supporting
0
Information). These results indicate that the oxidation of Q H
0
2
corresponds to k[O ] (= k ). On the one hand, from k and
by O proceeds in an autocatalytic fashion. In addition, there
2
obs
obs
2
−1 −1
fixed [O ], k was determined to be 0.28 M s . On the other
was no difference in the induction period when a stoichiometric
2
+
hand, the slope of the linear plot of r versus concentration of O2
in Figure 12 corresponds to k[Q ] (= k ).
amount of 1-H was added to this reaction solution (Figure 14
and Figure S8 in Supporting Information), indicating that the
0
obs
F
DOI: 10.1021/acs.inorgchem.6b01220
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
CONCLUSION
In conclusion, a water-soluble iridium(III) complex 1 catalyzes
the reduction of coenzyme Q as a ubiquinone analogue by
■
0
NADH in water at room temperature via the reduction of 1 by
NADH to produce the hydride complex 2, which reduces Q to
0
QH , acting as a functional mimic of respiratory chain complex
2
I. O was reduced by Q H via an autocatalytic pathway to
2
0
2
generate the stoichiometric amount of H O . Thus, the overall
2
2
catalytic reduction of dioxygen by NADH proceeds to generate
hydrogen peroxide in the presence of 1 and a ubiquinone
coenzyme analogue, Q , acting as cocatalysts (Scheme 2).
0
+
Because NADH is regenerated by the reduction of NAD by H
2
+
29
Figure 14. Time course of change in absorbance at λ = 269 nm for the
formation of Q in the reduction of O (1.4 mM) with Q H (25 μM)
with 1-H , H O can be produced by the reduction of O by
2
2
2
+
H using 1-H as an inorganic catalyst instead of an industrial
0
2
0
2
2
+
in the absence and presence of a stoichiometric amount of 1-H (0
and 25 μM; red and green, respectively) in an aqueous borate buffer
solution (pH 8.0) at 298 K.
anthranquinone method for the production of H O by H .
2
2
2
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.6b01220.
■
*
S
iridium complex has nothing to do with the oxidation of Q H2
0
by O . In addition, the rate of formation of H O by the
2
2
2
reduction of O (1.4 mM) by Q H (25 μM), which is
2
0
2
−8
−1
−1
calculated to be 1.0 × 10 M s using the k value (0.28 M
s ), agrees with the initial rate without Q in Figure 13 (1.2 ×
−
1
UV-vis absorption spectra, plot of H O concentration
2 2
0
−
8
−1
10
M s ).
versus time as O is reduced by NADH in presence of Q
2
0
The autocatalytic cycle for the oxidation of Q H by O may
catalyzed by 1, discussion of derived equations (PDF)
0
2
2
•
proceed as shown in Scheme 3, where Q H is formed in the
0
AUTHOR INFORMATION
Corresponding Authors
E-mail: fukuzumi@chem.eng.osaka-u.ac.jp. (S.F.)
E-mail: suenobu@chem.eng.osaka-u.ac.jp. (T.S.)
■
*
*
Scheme 3. Autocatalytic Radical Chain Reactions for
Oxidation of Q H by O
0
2
2
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by ALCA and SENTAN projects
from JST (to S.F.) and JSPS KAKENHI (Grant No. 16H02268
to S.F. and No. 16K05721 to T.S.), Japan.
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