One-step versus stepwise mechanism in protonated amino acid-promoted electron-transfer reduction of a quinone by electron donors and two-electron reduction by a dihydronicotinamide adenine dinucleotide analogue. Interplay between electron transfer and hyd

Article abstract of DOI:10.1021/ja8001452

Semiquinone radical anion of 1-(p-tolylsulfinyl)-2,5-benzoquinone (TolSQ^?-) forms a strong hydrogen bond with protonated histidine (TolSQ^?-/His·2H⁺), which was successfully detected by electron spin resonance. Strong hydro

Full text of DOI:10.1021/ja8001452

Published on Web 04/04/2008
One-Step versus Stepwise Mechanism in Protonated Amino
Acid-Promoted Electron-Transfer Reduction of a Quinone by
Electron Donors and Two-Electron Reduction by a
Dihydronicotinamide Adenine Dinucleotide Analogue.
Interplay between Electron Transfer and Hydrogen Bonding
Junpei Yuasa,† Shunsuke Yamada, and Shunichi Fukuzumi*
Department of Material and Life Science, DiVision of AdVanced Science and Biotechnology,
Graduate School of Engineering, Osaka UniVersity, SORST, Japan Science and Technology
Agency (JST), Suita, Osaka 565-0871, Japan
Received January 8, 2008; E-mail: fukuzumi@chem.eng.osaka-u.ac.jp
Abstract: Semiquinone radical anion of 1-(p-tolylsulfinyl)-2,5-benzoquinone (TolSQ•-) forms a strong
hydrogen bond with protonated histidine (TolSQ•-/His·2H+), which was successfully detected by electron
spin resonance. Strong hydrogen bonding between TolSQ•- and His ·2H+ results in acceleration of electron
transfer (ET) from ferrocenes [R2Fc, R ) C5H5, C5H4(n-Bu), C5H4Me] to TolSQ, when the one-electron
reduction potential of TolSQ is largely shifted to the positive direction in the presence of His·2H+. The
rates of His·2H+-promoted ET from R2Fc to TolSQ exhibit deuterium kinetic isotope effects due to partial
dissociation of the N-H bond in His·2H+ at the transition state, when His·2H+ is replaced by the deuterated
compound (His·2D+-d6). The observed deuterium kinetic isotope effect (kH/kD) decreases continuously
with increasing the driving force of ET to approach kH/kD ) 1.0. On the other hand, His ·2H+ also promotes
a hydride reduction of TolSQ by an NADH analogue, 9,10-dihydro-10-methylacridine (AcrH2). The hydride
reduction proceeds via the one-step hydride-transfer pathway. In such a case, a large deuterium kinetic
isotope effect is observed in the rate of the hydride transfer, when AcrH2 is replaced by the dideuterated
compound (AcrD2). In sharp contrast to this, no deuterium kinetic isotope effect is observed, when His ·2H+
is replaced by His·2D+-d6. On the other hand, direct protonation of TolSQ and 9,10-phenanthrenequinone
(PQ) also results in efficient reductions of TolSQH+ and PQH+ by AcrH2, respectively. In this case, however,
the hydride-transfer reactions occur via the ET pathway, that is, ET from AcrH2 to TolSQH+ and PQH+
•+
occurs in preference to direct hydride transfer from AcrH2 to TolSQH+ and PQH+, respectively. The AcrH2
produced by the ET oxidation of AcrH2 by TolSQH+ and PQH+ was directly detected by using a stopped-
flow technique.
Scheme 1
Introduction
Proton uptake and release often entail an improvement of
the driving force of electron transfer (ET) in biological redox
processes such as photosynthesis and respiration, which are
essential for life.1,2 When an electron acceptor (A) undergoes
protonation (AH+), the rate of ET is accelerated by the
protonation.3 Even when no protonation of A takes place, the
rate of ET is accelerated by protonation of the resulting radical
anion (A•-), which results in stabilization of the transition state
as well as the ET product (A•-).4 In such a case, the ET is
coupled with protonation of A•-, that is, one-electron reduction
and protonation of A occur at the same time (Scheme 1a green
arrow). On the other hand, the promoting effects of H+ on ET
are often regulated by Brønsted bases (:B) such as amino acid
residues in the protein environment.5–8 In such a case, A•- forms
† Present address: Graduate School of Materials Science, Nara Institute
of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192,
Japan.
(1) (a) The Photosynthetic Reaction Center; Deisenhofer, J., Norris, J. R.,
Eds.; Academic Press: San Diego, CA, 1993. (b) Anoxygenic
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C. E., Eds.; Kluwer Academic Publishing: Dordrecht, The Netherlands,
1995.
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Soc., Perkin Trans. 2 1987, 751. (b) Fukuzumi, S.; Mochizuki, S.;
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9
5808 J. AM. CHEM. SOC. 2008, 130, 5808–5820
10.1021/ja8001452 CCC: $40.75
2008 American Chemical Society

Electron-Transfer Reduction of a Quinone
A R T I C L E S
a hydrogen bond with H+:B instead of direct protonation, when
:B acts as the stronger base than A•- (Scheme 1b).9–13 The
interplay between ET and hydrogen bonding plays a crucial role
in biological redox systems, for example, specific hydrogen
bonds between nearby protonated amino acid residues and two
quinones (termed QA and QB) determines the direction of an
electron flow in photosynthetic reaction center.14,15
Scheme 2
One may regard such H+ and H+:B-promoted ET as proton-
coupled electron transfer (PCET). Although the precise defini-
tion of PCET has yet to be widely accepted,16 this term is often
applied to the mechanism in which the proton and electron are
transferred in one single kinetic step.17 It is therefore to be
contrasted with the stepwise pathway that involves mechanisti-
cally distinct ET and proton transfer (PT) steps. The one-step
pathway is generally thermodynamically more favorable than
the stepwise pathway, because the one-step pathway avoids
high-energy intermediates through the concerted electron–proton
transfer.16 Similarly, ET coupled with protonation (or hydrogen-
bond formation) [Scheme 1 green arrows] should be thermo-
dynamically more favorable than ET followed by protonation
(or hydrogen-bond formation) [Scheme 1 red and blue arrows].3,4
However, the one-step ET mechanism would be changed to the
stepwise mechanism, when the driving force of the initial ET
(Scheme 1 blue arrows) significantly increases. With regard to
such a mechanistic dichotomy,18 an important question arises:
are both pathways employed simultaneously? Alternatively, is
there a mechanistic continuity? However, such a mechanistic
dichotomy, including the interplay between ET and hydrogen
bonding (Scheme 1b), has yet to be scrutinized.
There is also a mechanistic dichotomy in hydride transfer of
dihydronicotinamide adenine dinucleotide (NADH) and ana-
logues, that is, one-step hydride transfer (green arrows) and ET
followed by proton–electron (blue and red arrows) [or hydrogen
(black arrows)] transfer as shown in Scheme 2.19–22 NADH is
an important source of two electrons and a proton in biological
redox reactions,23 and thereby always has been of general
interest to chemists.19–22,24–30 Hydride-transfer reactions of
¨
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S. J. Phys. Chem. B. 2005, 109, 7713.
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see: (a) Sjödin, M.; Styring, S.; Wolpher, H.; Xu, Y.; Sun, L.;
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an osmium complex containing a phosphorus-hydrogen bond; see:
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and stepwise mechanisms in Sc3+-promoted hydride transfer and
hydrogen transfer of an NADH analogue; see: (a) Yuasa, J.; Yamada,
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M. A., Chanon, M., Eds.; Elsevier: Amsterdam, The Netherlands, 1988;
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sulfinyl)-2,5-benzoquinone and protonated histidine (TolSQ•-
/
His·2H+) has appeared; see : Yuasa, J.; Yamada, S.; Fukuzumi, S.
Angew. Chem., Int. Ed. 2007, 46, 3553.
(16) For various definitions and examples of PCET, see: (a) Mayer, J. M.
Annu. ReV. Phys. Chem. 2004, 55, 363. (b) Cukier, R. I.; Nocera, D. G.
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(17) Such concerted proton-electron transfer reactions have recently been
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Soc. 2005, 127, 12490.
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A R T I C L E S
Yuasa et al.
NADH and analogues are often promoted by acids (H+), when
protonation of A (AH+) enhances electrophilicity as well as
the electron acceptor ability of A (Scheme 2a).3,4,19–21 In the
same way, the hydrogen-bond donor (H+:B) would also promote
hydride transfer of NADH and analogues via the one-step or
stepwise pathways (Scheme 2b). Hydrogen-bond donors in the
native enzymatic system, that is, the amino acid residues, play
an important role in the redox reactions of nicotinamide
coenzymes.31 However, promoting effects of the hydrogen-bond
donor on hydride transfer of NADH analogues through two
distinct mechanisms have yet to be examined.
We report herein for the first time extensive analysis on a
mechanistic dichotomy in a protonated histidine (His · 2H+)-
promoted ET reduction of 1-(p-tolylsulfinyl)-2,5-benzoquinone
(TolSQ) by electron donor as well as a two-electron reduction
(formally hydride reduction) of TolSQ by an acid-stable NADH
analogue, 9,10-dihydro-10-methylacridine (AcrH2). The promot-
ing effects of His ·2H+ on the ET and hydride-transfer reactions
are also extensively compared with acid-promoted hydride
reductions of TolSQ and 9,10-phenanthrenequinone (PQ) by
AcrH2.32 We employed the protonated amino acid as a
hydrogen-bond donor because of its important role in biological
redox reactions (vide supra).5–8,14,31,33,34 p-Quinone and o-
quinone derivatives (TolSQ and PQ, respectively) are used as
hydrogen-bond acceptors that have a multiple hydrogen-bonding
site for His ·2H+.35,36 The one-electron reduced species: a
hydrogen-bonded complex of TolSQ•- with His ·2H+ (TolSQ•-/
His·2H+), the semiquinone radical of TolSQ (TolSQH•), and
the hydroquinone radical cation (PQH2•+), were successfully
detected by electron spin resonance (ESR).15,32 Direct ESR
detection of the ET products, combined with the extensive
kinetic analysis of the His ·2H+-promoted ET and hydride
transfer provides valuable insight into the mechanistic dichotomy
in the His ·2H+-promoted ET reduction of the quinone as well
as the two-electron reduction by an NADH analogue.
9,10-dihydroacridine (AcrH2) and the dideuterated compound
(AcrD2) was described previously.38 The synthesis of 1-benzyl-
1,4-dihydronicotinamide dimer [(BNA)2] was also reported previ-
ously.39 10,10′-Dimethyl-9,9′-biacridine [(AcrH)2] was prepared by
the one-electron reduction of 10-methylacridinium perchlorate by
hexamethylditin.40 Histidine (His) was obtained from Aldrich.
Perchloric acid [HClO4] (70%) and deuterated HClO4 (DClO4)
[66%] were also obtained from Aldrich. For safety reasons, HClO4
(70%) containing 30% water was used in this work. Decamethyl-
ferrocene [(C5Me5)2Fc] was obtained from Wako Pure Chemical
Co., Ltd. 1,1′-Dimethylferrocene [(C5H4Me)2Fc], 1,1′-di-n-butyl-
ferrocene ([C5H4(n-Bu)]2Fc), and ferrocene [(C5H5)2Fc] were
obtained from Aldrich. 9,10-Phenanthrenequinone (PQ) was ob-
tained commercially and purified by the standard methods.41
Acetonitrile (MeCN) and propionitrile (EtCN) used as solvents were
purified and dried by the standard procedure.41 [2H3]Acetonitrile
(CD3CN) was obtained from EURI SO-TOP, CEA, France.
[2H2]Water (D2O) was purchased from Cambridge Isotope Labo-
ratories. Tetra-n-butylammonium perchlorate (TBAP) was pur-
chased from Fluka Chemical Co., twice recrystallized from absolute
ethanol, and dried in a vacuum at 45 °C prior to use. Deuterated
His·2H+ (His·2D+-d6) was prepared by the following procedure:
First, His (1.55 g) was dissolved in D2O (50 mL) and stirred by
the magnetic stirrer for 13 h. Then, deuterated His (His-d4) was
obtained by evaporation of the resulting aqueous solution. His·2D+-
d6 was obtained by addition of 2 equiv of DClO4 to a MeCN
solution of His-d4.
Reaction Procedures and Analysis. Typically, AcrH2 (8.5 ×
10-3 M) was added to an NMR tube that contained an
[2H3]acetonitrile (CD3CN) solution (0.6 mL) of TolSQ (8.5 × 10-3
M) in the presence of HClO4 (3.0 × 10-2 M) under an atmospheric
pressure of argon. Then the solution was deaerated with argon gas
for 5 min, and the NMR tube was sealed with a rubber septum.
The reaction was complete in one minute under these conditions.
The products of hydride reductions of TolSQ and PQ (TolSQH2
and PQH2) were identified by comparing the 1H NMR spectra with
those in the literatures.22a,42 The total yield of TolSQH2 in the
hydride-transfer reaction of TolSQ in the presence of HClO4 and
that in the presence of His ·2H+ were determined to be 97% and
1
Experimental Section
98%, respectively, from the H NMR spectra in comparison with
the internal standard, 1,4-dioxane (2.0 × 10-2 M). The total yield
Materials. 1-(p-Tolylsulfinyl)-2,5-benzoquinone (TolSQ) was
of PQH2 in the hydride-transfer reaction of PQ in the presence of
prepared according to the literature.37 Preparation of 10-methyl-
1
HClO4 was also determined to be 97% from the H NMR spectra
in comparison with the internal standard, 1,4-dioxane (2.0 × 10-2
M). 1H NMR measurements were performed with a JMN-AL-300
(300 MHz) NMR spectrometer at 298 K.
(30) For direct observation of sequential electron-proton transfer in
photoinduced hydrogen transfer from NADH analogues to triplet
species, 22b, see: (a) Schaefer, C. G.; Peters, K. S. J. Am. Chem. Soc.
1980, 102, 7566. (b) Peters, K. S.; Pang, E.; Rudzki, J. J. Am. Chem.
Soc. 1982, 104, 5535. (c) Manring, L. E.; Peters, K. S. J. Am. Chem.
Soc. 1985, 107, 6452. (d) Peters, K. S.; Cashin, A.; Timbers, P. J. Am.
Chem. Soc. 2000, 122, 107. (e) Dreyer, J.; Peters, K. S. J. Phys. Chem.
1996, 100, 19412.
Kinetic Measurements. Kinetic measurements were performed
by using a UNISOKU RSP-601 stopped-flow spectrophotometer
with an MOS-type high sensitive photodiode array. Rates of electron
transfer from R2Fc (1.0 × 10-4 M) to TolSQ (0 to 4.0 × 10-3 M)
in the presence of His ·2H+ (0 to 5.0 × 10-2 M) and His ·2D+-d6
(0 to 5.0 × 10-2 M) were monitored by the rise of the absorption
band at 620 nm due to R2Fc+ in deaerated MeCN at 298 K. Rates
of hydride transfer from AcrH2 to TolSQ (0 to 2.0 × 10-3 M) in
the presence of His ·2H+ (0 to 2.0 × 10-2 M) were monitored by
an increase in the absorption band due to 10-methylacridinium ion
(31) Brondijk, T. H. C.; van Boxel, G. I.; Singh, A.; Mather, O. M.; White,
H. A.; Quirk, P. G.; White, S. A.; Jackson, J. B. J. Biol. Chem. 2006,
281, 13345.
(32) A part of preliminary results on direct detection of the radical cation
of an NADH analogue, 9,10-dihydro-10-methylacridine (AcrH2) has
appeared; see: Yuasa, J.; Yamada, S.; Fukuzumi, S. Angew. Chem.,
Int. Ed. 2008, 47, 1068.
(33) It has been suggested that His-H126 and His-H128 nearby QB facilitate
proton transfer into the RC; see: (a) Paddock, M. L.; Graige, M. S.;
Feher, G.; Okamura, M. Y. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
(37) (a) García Ruano, J. L.; Alemparte, C. J. Org. Chem. 2004, 69, 1405.
(b) Carreño, M. C.; García Ruano, J. L.; Toledo, M. A.; Urbano, A.
Tetrahedron Lett. 1994, 35, 9759. (c) Carreño, M. C.; García Ruano,
J. L.; Lafuente, C.; Toledo, M. A. Tetrahedron: Asymmetry 1999, 10,
1119.
¨
6183. (b) Adelroth, P.; Paddock, M. L.; Tehrani, A.; Beatty, J. T.;
Feher, G.; Okamura, M. Y. Biochemistry 2001, 40, 14538.
(34) Muramoto, K.; Hirata, K.; Shinzawa-Itoh, K.; Yoko-o, S.; Yamashita,
E.; Aoyama, H.; Tsukihara, T.; Yoshikawa, S. Proc. Natl. Acad. Sci.
U.S.A. 2007, 104, 7881.
(38) Fukuzumi, S.; Koumitsu, S.; Hironaka, K.; Tanaka, T. J. Am. Chem.
Soc. 1987, 109, 305.
(35) (a) Ge, Y.; Lilienthal, R. R.; Smith, D. K. J. Am. Chem. Soc. 1996,
118, 3976. (b) Ge, Y.; Miller, L.; Ouimet, T.; Smith, D. K. J. Org.
Chem. 2000, 65, 8831.
(39) Wallenfels, K.; Gellerich, M. Chem. Ber. 1959, 92, 1406.
(40) Fukuzumi, S.; Kitano, T.; Mochida, K. J. Am. Chem. Soc. 1990, 112,
3246.
(36) Quinones act as biological electron acceptors that can undergo either
one- or two-electron reductions coupled with protonation to yield the
corresponding semiquinones and hydroquinones, respectively; see:
Functions of Quinones in Energy ConserVing Systems; Trumpower,
B. I., Ed.; Academic Press: New York, 1986.
(41) Armarego, W. L. F.; Chai, C. L. L. Purification of Laboratory
Chemicals, 5th ed.; Butterworth Heinemann: Amsterdam, The Neth-
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Electron-Transfer Reduction of a Quinone
A R T I C L E S
Scheme 3
(AcrH+: λmax ) 358 nm, ꢀmax ) 1.80 × 104 M-1 cm-1) in deaerated
MeCN at 298 K in the dark. All kinetic measurements were carried
out under pseudo-first-order conditions where the concentrations
of TolSQ were maintained at more than 10-fold excess of the
concentrations of R2Fc and AcrH2 at 298 K. Pseudo-first-order rate
constants were determined by least-squares curve fits using a
personal computer.
electrode. The second-harmonic alternating current voltammetry
(SHACV) measurements of TolSQ (1.0 × 10-2 M) in the presence
of His ·2H+ (5.0 × 10-3 M) and HClO4 (1.0 × 10-2 M) were
carried out with a BAS 100B electrochemical analyzer in deaerated
MeCN containing 0.10 M TBAP as a supporting electrolyte at 298
K. All potentials (vs Ag/Ag+) were converted to values vs SCE
by adding 0.29 V.43 All electrochemical measurements were carried
out under an atmospheric pressure of argon.
Spectral Measurements. Protonation of TolSQ and PQ were
examined from the UV–vis spectral changes of TolSQ (1.0 × 10-3
M) and PQ (1.0 × 10-3 M), respectively, in the presence of various
concentrations of HClO4 (0–1.4 M) by using a Hewlett-Packard
8453 diode array spectrophotometer.
Theoretical Calculations. Density-functional theory (DFT)
calculations were performed on a 8CPU workstation (PQS,
Quantum Cube QS8–2400C-064). Geometry optimizations were
carried out using BeckeLYP functional and 6-31G** basis set44
with the restricted Hartree–Fock (RHF) formalism or the unre-
stricted Hartree–Fock (UHF) and as implemented in the Gaussian
03 program, revision C.02.
ESR Measurements. ESR detection of TolSQ•-/His·2H+,
TolSQH•, and PQH2•+ were performed as follows. Typically, TolSQ
(8.0 × 10-3 M) was dissolved in deaerated MeCN and purged with
argon for 10 min. His (8.0 × 10-3 M) and HClO4 (1.6 × 10-2 M)
were dissolved in deaerated MeCN. The TolSQ (200 µL) and
His ·2H+ (200 µL) solutions were introduced into an ESR cell (1.8
mm i.d.) containing (AcrH)2 (1.6 × 10-2 M) and mixed by bubbling
with an Ar gas through a syringe with a long needle. The ESR
spectra of TolSQ•-/His·2H+, TolSQH•, and PQH2•+ were recorded
on a JEOL JES-RE1XE spectrometer under irradiation of a high
pressure mercury lamp (USH-1005D) focusing at the sample cell
•+
in the ESR cavity. ESR detection of thermally generated AcrH2
was performed by using a JEOL ES-EMCNT1 rapid mixing flow
apparatus. Deaerated MeCN solutions of AcrH2 (2.9 × 10-3 M)
and TolSQ (2.8 × 10-3 M) in the presence of HClO4 (7.0 × 10-2
M) under an atmospheric pressure of argon were mixed in a flat
ESR cell at the flow rate of 1.9 cm3 s-1. The mixing time before
reaching the ESR cell is about several hundred milliseconds, which
is short enough to detect AcrH2•+. The magnitude of modulation
was chosen to optimize the resolution and signal-to-noise (S/N)
ratio of the observed spectra under nonsaturating microwave power
conditions. The g values were calibrated using an Mn2+ marker.
Computer simulation of the ESR spectra was carried out by using
Calleo ESR version 1.2 (Calleo Scientific Publisher) on a personal
computer.
Cyclic Voltammetry. Cyclic voltammetry measurements were
performed on a BAS 100W electrochemical analyzer in deaerated
MeCN containing 0.1 M TBAP as a supporting electrolyte at 298
K. A conventional three-electrode cell was used with a platinum
working electrode (surface area of 0.3 mm2) and a platinum wire
as the counter electrode. The Pt working electrode (BAS) was
routinely polished with a BAS polishing alumina suspension and
rinsed with acetone before use. The measured potentials were
recorded with respect to the Ag/AgNO3 (0.01 M) reference
Results and Discussion
ESR Detection of a Hydrogen-Bonded Complex between a
Semiquinone Radical Anion and a Protonated Amino Acid.
Hydrogen-bond formation of semiquinone radical anions with
protonated amino acids were examined by ESR in photoinduced
electron transfer (ET) from 10,10′-dimethyl-9,9′-biacridine
[(AcrH)2] to quinones (Scheme 3). The (AcrH)2 is known to
act as a two electron donor to produce 2 equiv of the radical
anion of electron acceptor.45 Steady-state photoirradiation of
an acetonitrile (MeCN) solution of (AcrH)2 (1.6 × 10-2 M)
and 1-(p-tolylsulfinyl)-2,5-benzoquinone (TolSQ) (4.0 × 10-3
(43) Mann, C. K.; Barnes, K. K. Electrochemical Reactions in Nonaqueous
Systems; Marcel Dekker: New York, 1970.
(44) Becke, A. D. J. Chem. Phys. 1993, 98, 5648.
(45) (a) Fukuzumi, S.; Kitano, T.; Mochida, K. J. Am. Chem. Soc. 1990,
112, 3246. (b) Fukuzumi, S.; Tokuda, Y. J. Phys. Chem. 1992, 96,
8409.
9
J. AM. CHEM. SOC. VOL. 130, NO. 17, 2008 5811

A R T I C L E S
Yuasa et al.
ESR spectrum in Figure 1a allows us to determine the hyperfine
coupling constants (hfc) due to three protons [a(3H) ) 0.88,
5.31, and 6.08 G] of TolSQ•- and superhyperfine splitting due
to one nitrogen and three equivalent protons [a(N) ) 1.35 G
and a(3H) ) 2.97 G] of His·2H+ (Figure 1b). The complete
agreement of the observed ESR spectrum (Figure 1a) with the
computer simulation spectrum (Figure 1b) clearly indicates
formation of the TolSQ•-/His·2H+ complex (Scheme 3a). The
g value (2.0026) and the hfc values [a(3H) ) 0.88, 5.31, and
6.08 G] of the TolSQ•-/His·2H+ complex are drastically
changed from those of TolSQ•- in the absence of His ·2H+,
which are g ) 2.0057 and a(3H) ) 2.00, 2.20, and 3.35 G.22a,47
When p-benzoquinone (Q) is employed instead of TolSQ,
only the protonated species (semiquinone radical: QH•) is
detected by ESR in photoinduced ET from (AcrH)2 to Q in the
presence of His ·2H+ (Figure 1c, Scheme 3b). Similarly, only
the semiquinone radical of TolSQ (TolSQH•) can be detected
by ESR (Figure 1e), when His ·2H+ is replaced by protonated
phenyl alanine (Phe ·H+) (Scheme 3c).48 Thus, the SdO oxygen
in TolSQ as well as the N-H proton of imidazole ring in
His·2H+ play crucial roles for hydrogen-bond formation
between TolSQ•- and His·2H+.49 Virtually the same ESR
spectrum as Figure 1e can be detected in photoinduced ET from
dimeric 1-benzyl-1,4-dihydronicotinamide [(BNA)2]50 to TolSQ
in the presence of HClO4 (6.0 × 10-1 M) in propionitrile (EtCN)
at 193 K (Figure 1g). In this case (BNA)2 also acts as a two
electron donor to produce 2 equiv of the semiquinone radical
anions.51,52 When the light is cut off, the ESR signal due to
TolSQH• disappears immediately as in the case of TolSQ•-
/
His·2H+. This is attributed to a fast disproportionation of
TolSQH• (2TolSQH• f TolSQH2 + TolSQ). It should be
emphasized that there is no ESR signal due to the diprotonated
species, that is, TolSQH2•+ even in the presence of the extremely
high concentration of HClO4 (6.0 × 10-1 M) (Figure 1g). This
indicates that no further protonation of TolSQH• takes place.
The optimized structure and the hfc values of TolSQ•-
/
His·2H+ obtained by using density functional theory (DFT) at
the BLYP/6–31G** basis are shown in Figure 2a.53 The
optimized structure of TolSQ•-/His·2H+ shows multiple hy-
drogen bonding between TolSQ•- and His·2H+ (hydrogen
bonds between the CdO oxygen of TolSQ•- and the COOH
Figure 1. (a) ESR spectrum of a deaerated MeCN solution of TolSQ (4.0
× 10-3 M) and (AcrH)2 (1.6 × 10-2 M) in the presence of His (4.0 ×
10-3 M) and HClO4 (8.0 × 10-3 M) under photoirradiation at 298 K and
(b) the computer simulation spectrum. (c) ESR spectrum of a deaerated
MeCN solution of Q (8.3 × 10-3 M) and (AcrH)2 (1.6 × 10-2 M) in the
presence of His (8.3 × 10-3 M) and HClO4 (1.7 × 10-2 M) under
photoirradiation at 298 K and (d) the computer simulation spectrum. (e)
ESR spectrum of a deaerated MeCN solution of TolSQ (9.1 × 10-3 M)
and (AcrH)2 (1.6 × 10-2 M) in the presence of Phe (9.1 × 10-3 M) and
HClO4 (9.1 × 10-3 M) under photoirradiation at 233 K and (f) the computer
simulation spectrum. (g) ESR spectrum of a deaerated EtCN solution of
TolSQ (2.1 × 10-2 M) and (BNA)2 (1.6 × 10-2 M) in the presence of
HClO4 (6.0 × 10-1 M) under photoirradiation at 193 K and (h) the computer
simulation spectrum. (i) ESR spectrum of a deaerated EtCN solution of
PQ (8.0 × 10-2 M) and (BNA)2 (1.6 × 10-2 M) in the presence of His
(5.2 × 10-1 M) and HClO4 (1.04 M) under photoirradiation at 193 K and
(j) the computer simulation spectrum. (k) ESR spectrum of a deaerated
EtCN solution of PQ (5.0 × 10-2 M) and (BNA)2 (1.6 × 10-2 M) in the
presence of HClO4 (2.5 × 10-2 M) under photoirradiation at 193 K and (l)
the computer simulation spectrum.
(46) Although His is hardly dissolved in MeCN, His becomes soluble in
the presence of 2 equiv of HClO4 in MeCN. Similarly, phenyl alanine
(Phe) also becomes soluble in the presence of 1 equiv of HClO4 in
MeCN.
(47) The significantly smaller g value of TolSQ•-/His·2H+ than TolSQ•-
(2.0057) indicates that the spin density on oxygen nuclei in TolSQ•-
is reduced significantly due to the strong hydrogen bonding with
His·2H+.
(48) Un-protonated His could not be employed as the control experiment,
because His itself is hardly dissolved in MeCN (see ref 46).
(49) His·2H+ acts as a multiple hydrogen-bond donor toward the semi-
quinone radical anion (TolSQ•-). Multiple hydrogen-bonds between
nearby protonated amino acid residues and semiquinone radical anions
play an important role in the direction of an electron flow in the
photosynthetic reaction center. Nevertheless, His ·2H+ is less physi-
ological as the carboxylate and amide are typically involved in peptide
bonds.
(50) Patz, M.; Kuwahara, Y.; Suenobu, T.; Fukuzumi, S. Chem. Lett. 1997,
567.
(51) Fukuzumi, S.; Suenobu, T.; Patz, M.; Hirasaka, T.; Itoh, S.; Fujitsuka,
M.; Ito, O. J. Am. Chem. Soc. 1998, 120, 8060.
M) in the presence of protonated histidine (His ·2HClO4) (4.0
× 10-3 M) afforded an ESR spectrum as shown in Figure
1a.15,46
Without steady-state photoirradiation, no ESR was detected
because of instability of a hydrogen-bonded complex between
TolSQ•- and His ·2H+ (TolSQ•-/His·2H+). The well-resolved
(52) The (BNA)2 acting as stronger electron donor than (AcrH)2 was used
in the ESR detection of TolSQH• (Figure 1g), because the TolSQH•
is quite unstable in the presence of extremely high concentration of
HClO4 (6.0 × 10-1 M). For the same reason, we employ (BNA)2 in
order to detect a hydroquinone radical cation of 9,10-phenanthrene-
quinone (PQH2•+) by ESR (Figure 1, panels I and k).
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5812 J. AM. CHEM. SOC. VOL. 130, NO. 17, 2008

Electron-Transfer Reduction of a Quinone
A R T I C L E S
of TolSQH• (vide supra). The ESR spectrum of PQH2•+ is also
detected by ESR in photoinduced ET from (BNA)2 to PQ in
the presence of a high concentration of HClO4 (6.0 × 10-1 M)
[Figure 1k].52,60,61
Large Positive Shifts in One-Electron Reduction Potentials
of TolSQ and PQ in the Presence of His ·2H+ or HClO4. Strong
hydrogen-bond formation between TolSQ•- and His ·2H+
(TolSQ•-/His · 2H+) as well as protonation of TolSQ•-
(TolSQH•) are expected to result in positive shifts of the one-
electron reduction potential (Ered) of TolSQ,9,10 which were
verified by the electrochemical measurements (vide infra). In
contrast to a reversible redox wave of TolSQ in the absence of
His·2H+ (or HClO4) [Figure 3a], the cyclic voltammogram of
TolSQ in the presence of His ·2H+ (Figure 3b)15 and that in
the presence of HClO4 (Figure 3d) exhibit irreversible cathodic
waves due to instability of TolSQ•-/His·2H+ and TolSQH•,
respectively. Thus, the Ered value of TolSQ in the presence of
His·2H+ and the value in the presence of HClO4 were
determined by second-harmonic alternating current voltammetry
(SHACV) [Figure 3c and 3e, respectively]. In the presence of
5.0 × 10-2 M of His·2H+, the Ered value of TolSQ (-0.26 V
versus SCE) is shifted to 0.29 V versus SCE (Figure 3c),15
which is further shifted to 0.69 V versus SCE in the presence
of 5.0 × 10-2 M of HClO4 (Figure 3e).32
Figure 2. Optimized structures, spin-density plots, and the calculated hfc
values of (a) TolSQ•-/His · 2H+, (b) (c) TolSQH• calculated by using a
density functional theory at the BLYP/6-31G** (observed hfc values are
given in parentheses).
+
proton as well as the NH3 protons of His·2H+, and also
between the SdO oxygen of TolSQ•- and the NH+ proton of
the imidazole ring of His ·2H+) [Figure 2a]. Such multiple
Since the formation constants of TolSQ/His · 2H+ and
TolSQH+ are significantly smaller than those of the one-electron
reduced products, TolSQ•-/His·2H+ (K1) and TolSQH• (K2),
the positive shift in the one-electron reduction potential of TolSQ
in the presence of His ·2H+ and that in the presence of HClO4
hydrogen bonding may result in stabilization of TolSQ•-
/
His·2H+ (for the hydrogen-bond lengths; see Supporting
Information S1).54 The superhyperfine due to the hydrogen-
+
bonded NH3 proton of His ·2H+ is estimated as 6.61 G. The
+
are expressed by eq 1 and 2, respectively, where E0 is the
averaged hfc value (2.20 G) due to the hydrogen-bonded NH3
red
reduction potential of TolSQ in the absence of His ·2H+ (or
three protons agrees with the observed value (Figure 1b;
parentheses in Figure 2a).55 This indicates the rapid exchange
in the NH3+ protons that is the hydrogen-bonded proton in the
ESR time scale. The existence of the strong hydrogen bond
between TolSQ•- and His ·2H+ is firmly supported by the
existence of the superhyperfine due to the hydrogen-bonded
Ered ) E0red + (2.3RT/F) log{K1[His·2H+]}
Ered ) E0red + (2.3RT/F) log{K2[H+]}
(1)
(2)
HClO4). The K1 and K2 values were determined as 4.2 × 1010
M-1 and 2.5 × 1017 M-1 from the Ered value of TolSQ in the
presence of His·2H+ (5.0 × 10-2 M) and the value in the
presence of HClO4 (5.0 × 10-2 M), respectively. Such large
K1 and K2 values indicate the existence of strong hydrogen
+
protons and nitrogen of NH3 (Figure 1b). The optimized
structures and the hfc values of TolSQH• are also obtained by
DFT at the BLYP/6-31G** basis (Figures 2b and 2c).53 The
hfc values in Figure 2b agrees better with the observed values
(Figure 1h and parentheses in Figure 2b) than those in Figure
2c.55,56 Such agreement indicates that the proton from HClO4
may be bound to the CdO oxygen on the opposite side of the
SdO oxygen in contrast to the case of TolSQ•-/His·2H+.57
When 9,10-phenanthrenequinone (PQ) is employed as a
multiple hydrogen-bond acceptor,35 however, the diprotonated
species (hydroquinone radical cation: PQH2•+) is detected by
ESR in photoinduced ET from (BNA)2 to PQ in the presence
of His ·2H+ (Figure 1i) (Scheme 4).52,58,59
(56) The ESR-measured hfc values (parentheses in Figure 2b) for TolSQH•
are not an average of the DFT-determined values for b and c in Figure
2, because two large hfc values [a(2H) ) 4.57 and 5.67 G] due to
two protons in Figure 2c are inconsistent with the existence of only
one large hfc value [a(H) ) 4.90 G] due to one proton (parentheses
in Figure 2b).
(57) The structure of TolSQH• in Figure 2c is enthalpically more favorable
but entropically less favorable than that in Figure 2b. This may be
the reason why H+ binds to the CdO oxygen on the opposite side of
the SdO oxygen as shown in Figure 2b in which the CdO oxygen
far from the SdO oxygen is involved.
•+
Formation of PQH2 indicates the high basicity of mono-
protonated species (PQH•), showing sharp contrast with the case
•+
(58) The existence of two protons binding to two CdO oxygens in PQH2
was confirmed by a drastic change of the ESR spectrum (Figure 1i)
by deuterium substitution of HClO4 by DClO4 (see Supporting
Information S2). This affords a clear assignment of the observed hfc
values of PQH2•+, because a single deuteron gives a triplet (instead
of doublet) hyperfine pattern and the deuteron splitting should decrease
by the magnetogyric ratio of proton to deuterium (0.153).
(59) We have previously reported the 1:2 complex between PQ•- and La3+
[PQ•--(La3+)2]; see: Yuasa, J.; Suenobu, T.; Fukuzumi, S.
ChemPhysChem 2006, 7, 942.
(53) The calculated hfc values of semiquinone radical (QH•) by using BLYP
methods have been reported to agree well with experimental data, see:
Nonella, M. J. Phys. Chem. B 1997, 101, 1235.
+
(54) The hydrogen-bonded proton of NH3 is not covalently bound to
TolSQ•- but electrostatically bound to TolSQ•- through the hydrogen
bond. The distance of the N-H bond in NH3+ (1.57 Å) is longer than
that between the NH3+ proton and the CdO oxygen of TolSQ•- (1.05
Å) in the optimized structure of TolSQ•-/His·2H+ (see Supporting
Information S2). This indicates that the binding of the N-H bond in
(60) The ESR spectrum due to PQH2•+ (Figure 1k) is changed to PQH• in
the presence of a low concentration of HClO4 (2.5 × 10-2 M) (see
Supporting Information S3).
+
NH3 is significantly weakened by strong hydrogen-bond formation
with TolSQ•-
.
(55) Some deviations of the calculated hfc values of TolSQ•-/His·2H+
(Figure 2a) and TolSQH• (Figure 2b) from those of observed values
(Figures 1b and 1h, respectively) indicate that the spin distribution is
somewhat affected by the solvent.
(61) For ESR spectra of o-semiquinone radicals, see: (a) Lucarini, M.;
Mugnaini, V.; Pedulli, G. F. J. Org. Chem. 2002, 67, 928. (b) Ci, X.;
da Silva, R. S.; Nicodem, D.; Whitten, D. G. J. Am. Chem. Soc. 1989,
111, 1337.
9
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A R T I C L E S
Yuasa et al.
Scheme 4
bonding in TolSQ•-/His·2H+ and the high basicity of TolSQ•-,
respectively. Similarly, the Ered value of PQ in the presence of
HClO4 was determined by SHACV (Figure 3g), because the
cyclic voltammogram of PQ in the presence of HClO4 also
exhibits an irreversible cathodic wave due to the instability of
ylferrocene [(C5H4Me)2Fc] (Eox ) 0.26 V vs SCE) to TolSQ
(Ered ) –0.26 V vs SCE)22a in the absence of His ·2H+ is highly
endergonic (∆G0 ) 0.52 eV), no ET reaction occurs in the
et
absence of His ·2H+. In the presence of His ·2H+ (5.0 × 10-2
M), however, the Ered value of TolSQ is shifted to 0.29 V vs
SCE (vide supra). ET from (C5H4Me)2Fc to TolSQ therefore
occurs in the presence of His ·2H+ (eq 3), as expected from
the negative free-energy change of ET (∆Get ) –0.03 eV).
The rates of His ·2H+-promoted ET from (C5H4Me)2Fc to
•+
PQH2 (Figure 3f). The Ered value of PQ (-0.65 V versus
SCE)59 is shifted to 0.51 V versus SCE in the presence of 5.0
× 10-2 M HClO4 (Figure 3g). The significant positive shift of
the Ered value of PQ (+1.16 V) in the presence of HClO4 (4.9
× 10-2 M) indicates that PQ•- is significantly stabilized by
diprotonation to form PQH2•+ (see the ESR spectrum in Figure
1k).
His·2H+-Promoted ET from R2Fc to TolSQ. The positive
shift of the Ered value of TolSQ in the presence of His ·2H+
(Figure 3c) should result in enhancement of the reactivity of
the ET reduction of TolSQ, which was examined by ET from
ferrocenes (R2Fc) to TolSQ in the presence of His ·2H+ (vide
infra). Since the free-energy change of ET from 1,1′-dimeth-
TolSQ obeyed pseudo-first-order kinetics in the presence of a
large excess TolSQ and His ·2H+ relative to the concentration
of (C5H4Me)2Fc (see the first-order plot in Supporting Informa-
tion S4). The observed pseudo-first-order rate constant (kobs
)
increases proportionally with increasing TolSQ concentration
(see Supporting Information S5). The second-order rate constant
(kH) also increases linearly with increasing the His ·2H+
concentration ([His ·2H+]) [Figure 4a red circles].15 Virtually
the same results are obtained, when ferrocene [(C5H5)2Fc] (Eox
) 0.37 V versus SCE) and 1,1′-di-n-butylferrocene ([C5H4(n-
Bu)]2Fc) [Eox ) 0.31 V vs SCE] are employed instead of
(C5H4Me)2Fc (red circles in Figures 4b and 4c, respectively;
see Supporting Information S5).62
∆Get ) ∆G0et - (2.3RT/F) log{K1[His·2H+]}
(4)
Since His ·2H+ has no effect on the oxidation potential of
R2Fc, the free energy change of ET from R2Fc to TolSQ in the
presence of His·2H+ (∆Get) can be expressed by eq 4, where
∆G0 is the free energy change in the absence of His·2H+.
et
Such a change in ∆Get has previously been well evaluated in
light of the Marcus theory of ET63 for metal ion-promoted ET
from ferrocene to naphthoquinone (NQ) moiety of ferrocene-
naphthoquinone (Fc-NQ) linked dyad, when the second-order
rate constant of ET (ket) increases linearly with increasing the
concentration of metal ion.64 In the same way, the kH value
also increases linearly with [His ·2H+] in His·2H+-promoted
Figure 3. (a) Cyclic voltammogram of TolSQ (1.0 × 10-2 M) in the
absence of His ·2H+, (b) cyclic voltammogram and (c) second-harmonic
alternating current voltammogram of TolSQ (5.0 × 10-3 M) in the presence
of His·2H+ (5.0 × 10-2 M) in deaerated MeCN containing tetra-n-
butylammonium perchlorate (TBAP) [0.10 M] with Pt working electrode
at 298 K; sweep rate 0.1 V s-1. (d) Cyclic voltammogram and (e) second-
harmonic alternating current voltammogram of TolSQ (5.0 × 10-3 M) in
the presence of HClO4 (5.0 × 10-2 M) in deaerated MeCN containing TBAP
(0.10 M) with Pt working electrode at 298 K; sweep rate 0.1 V s-1. (f)
Cyclic voltammogram and (g) second-harmonic alternating current volta-
mmogram of PQ (7.0 × 10-3 M) in the presence of HClO4 (5.0 × 10-2
M) in deaerated MeCN containing TBAP (0.10 M) with Pt working
(62) Although ET from (C5H5)2Fc (Eox ) 0.37 V vs SCE) and [C5H4(n-
Bu)]2Fc (Eox ) 0.31 V vs SCE) to TolSQ in the presence of 5.0 ×
10-2 M of His·2H+ (Ered ) 0.29 V vs SCE) are still slightly uphill
(∆Get ) 0.08 eV and ∆Get ) 0.02 eV, respectively), the follow-up
disproportionation of the TolSQ•-/His·2H+ complex makes the one-
electron oxidation of (C5H5)2Fc and [C5H4(n-Bu)]2Fc undergo to
completion.
(63) Marcus, R. A. Angew. Chem., Int. Ed. Engl. 1993, 32, 1111.
(64) Okamoto, K.; Imahori, H.; Fukuzumi, S. J. Am. Chem. Soc. 2003,
125, 7014.
electrode at 298 K; sweep rate 0.1 V s-1
.
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5814 J. AM. CHEM. SOC. VOL. 130, NO. 17, 2008

Electron-Transfer Reduction of a Quinone
A R T I C L E S
Figure 5. Plots of (a) kH/kD and (b) log kH vs -∆G0 for ET from R2Fc
et
to TolSQ in the presence of His ·2H+ (5.0 × 10-2 M) in deaerated MeCN
at 298 K. Numbers at closed circles correspond to Table 1.
NH3+ of His ·2H+ at the transition state, when the ET is tightly
coupled with hydrogen-bond formation (Scheme 5a).12 The
existence of the strong hydrogen bond between TolSQ•- and
His·2H+ has been well supported by the existence of the
superhyperfine coupling due to the hydrogen-bonded protons
and nitrogen of NH3+ in the ESR spectrum of TolSQ•-/His·2H+
(Figure 1a) (vide supra). The kH/kD values for ET from R2Fc to
TolSQ in the presence of His·2H+ (5.0 × 10-2 M) are listed
in Table 1 together with the ET driving force (-∆G0et) in the
absence of His ·2H+. The plot of kH/kD versus -∆G0et is shown
in Figure 5a, combined with the plot of log kH vs -∆G0et (Figure
5b).67 The kH/kD value decreases with increasing the -∆G0
et
value to approach kH/kD ) 1.0 (Figure 5a) with a concomitant
increase of the log kH value (Figure 5b).68 In contrast to the ET
coupled with hydrogen-bond formation (Scheme 5a), the rate-
determining ET followed by fast hydrogen-bond formation
(Scheme 5b) would exhibit no deuterium kinetic isotope effect
(kH/kD ) 1.0). Thus, the continuous decrease of the deuterium
kinetic isotope effect (kH/kD) with an increase in the ET driving
force (-∆G0et) [Figure 5a] indicates that there is a mechanistic
continuity in two reaction pathways, that is, the one-step
pathway (Scheme 5a) is continuously changed to the stepwise
pathway (Scheme 5b) with increasing the ET driving force
(-∆G0et). If two reaction pathways were employed simulta-
neously, the deuterium kinetic isotope effect (kH/kD) would be
constant irrespective of the ET driving force (-∆G0et) above
the changeover to the stepwise mechanism (Scheme 5b).69
His·2H+ and HClO4-Promoted Hydride Reduction of
Quinones by an NADH Analogue. When R2Fc (electron donor)
is replaced by an NADH analogue, AcrH2 (Eox ) 0.81 V vs
SCE)70 that is a source of two electrons and a proton (equivalent
to a hydride ion), no hydride transfer from AcrH2 to TolSQ
occurs in the absence of His ·2H+. However, an efficient hydride
transfer from AcrH2 to TolSQ occurs to yield AcrH+ and
TolSQH2 in the presence of His ·2H+ (eq 5; for the products
analysis, see Experimental Section).71 The rates of hydride
Figure 4. Dependence of kH (red circles) and kD (blue circles) on [His ·2H+]
and [His ·2D+-d6] for ET from (a) (C5H4Me)2Fc (1.0 × 10-4 M), (b)
[C5H4(n-Bu)]2Fc (1.0 × 10-4 M), and (c) (C5H5)2Fc (1.0 × 10-4 M) to
TolSQ in the presence of His ·2H+ and His ·2D+-d6 in deaerated MeCN at
298 K.
ET from R2Fc to TolSQ (Figure 4 red circles).65 One may think
that the linear correlation of kH with [His ·2H+] (Figure 4) results
from the TolSQ/His ·2H+ complex (formation constant of
TolSQ/His·2H+ is too small to be determined), albeit it was
not detected, which increases with increasing the His ·2H+
concentration. However, this is certainly not the case of ET
reactions,65 because hydrogen-bond formation of the product
of radical anion with the hydrogen-bond donor (A•- · · ·H+:B),
which was detected by ESR, results in acceleration of ET (vide
supra) without formation of the TolSQ/His ·2H+ complex,10
when the Ered value of A is shifted to positive direction.9,10 This
is quite different from the case of the one-step hydride transfer
from AcrH2 to TolSQ, which requires formation of the TolSQ/
His·2H+ complex prior to the hydride transfer (vide infra).
The rates of ET from R2Fc to TolSQ exhibit deuterium kinetic
isotope effects (1.3 < kH/kD < 1.9), when His ·2H+ is replaced
by the deuterated compound (His ·2D+-d6) as shown in Figure
4 blue circles; see the structure of His ·2D+-d6 in eq 3.66 The
observed deuterium kinetic isotope effects may result from
partial dissociation of the N-H bond in the hydrogen-bonded
(67) The kH/kD value is plotted against the ET driving force (-∆G0et) in
the absence of His ·2H+, which are highly negative.
(68) The K1 values are assumed to be the same between the protiated and
dueterated His. If the KIE results from the isotope effect in K1, the
KIE value would be constant irrespective of the driving force in
contrast to the results in Figure 5. Unfortunately the determination of
the K1 values based on Ered values was not accurate enough to
recognize a small isotope effect.
(65) Fukuzumi, S. Org. Biomol. Chem. 2003, 1, 609.
(66) The deuteration ratio of His ·2D+-d6 was determined to be 98% from
the 1H NMR spectrum.
9
J. AM. CHEM. SOC. VOL. 130, NO. 17, 2008 5815

A R T I C L E S
Yuasa et al.
Scheme 5. (a) ET Coupled with Hydrogen-Bond Formation; (b) Rate-Determining ET Followed by Fast Hydrogen-Bond Formation
Table 1. One-Electron Oxidation Potentials of R2Fc (Eox), One-Electron Reduction Potentials of TolSQ in the Absence (Ered0) and Presence
of 5.0 × 10-2 M of His·2H+ (Ered), Free Energy Change of ET in the Absence (∆Get0) and Presence of 5.0 × 10-2 M of His·2H+ (∆Get),
Rate Constants (kH) of ET from R2Fc to TolSQ in the presence of His ·2H+ (5.0 × 10-2 M), and the Deuterium Kinetic Isotope Effects
(kH/kD) in Deaerated MeCN at 298 K
0
Ered (V vs SCE)
a
Ered (V vs SCE)
0
b
no.
R2Fc
Eox (V vs SCE)
∆Get (eV)
∆Get (eV)
kH (M-1 s-1
)
kH/kD
1
2
3
(C5H5)2Fc
[C5H4(n-Bu)]2Fc
(C5H4Me)2Fc
0.37
0.31
0.26
-0.26
-0.26
-0.26
0.29
0.29
0.29
0.63
0.57
0.52
0.08
0.02
-0.03
3.7 × 102
1.3 × 103
4.1 × 103
1.9 ( 0.1
1.5 ( 0.1
1.3 ( 0.1
a Determined by second-harmonic alternating current voltammetry. b Determined from the slopes of plots of kH and kD vs [His ·2H+].
transfer obeyed pseudo-first-order kinetics in the presence of a
large excess TolSQ and His ·2H+ relative to the concentration
of AcrH2 (see the first-order plot in Supporting Information S6).
The observed pseudo-first-order rate constant (kobs) increases
proportionally with an increase in [TolSQ] (S7). The second-
circles in Figure 6a). The rates of hydride transfer exhibit a
deuterium kinetic isotope effect (kHH/kDH ) 1.7 ( 0.1, when
AcrH2 is replaced by the dideuterated compound (AcrD2, kDH
denotes the rate constant of hydride transfer from AcrD2 to
TolSQ with His ·2H+) (blue circles in Figure 6b). In sharp
contrast to this, no deuterium kinetic isotope effect (kHH/kHD
)
1.0 and kDH/kDD ) 1.0) is observed in hydride transfer from
AcrH2 and AcrD2 to TolSQ, when His·2H+ is replaced by
His·2D+-d6 (kHD denotes the rate constant of hydride transfer
from AcrH2 to TolSQ with His ·2D+-d6); see red and blue closed
triangles in Figure 6, respectively. If the hydride transfer
proceeds via ET from AcrH2 (Eox ) 0.81 V vs SCE)70 to TolSQ
(Ered ) –0.26 V vs SCE) as shown by broken arrow in Scheme
6, the rates of the formal hydride reactions would exhibit
deuterium kinetic isotope effects as in the case of His ·2H+-
promoted ET from R2Fc to TolSQ (Figure 4), when His ·2H+
is replaced by His ·2D+-d6. Thus, the observed deuterium kinetic
isotope effect (kHH/kDH ) 1.7 ( 0.1) by deuterium substitution
of AcrH2 by AcrD2 and the absence of the deuterium kinetic
isotope effect (kHH/kHD ) 1.0 and kDH/kDD ) 1.0) by deuterium
substitution of His ·2H+ by His ·2D+-d6 indicate that the hydride
transfer proceeds via the one-step pathway (Scheme 6). It should
be noted that no absorption band due to AcrH2•+ is observed in
the His ·2H+-promoted hydride transfer from AcrH2 to TolSQ.
The linear correlation between kHH and [His·2H+] (Figure 6a
red circles) may result from formation of the hydrogen-bonded
order rate constant of hydride transfer from AcrH2 to TolSQ
with His·2H+ (kHH) also increases linearly with [His·2H+] (red
(69) The ET mechanism would be completely changed to the stepwise
pathway (Scheme 5b) with further increasing the ET driving force
(-∆G0et), in which the rates of ET exhibit no deuterium kinetic isotope
effect (kH/kD ) 1.0). However, the rate of such ET with a large driving
force was too fast to be determined. In the case of hydride-transfer
reactions, the deuterium kinetic isotope effect increases with increasing
the driving force when the transition state is productlike or later than
symmetirical. In Figure 5, however, the KIE value decreases with
increasing the driving force in the region where the driving force is
still negative and thereby the transition state is productlike. Thus, the
driving force dependence of deuterium kinetic isotope effect for ET
from R2Fc to TolSQ in the presence of His ·2H+ (Figure 5) is totally
different from the case of hydride-transfer reactions; see: Hermes, J. D.;
Morrical, S. W.; O’Leary, M. H.; Cleland, W. W. Biochemistry 1984,
23, 5479.
(70) Fukuzumi, S.; Ohkubo, K.; Tokuda, Y.; Suenobu, T. J. Am. Chem.
Soc. 2000, 122, 4286.
(71) The stoichiometry in eq 5 and 6 is confirmed by 1H NMR, where 1
equiv of AcrH2 reacts with 1 equiv of TolSQ (or PQ) to yield 1 equiv
of AcrH+ and TolSQH2 (or PQH2) in the presence of His ·2H+ (or
HClO4).
9
5816 J. AM. CHEM. SOC. VOL. 130, NO. 17, 2008

Electron-Transfer Reduction of a Quinone
A R T I C L E S
Stopped-flow mixing of a deaerated MeCN solution of AcrH2
(1.2 × 10-2 M) and that of TolSQ (9.2 × 10-4 M) containing
HClO4 (9.8 × 10-2 M) results in instant appearance of a
transient absorption band at λmax ) 640 nm (Figure 7b red
line),32which is ascribed to formation of AcrH2•+ that had been
fully characterized including the ESR detection.29a To make sure
•+
of this, AcrH2 have also been detected by applying a rapid-
mixing ESR technique in the thermal oxidation of AcrH2 (2.9
× 10-3 M) with TolSQ (2.8 × 10-3 M) in the presence of
HClO4 (7.0 × 10-2 M). The resulting ESR spectrum (Figure
7c) can be well reproduced by the computer simulation spectrum
(Figure 7d) with the previously reported hfc values [aH (C-9)
) 24.2, aN (N-CH3) ) 14.0, aH (N-CH3) ) 10.4, aH (C-2,7) )
3.4, and aH (C-4,5) ) 1.0 G] due to AcrH2 .
•+ 29a,74 This strongly
supports the formation of AcrH2•+ in the reduction of TolSQH+
by AcrH2. Instant appearance of the transient absorption band
due to AcrH2•+ is also observed, when PQ is employed instead
Figure 6. (a) Dependence of kHH (red circles) on [His ·2H+] for hydride
transfer from AcrH2 (1.0 × 10-4 M) to TolSQ in the presence of His ·2H+,
and that of kHD (red triangles) on [His ·2D+-d6] for hydride transfer from
AcrH2 (1.0 × 10-4 M) to TolSQ in the presence of His ·2D+-d6 in deaerated
MeCN at 298 K. (b) Dependence of kDH (blue circles) on [His ·2H+] for
hydride transfer from AcrD2 (1.0 × 10-4 M) to TolSQ in the presence of
His · 2H+, and that of kDD (blue triangles) on [His · 2D+-d6] for hydride
transfer from AcrD2 (1.0 × 10-4 M) to TolSQ in the presence of His ·2D+-
d6 in deaerated MeCN at 298 K.
•+
of TolSQ (Figure 7e red line). Formation of AcrH2 clearly
indicates the occurrence of ET from AcrH2 to TolSQH+ and
PQH+ (Scheme 7 paths a and b, respectively). No ET from
AcrH2 (Eox ) 0.81 V vs SCE)70 to TolSQ (Ered ) –0.26 V vs
SCE) and PQ (Ered ) –0.65 V vs SCE)59 occurs in the absence
of HClO4, as expected from the highly positive free energy
changes of ET (∆Get ) 1.07 and 1.46 eV, respectively).
However, the Ered values of TolSQ and PQ are shifted to 0.69
and 0.51 V vs SCE, respectively, in the presence of HClO4 (5.0
× 10-2 M) (vide supra). The free energy changes of ET from
AcrH2 to TolSQH+ and PQH+ are still slightly positive (∆Get:
0.12 and 0.30 eV, respectively), which implies occurrence of
the subsequent chemical processes. The efficient ET from AcrH2
to TolSQH+ may be followed by rapid disproportionation of
TolSQH• (Scheme 7a, green arrow), which makes ET oxidation
of AcrH2 undergo to completion. The absence of ESR signal
due to TolSQH• (Figure 7c) suggests occurrence of rapid
disproportionation of TolSQH• that is produced by ET from
AcrH2 to TolSQH+.
Scheme 6
complex between TolSQ and His ·2H+ (TolSQ/His·2H+) which
increases with increasing the His ·2H+ concentration.65,72
On the other hand, HClO4 also promotes reductions of TolSQ
and PQ by AcrH2, however, the mechanism of this is quite
different from that of His ·2H+-promoted hydride transfer from
AcrH2 to TolSQ (Scheme 6) (vide infra). Efficient reductions
of TolSQ and PQ by AcrH2 also occur in the presence of HClO4
(eq 5 and 6, respectively),73 whereas no hydride reduction occurs
in the absence of HClO4.
In the case of the reduction of TolSQH+ by AcrH2 (Scheme
•+
7a), the absorption at 640 nm due to AcrH2 decays ac-
companied by the rise in absorption at 420 nm due to AcrH+
as shown in Figure 7b (red line-blue line).32,75 The resulting
AcrH2•+ decays (and AcrH+ rises) through both first-order and
second-order processes (red and blue circles in inset of Figure
7b), which correspond to the deprotonation and disproportion-
Stoichiometry of eq 5 is confirmed by the spectral titration
of TolSQ by AcrH2 in the presence of HClO4 (Figure 7a), in
•+
ation of AcrH2 as shown by blue and red solid arrows in
Scheme 7a, respectively.32,76 There are large primary kinetic
isotope effects in both the first-order and second-order processes
(kH/kD ) 3.2 and 10, respectively), when AcrH2 is replaced by
(72) TolSQ/His ·2H+ could not be detected, because the formation constant
of the TolSQ/His · 2H+ complex is too small to be detected (vide
supra).
(73) Virtually no protonation of AcrH2 occurs in the presence of HClO4
(4.9 × 10-2 M) containing 30% water. Even when higher concentra-
tion of HClO4 was employed in this system, protonation of AcrH2
hardly occurred in the presence of water in MeCN. Nevertheless,
protonated NADH analogues are generally still capable of donating
electrons to quinones, when the resulting semiquinone radical anion
(Q•-) is stabilized by the protonation (QH•) [see ref 3,4].
which all TolSQ molecules are consumed by addition of 1 equiv
of AcrH2 to yield 1 equiv of AcrH+.32,71 The promoting effects
of HClO4 on reductions of TolSQ and PQ by AcrH2 should
result from protonation of TolSQ (TolSQH+) and PQ (PQH+),
and protonation of which is confirmed by UV–vis spectral
changes of TolSQ and PQ in the presence of various concentra-
tions of HClO4, respectively (see Supporting Information S8).
The dynamics of the reductions of TolSQ and PQ by AcrH2
were examined by using a stopped-flow technique (vide infra).
(74) It should be noted that water contained in HClO4 (70%) significantly
reduces the sensitivity of ESR.
(75) The differential absorption spectra were recorded by subtracting the
final absorption spectrum from the observed spectra during the
reduction of TolSQH+ by AcrH2 as shown in Figure 7b. Thus,
formation of AcrH+ is represented by the disappearance of the negative
absorption band due to AcrH+ (red line-blue line).
(76) Virtually the same first-order and second-order processes were observed
•+
in the decay dynamics of AcrH2 produced by the ET oxidation of
AcrH2 by one-electron oxidants; see: ref 29a.
9
J. AM. CHEM. SOC. VOL. 130, NO. 17, 2008 5817

A R T I C L E S
Yuasa et al.
Figure 7. (a) Absorption spectral changes observed upon addition of AcrH2 (0 to 1.9 × 10-4 M) to a deaerated MeCN solution of TolSQ (1.0 × 10-4 M)
in the presence of HClO4 (1.0 × 10-1 M) at 298 K. (b) Differential spectral changes in the reduction of TolSQ (4.6 × 10-4 M) by AcrH2 (6.0 × 10-3 M)
in the presence of HClO4 (4.9 × 10-2 M) in deaerated MeCN at 298 K. (c) ESR spectrum of AcrH2•+ generated by oxidation of AcrH2 (2.9 × 10-3 M) with
TolSQ (2.8 × 10-3 M) in the presence of HClO4 (7.0 × 10-2 M) in deaerated MeCN at 298 K and (d) the computer simulation spectrum. (e) Differential
spectral changes in the reduction of PQ (4.9 × 10-4 M) by AcrH2 (4.8 × 10-3 M) in the presence of HClO4 (4.9 × 10-2 M) in deaerated MeCN at 298
K. Insets: (a) Plot of [AcrH+]/[TolSQ]0 vs [AcrH2]/[TolSQ]0, where [TolSQ]0 is the initial concentration of TolSQ (1.0 × 10-4 M); time course of the
absorption change at λ ) 640 nm (red) and λ ) 420 nm (blue and green) for the reduction of (b) TolSQ and (e) PQ by AcrH2 (red and blue circles) and
AcrD2 (green triangles). A0 ) the initial absorbance.
AcrD2 (green triangles in inset of Figure 7b)32,77 Since AcrH•
that is formed by deprotonation of AcrH2 (Scheme 7a, blue
line in Figure 7e) prior to appearance of the absorption band at
420 nm due to AcrH+. This suggests that an adduct between
AcrH2•+ and PQH• (or PQH2•+), AcrH-PQH, is initially formed
prior to formation of the final products (green arrow in Scheme
7b).78–81 Such an adduct formation of an NADH analogue with
a p-quinone derivative has been reported previously in cycload-
dition of 1-benzyl-4-tert-butyl-1,4-dihydronicotinamide with
p-benzoquinone.82 The decay time profile of AcrH2•+ (red circles
in inset of Figure 7e) obeys second-order kinetics, exhibiting a
•+
arrow) acts as a much stronger reductant than AcrH2, the rapid
ET from AcrH• (Eox ) –0.46 V vs SCE)70 to TolSQH+ occurs
to yield AcrH+ and TolSQH• (black solid arrow in Scheme 7a).
As a result, 1 equiv of TolSQH+ is reduced by 1 equiv of AcrH2
to yield 1 equiv of AcrH+ and TolSQH2 (Scheme 7a). The
proton transfer from AcrH2•+ to TolSQH• (red broken arrow in
Scheme 7a) is unlikely to occur, because no protonation of
TolSQH• takes place even in the presence of the extremely high
concentration of HClO4 as shown in Figure 1g (vide supra).
In contrast to the reduction of TolSQH+ by AcrH2, the
absorbance at 640 nm due to AcrH2•+ decays immediately (red
circles in inset of Figure 7e) in the reduction of PQH+ by AcrH2
(Scheme 7b). This does not coincide with the slower rise in
absorbance at 420 nm due to AcrH+ (blue circles in inset of
Figure 7e) in the reduction of PQH+ by AcrH2. In such a case,
the absorption band at 640 nm due to AcrH2•+ (red line in Figure
7e) is rapidly replaced by the absorption band at 469 nm (green
(78) Although the transient adduct (AcrH-PQH) could not be fully
characterized, Scheme 7b seems to be the most likely mechanism of
the reduction of PQH+ by AcrH2, judging from other NADH model
reactions with quinones.3,4,82
.
(79) The optimized structure of AcrH-PQH was obtained by DFT calcula-
tions with the BLYP/6-31G** basis set, where the long bond length
(1.68 Å) is found between AcrH and PQ moieties (see Supporting
Information S11).
(80) The slower decay of absorbance changes at 640 nm (red circles in
inset of Figure 7e) may result from the decay of AcrH-PQH that has
a small absorption band at 640 nm. Time constant of the slower decay
of absorbance changes at 640 nm is therefore quite similar to that of
formation of AcrH+ (blue circles in inset of Figure 7e).
(81) The absence of induction period in the formation of AcrH+ (blue circles
in inset of Figure 7e may be ascribed to the deprotonation and
disproportionation of AcrH2•+ prior to formation of the transient adduct
(AcrH-PQH).
(77) The first-order decay rate constant (k1) and the second-order decay
rate constant of AcrH2•+ (k2) were determined as 1.1 × 10-1 s-1 and
6.6 × 103 M-1 s-1 from the first-order and second-order plots,
respectively (see Supporting Information S9).
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5818 J. AM. CHEM. SOC. VOL. 130, NO. 17, 2008

Electron-Transfer Reduction of a Quinone
A R T I C L E S
Figure 8. Electrostatic potential maps for (a) TolSQ, (b) TolSQH+, (c) PQ, and (d) PQH+, calculated by using the density functional theory at the BLYP/
6-31G** level.
Scheme 7
deuterium kinetic isotope effect (kH/kD ) 1.5) (see Supporting
Information S10). The observation of the deuterium kinetic
isotope effect indicates that deprotonation of AcrH2•+ is involved
in the adduct formation (green arrow in Scheme 7b). Heterolysis
of AcrH-PQH (blue arrow in Scheme 7b) affords the final
products, AcrH+ and PQH2.79,83 In fact, the formation dynamics
of AcrH+ obeys first-order kinetics without deuterium kinetic
isotope effect (blue circles in inset of Figure 7e; see Supporting
Information S10).
Protonation of TolSQ and PQ are also expected to result in
enhancement of electrophilicity of TolSQ and PQ to accelerate
one-step hydride transfer from AcrH2 to TolSQH+ and PQH+,
respectively (Scheme 7 paths a and b, black broken arrows,
respectively), as His·2H+-promoted hydride transfer from
AcrH2 to TolSQ (Scheme 6). Electrostatic potential maps for
TolSQH+ (Figure 8b) and PQH+ (Figure 8d) indicate that the
positive charges (blue) due to protonation of TolSQ and PQ
are fully delocalized over the entire ring systems as compared
with those of neutral species (Figure 8, panels a and c,
respectively).84 In such a case, the delocalization of the positive
charge (due to H+) in the protonated species (TolSQH+ and
PQH+) does not lead to the expected increase of electrophilicity
that would promote the ET pathways, when the Ered values of
TolSQ and PQ are shifted to the positive direction in the
presence of HClO4 (Figure 3, panels e and g, respectively) (vide
supra). This may be the reason why ET from AcrH2 to TolSQH+
and PQH+ (Scheme 7 paths a and b, respectively) occurs instead
of the one-step hydride transfer from AcrH2 to TolSQH+ and
PQH+. In the case of His ·2H+-promoted hydride transfer, the
Ered value of TolSQ is also shifted to positive direction (Ered
)
0.29 V vs SCE) (vide supra). However, the free energy change
of ET from AcrH2 (Eox ) 0.81 V vs SCE)70 to TolSQ is still
highly positive (∆Get ) 0.52 eV), when the ET reaction is
thermodynamically infeasible (broken arrow in Scheme 6).
Summary and Conclusions
We have demonstrated a one-step versus stepwise mechanism
in a protonated histidine (His ·2H+)-promoted electron-transfer
(82) Fukuzumi, S.; Fujii, Y.; Suenobu, T. J. Am. Chem. Soc. 2001, 123,
10191.
(84) In contrast to TolSQH•, H+ may be bound to the SdO oxygen, since
the larger negative charge (red) is located on the SdO oxygen as
compared with the CdO oxygens (Figure 8a).
(83) Heterolysis of AcrH-PQH may be accelerated under acidic conditions.
9
J. AM. CHEM. SOC. VOL. 130, NO. 17, 2008 5819

A R T I C L E S
Yuasa et al.
(ET) reduction of 1-(p-tolylsulfinyl)-2,5-benzoquinone (TolSQ)
by ferrocenes (R2Fc) as well as a two-electron reduction by an
NADH analogue, 9,10-dihydro-10-methylacridine (AcrH2). Strong
hydrogen bonding between the semiquinone radical anion of
TolSQ (TolSQ•-) and His ·2H+ was revealed by the presence
of superhyperfine splitting due to NH3+ of His·2H+ in the ESR
spectrum of a hydrogen-bonded complex between TolSQ•- and
His·2H+ (TolSQ•-/His·2H+). Strong hydrogen bonding of
TolSQ•- with His ·2H+ results in a large positive shift of the
one-electron reduction potential of TolSQ, when ET from R2Fc
to TolSQ becomes possible in the presence of His ·2H+. The
ET proceeds in a one-step (concerted) mechanism, that is, ET
from R2Fc to TolSQ is coupled with hydrogen-bond formation
of TolSQ•- with His ·2H+. In such a case, the rates of ET exhibit
deuterium kinetic isotope effects (1.3 < kH/kD < 1.9) due to
partial dissociation of the N-H bond in His ·2H+ by strong
hydrogen bonding of TolSQ with His ·2H+ at the transition state,
whenHis ·2H+ isreplacedbythedeuteratedcompound(His ·2D+-
d6). The one-step mechanism, that is, ET coupled with hydrogen-
bond formation, is continuously changed to the stepwise
mechanism, that is, the rate-limiting ET followed by fast
hydrogen-bond formation, with increasing the ET driving force.
In such a case, the observed deuterium kinetic isotope effect
continuously decreases with increasing the ET driving force to
approach kH/kD ) 1.0. On the other hand, His ·2H+ promotes
the hydride reduction of TolSQ by AcrH2 via the one-step
hydride-transfer mechanism. In such a case, the rates of
His·2H+-promoted hydride transfer exhibit no deuterium kinetic
isotope effect, when His·2H+ is replaced by His·2D+-d6,
whereas large deuterium kinetic isotope effects are observed,
when AcrH2 is replaced by AcrD2. In contrast to His · 2H+-
promoted hydride transfer, perchloric acid (HClO4) without His
promotes hydride reductions of TolSQ and 9,10-phenanthrene-
quinone (PQ) by AcrH2 via ET from AcrH2 to TolSQH+ and
PQH+, respectively. Thus, His regulates ET from AcrH2 to
TolSQH+, leading to the change of the mechanism of the two-
electron reduction of the p-quinone derivative (TolSQ) by the
NADH analogue (AcrH2) from the ET pathway to the one-step
hydride-transfer pathway.
Acknowledgment. This work was partially supported by
Grants-in-Aid (No. 19205019) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
Supporting Information Available: Hydrogen-bond lengths
•+
in TolSQ•-/His·2H+ (S1); ESR spectra of PQD2 (S2), and
PQH• (S3); first-order plot (S4), and dependence of kobs on
[TolSQ] (S5) for His ·2H+-promoted ET from R2Fc to TolSQ;
first-order plot (S6) and dependence of kobs on [TolSQ] (S7)
for the His ·2H+-promoted hydride reduction of TolSQ by
AcrH2; UV–vis spectra of TolSQ and PQ in the presence of
HClO4 (S8); first-order and second-order plots for the reductions
of TolSQ (S9) and PQ (S10) by AcrH2 and AcrD2; and the
optimized structure of AcrH-PQH calculated by DFT at the
BLYP/6-31G** level (S11). This material is available free of
charge via the Internet at http://pubs.acs.org.
JA8001452
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5820 J. AM. CHEM. SOC. VOL. 130, NO. 17, 2008