Radiation-induced and photosensitized splitting of c5-c5′-linked dihydrothymine dimers. 2. conformational effects on the reductive splitting mechanism

Article abstract of DOI:10.1021/jp9939367

Radiation-induced and photosensitized reductive splitting of stereoisomeric C5-C5′-linked dihydrothymine dimers (1a,b[meso], meso compound of (5R,5′S)- and (5S,5′R)-bi-5,6-dihydrothymines; 1a,b[rac], racemic compound of (5R,5′R)- and (5S,5′S)-bi-5,6-dihydrothymines) in aqueous solution were studied to compare with the one-electron oxidative splitting mechanism and the photorepair reaction of cyclobutane pyrimidine photodimers. Reacting with radiation-chemically and photochemically generated hydrated electrons or with photoexcited reduced form of flavin adenine dinucleotide (*FADH^-), the C5-C5′-linked dihydrothymine dimers 1a,b produced the corresponding 5,6-dihydrothymine derivatives (3a,b) along with the thyrnine monomers (2a,b) in minor yields. Both the product and laser flash photolysis studies indicated that oneelectron adducts of the C5-C5′-linked dimers 1a,b undergo C5-C5′-bond cleavage to generate the 5,6-dihydrothymin-5-yl radicals (5a,b) and the 5,6-dihydrothymine C5-anions (6a,b) resulting in the formation of 3a,b by facile protonation at C5. In the reduction by *FADH^-, splitting of the 5,6-dihydro-1-methylthymine dimer 1a[meso] into the monomer 2a was more efficient than that of the racemic isomer 1a[rac]. Conformational analysis by NMR of 1a[meso] and 1a[rac] in solution suggested that 1a[meso] may favor a "closed-shell" conformation and undergo one-electron reduction to form 5a and 6a, whereas 1a[rac] may be in a "opened-shell" conformation and undergo successive two-electron reduction by *FADH^- to produce 2 equiv of 6a as a precursor of 3a.

Full text of DOI:10.1021/jp9939367

2886
J. Phys. Chem. A 2000, 104, 2886-2893
Radiation-Induced and Photosensitized Splitting of C5-C5′-Linked Dihydrothymine
Dimers. 2. Conformational Effects on the Reductive Splitting Mechanism
Takeo Ito,† Hideki Shinohara,† Hiroshi Hatta,† Shin-ichi Fujita,§ and Sei-ich Nishimoto*,†
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto UniVersity,
Sakyo-ku, Kyoto 606-8501, Japan, and Research Institute of AdVanced Technology, UniVersity of Osaka
Prefecture, Osaka 599-8231, Japan
ReceiVed: NoVember 5, 1999; In Final Form: January 24, 2000
Radiation-induced and photosensitized reductive splitting of stereoisomeric C5-C5′-linked dihydrothymine
dimers (1a,b[meso], meso compound of (5R,5′S)- and (5S,5′R)-bi-5,6-dihydrothymines; 1a,b[rac], racemic
compound of (5R,5′R)- and (5S,5′S)-bi-5,6-dihydrothymines) in aqueous solution were studied to compare
with the one-electron oxidative splitting mechanism and the photorepair reaction of cyclobutane pyrimidine
photodimers. Reacting with radiation-chemically and photochemically generated hydrated electrons or with
photoexcited reduced form of flavin adenine dinucleotide (*FADH-), the C5-C5′-linked dihydrothymine
dimers 1a,b produced the corresponding 5,6-dihydrothymine derivatives (3a,b) along with the thymine
monomers (2a,b) in minor yields. Both the product and laser flash photolysis studies indicated that one-
electron adducts of the C5-C5′-linked dimers 1a,b undergo C5-C5′-bond cleavage to generate the 5,6-
dihydrothymin-5-yl radicals (5a,b) and the 5,6-dihydrothymine C5-anions (6a,b) resulting in the formation
of 3a,b by facile protonation at C5. In the reduction by *FADH-, splitting of the 5,6-dihydro-1-methylthymine
dimer 1a[meso] into the monomer 2a was more efficient than that of the racemic isomer 1a[rac].
Conformational analysis by NMR of 1a[meso] and 1a[rac] in solution suggested that 1a[meso] may favor a
“closed-shell” conformation and undergo one-electron reduction to form 5a and 6a, whereas 1a[rac] may be
in a “opened-shell” conformation and undergo successive two-electron reduction by *FADH- to produce 2
equiv of 6a as a precursor of 3a.
Introduction
todimer radical anions, followed by electron transfer back to
the flavin, reproduces the monomeric pyrimidines. Previous laser
flash photolysis and photoinduced CIDNP spectroscopic studies
have detected the radical anion intermediates in the reductive
splitting reaction, indicating that the splitting of the photodimer
radical anion involves a concerted but nonsynchoronous frag-
mentation of C5-C5′ and C6-C6′ bonds with a rate constant
Ionizing radiation and ultraviolet (UV) light are the etiological
agents of gene carcinogenesis and mutagenesis that give rise to
a variety of DNA-base modifications.1,2 Pyrimidine cyclobutane
photodimers are among the most representative DNA-base
lesions, being produced as a consequence of [2 + 2] photocy-
cloaddition at a tandem pyrimidine moiety in the DNA duplex
upon exposure to UV light.2 The base residues thus modified
are subjected to an intracellular repair process of photoreacti-
vation by a photoenzyme, DNA photolyase, consisting of a
deprotonated reduced flavin adenine dinucleotide (FADH-) and
an antenna chromophore of methenyltetrahydrofolate (MTHF)
or 8-hydroxy-5-deazaflavin (8-HDF). While both radical cation
and radical anion of the photodimer undergo facile fragmentation
into monomers as confirmed by the model photochemical
electron-transfer reactions, the enzymatic photoreactivation may
favor a reductive fragmentation.3 Thus, a likely mechanism of
the photoreactivation has been drawn from extensive biochemi-
cal and photochemical studies:3 the antenna chromophore of
the photolyase complexed to the photodimer in DNA absorbs
light in the 300-500 nm range and transfers the excitation
energy to the reduced flavin FADH- moiety, thereby splitting
of the cyclobutane photodimer being induced via one-electron
transfer from the excited state of FADH-. Concerted C5-C5′
and C6-C6′ bond splitting of the resulting cyclobutane pho-
of the order 1 × 106 s-1 4,5
.
An alternative [2 + 2] photocycloaddition between adjacent
pyrimidines is simultaneously induced by UV exposure to result
in a similar mutagenic photolesion of pyrimidine(6-4)pyrimi-
done [(6-4)photoadduct]. A photoenzyme (6-4)photolyase that
shows a specific repairing ability toward the (6-4)photoadduct
has also been identified.6 As characterized more recently by a
sequence analysis of the genes, the (6-4)photolyase has a high
degree of structural identity to the DNA photolyase.6c These
results strongly suggest a similarity of the repair mechanism
between the (6-4)photolyase and DNA photolyase, involving
a common electron-transfer process from the excited state of
the enzyme to the UV-damaged DNA bases.6c-e
Recently, we have identified formation of stereoisomeric C5-
C5′-linked dihydrothymine dimers, as fractionated into the meso
compounds of (5R,5′S)- and (5S,5′R)-bi-5,6-dihydrothymines
(1a,b[meso]) and racemic mixtures of (5R, 5′R)- and (5S,5′S)-
bi-5,6-dihydrothymines (1a,b[rac]), in the radiolytic one-
electron reduction of thymine derivatives [1-methylthymine (2a)
and 1,3-dimethylthymine (2b)] in anoxic aqueous solution.7 The
X-ray crystallography demonstrated that the C5-C5′-linked
dihydrothymine dimers are structurally similar to the cyclobu-
tane thymine photodimers. Especially, as in the cis-syn cy-
* Corresponding author. Fax: +81(75)753-3351. E-mail: nishimot@
scl.kyoto-u.ac.jp.
† Graduate School of Engineering, Kyoto University.
§ Research Institute of Advanced Technology, University of Osaka
Prefecture.
10.1021/jp9939367 CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/11/2000

Splitting of C5-C5′-Linked Dihydrothymine Dimers
J. Phys. Chem. A, Vol. 104, No. 13, 2000 2887
CHART 1
C18 reversed-phase column (Wakosil 5C18, o.d. 4.6 mm × 150
mm, Wako). The phosphate buffer solutions (10 mM, pH 3.0)
containing various concentrations of methanol (10-25 vol %)
were delivered as the mobile phase. The column eluents were
monitored by the UV absorbance at 210 nm.
Radiolytic Reduction. Aqueous solutions of C5-C5′-linked
dihydrothymine dimers 1a,b (0.5 mM) containing 2-methyl-2-
propanol (50 mM) were buffered at pH 7.0 with phosphate
buffer, and then purged with Ar before γ-irradiation. Steady-
state γ-irradiation was performed in sealed ampules at room
temperature with a 60Co γ-ray source (dose rate: 0.75 Gy
min-1).
Photosensitized Reduction. Typically, solutions of 1a,b (0.5
mM) in phosphate buffer containing FAD (0.2 mM) were purged
with Ar before photoirradiation. For effective generation of the
reduced form of FAD (FADH-) in situ, EDTA (20 mM) was
added to the solutions. The solutions in sealed Pyrex glass tubes
were photoirradiated (λex > 280 nm) under magnetic stirring
(1000 rpm) at 24 °C with a high-pressure Hg arc (450 W, Eiko-
sha 400).
Nanosecond Laser Flash Photolysis. The laser flash pho-
tolysis experiments were carried out with a Unisoku TSP-601
flash spectrometer, as described previously.8 Aqueous solutions
of 1a,b[meso] and 1a,b[rac] (1.0 mM) at pH 6.7 containing
DMA (0.16-0.50 mM) were deaerated by Ar bubbling prior
to the laser flash photolysis experiments.
Fluorescence Quenching. Various kinds of quenchers (0.1-
4.0 mM) at varying concentrations were dissolved in phosphate
buffer (10 mM, pH 7.0) solutions of Ru(bpy)32+ (10 µM). Each
sample solution (2 mL) was purged with Ar for 10 min, sealed
in a quartz cell (10 × 10 × 35 mm) with a Teflon cap, and
then subjected to the measurement of the fluorescence spectrum
which was recorded on a Hitachi F-2000 fluorescence spectro-
photometer (slit width: 1.0 nm, λex ) 450 nm, λflu ) 600 nm).
clobutane thymine photodimers, the meso-dimer structure like
1a[meso] seemed to cause insufficient stacking of the pyrimidine
rings that will weaken the hydrogen bonds between the dimer
and the complementary adenine when formed by chance in a
DNA duplex. A remarkable observation with respect to the
reactivity was that the C5-C5′-linked dihydrothymine dimers
1a,b undergo oxidative splitting to regenerate the corresponding
thymine monomers 2a,b via one-electron transfer from the dimer
to radiation chemically or photochemically generated oxidants.8
Formally, this oxidative splitting of 1a,b is a reverse reaction
of the reductive dihydrodimerization of 2a,b.
In view of the fact that the splitting of cyclobutane photo-
dimers into monomers occurs in principle by both oxidative
and reductive pathways, further attempt has been made to
characterize reductive splitting reactivity of the C5-C5′-linked
dihydrothymine dimers 1a,b under conditions of generating
hydrated electrons (eaq-) or photoexcited state of dihydroflavin
FADH-. Laser flash photolysis study using N,N-dimethylaniline
as a reducing photosensitizer has also been conducted to identify
the mechanism and evaluate the kinetic parameters involved in
the reductive splitting of 1a,b.
Results
Experimental Section
Splitting of C5-C5′-Linked Dihydrothymine Dimers by
Hydrated Electrons Generated in the Radiolysis. γ-Radioly-
ses of deoxygenated solutions of 1-methyl-5,6-dihydrothymine
dimers (1a[meso] and 1a[rac]; 0.5 mM) and 1,3-dimethyl-5,6-
dihydrothymine dimers (1b[meso] and 1b[rac]; 0.5 mM) in
phosphate buffer containing 2-methyl-2-propanol (50 mM) were
carried out to characterize the reductive splitting reactivity of
the C5-C5′-linked dihydrothymine dimers. In the radiolysis of
a diluted aqueous solution, water radicals such as oxidizing
hydroxyl radicals (•OH), reducing hydrated electrons (eaq-), and
reducing hydrogen atoms (•H) are primarily generated with the
G values9 of G(•OH) ) 2.8 × 10-7 mol J-1, G(eaq-) ) 2.8 ×
10-7 mol J-1, and G(•H) ) 0.6 × 10-7 mol J-1 (reaction 1).
Under the present conditions, the OH radicals are scavenged
by excess amount of 2-methyl-2-propanol into substantially
unreactive 2-hydroxy-2-methylpropyl radicals (•CH2(CH3)2-
COH) as in reaction 2. Consequently, the hydrated electrons
eaq-, (E(nH2O/eaq-) ) -2.9 V vs NHE10 at pH 7.0) along with
smaller amount of the hydrogen atoms (E(H+/•H) ) -2.4 V vs
NHE10 at pH 7.0) are involved as the reductants in the reactions
of 1a,b.
Materials. 1-Methylthymine (2a) and flavin adenine dinucle-
otide (FAD) disodium salt were used as received from Sigma
Chemical. Purified 1,3-dimethylthymine (2b) was kindly sup-
plied by Fujii Memorial Research Institute, Ohtsuka Pharma-
ceutical. Ethylenediaminetetraacetic acid (EDTA), N,N-di-
methylaniline (DMA), and 2-methyl-2-propanol were purchased
from Nacalai Tesque and used without further purification.
N,N,N′,N′-Tetramethyl-p-phenylenediamine (TMPD), 2′-deoxy-
thymidine, and reagents for high-performance liquid chroma-
tography (HPLC) including solvents, sodium dihydrogen phos-
phate (NaH2PO4), and methanol (HPLC grade) were used as
received from Wako Pure Chemical Industries. For fluorescence
quenching experiments, tris(2,2′-bipyridyl)ruthenium(II) chloride
hexahydrate (Ru(bpy)3Cl2‚6H2O; Aldrich Chemical), dimeth-
ylbenzoquinone (Aldrich Chemical), terephthalaldehyde (Kanto
Chemical), 1-methylnicotinamide chloride (Tokyo Chemical
Industries), 5-nitrouracil (Tokyo Chemical Industries), methyl
viologen (Nacalai Tesque), anthraquinone-2-sulfonate (Nacalai
Tesque), and p-benzoquinone (Nacalai Tesque) were used as
obtained commercially. C5-C5′-linked dihydrothymine dimers
1a,b of 1-methylthymine 2a and 1,3-dimethylthymine 2b were
synthesized and purified following the methods reported previ-
ously.7 Aqueous solutions for all experiments were prepared
using water purified with Corning Mega-Pure System MP-190
(>16 MΩ cm).
H2O f •OH, •H, eaq-, H+, H2, H2O2
(1)
•OH + (CH3)3COH f H2O + •CH2(CH3)2COH (2)
HPLC Analysis. Analytical HPLC was performed with
Shimadzu 6A HPLC system equipped with Rheodyne 7725
sample injector. Sample solutions were injected onto a 5 µm
Analytical HPLC of the γ-irradiated solutions by reference
to authentic samples demonstrated that the C5-C5′ linkages

2888 J. Phys. Chem. A, Vol. 104, No. 13, 2000
Ito et al.
SCHEME 1
SCHEME 2
TABLE 1: Reductive Splitting of 1a,b (0.5 mM) in
Deoxygenated Aqueous Solution by Radiation-Chemically
Generated Hydrated Electrons
TABLE 2: Reductive Splitting of 1a,b (0.5 mM) by
Photoexcited Reduced Form of FAD (*FADH-) in Phosphate
Buffer
G values × 107/mol J-1
yield/%
conversion/%
-1
ratio/%
2/3
substrate
additive
FAD
FAD/EDTA
FAD
FAD/EDTA
FAD
FAD/EDTA
FAD
2
3
substrate
-1
2
3
isomerizn
1a[meso]
4
30
5
26
11
42
11
28
17
1
5
1
7
2
8
2
34
79
72
75
97
86
93
91
50
1
7
1
7
2
9
2
1a[meso]
1a[rac]
1b[meso]
1b[rac]
2.2
2.3
2.7
2.9
0.59 (13%)a
0.36 (8%)
0.80 (15%)
0.96 (17%)
2.1 (48%)
2.0 (43%)
2.8 (52%)
2.9 (50%)
0.25 (11%)
0.32 (14%)
0.17 (6%)
0.31 (11%)
1a[rac]
1b[meso]
1b[rac]
a Selectivities based on G values for decomposition of 1a,b.
FAD/EDTA
of stereoisomeric dimers 1a,b[meso] and 1a,b[rac], split via
one-electron reduction by hydrated electrons to produce the
corresponding 5,6-dihydrothymine derivatives (3a,b) as the
major products along with relatively smaller amounts of thymine
monomers 2a,b (Scheme 1). The one-electron reductive splitting
of 1a,b to 2a,b and 3a,b was accompanied to a lesser extent by
isomerization from meso dimers 1a,b[meso] to racemic dimers
1a,b[rac] or vice versa. Apart from the distribution of product
yields (see Table 1), the apparent reaction characteristics of one-
electron reductive splitting are essentially the same as in the
one-electron oxidative splitting reported recently.8 Figure 1
shows linear dose-responses of the dimer decomposition and
the product formation in the radiolytic reduction, the slopes of
which gave the respective G values as listed in Table 1.
Comparing with the G value of hydrated electrons (G(eaq-) )
2.8 × 10-7 mol J-1) as the substantial species for one-electron
reduction, it is apparent that the stereoisomeric 1,3-dimethyl-
5,6-dihydrothymine dimers 1b underwent reductive decomposi-
tion almost quantitatively (G(-1b) ) (2.7-2.8) × 10-7 mol
J-1), while the stereoisomeric 1-methyl-5,6-dihydrothymine
dimers 1a showed somewhat smaller reactivity toward hydrated
electrons (G(-1a) ) (2.2-2.3) × 10-7 mol J-1). In these
radiolytic reductions, the major products of 5,6-dihydrothymines
3a,b and thymines 2a,b accounted for about 50% and 15% of
the decomposed dimers, respectively. The minor isomerization
occurred in 6-14% yields, which is comparable to the isomer-
ization in the previously reported one-electron oxidative split-
ting.8
gave only 3a as a major product, suggesting that the intermediate
5-yl radical is one-electron reduced almost quantitatively by
TMPD into C5-anion which undergoes protonation at C5 to form
3a (Scheme 2). Consequently, the restoration of 2a and the
isomerization may be suppressed in the presence of TMPD.
Splitting of C5-C5′-Linked Dihydrothymine Dimers by
Photoexcited Reducing Sensitizer. It has been suggested that
in the DNA photorepair process the cyclobutane photodimer
flips out of the DNA helix to fit into the “hole” domain of
photolyase,3k-o and the fully reduced form of FAD cofactor
(FADH-) can catalyze the splitting reaction. In view of the
photorepair reaction, FADH--sensitized photoreductive splitting
reaction of the C5-C5′-linked dihydrothymine dimers 1a,b was
performed as a related model reaction. Upon photoexcitation
of FAD (0.2 mM) in deoxygenated phosphate buffer containing
1a,b (0.5 mM) in the presence of EDTA (20 mM), splitting of
1a,b occurred to afford 5,6-dihydrothymine derivatives 3a,b in
considerably high yield (> 75%) along with the minor yield
(<10%) of monomeric thymines 2a,b, in which the formation
of 3a,b were more efficient than that in the radiation chemical
reductions (Table 2). In the presence of a secondary reductant
EDTA that converts FAD into a fully reduced form FADH- in
situ, hydrogen transfer from EDTA to intermediate 5,6-
dihydrothymin-5-yl radicals 5a,b is possibly involved in the
enhanced formation of 3a,b. To remove the influence of the
secondary reaction by EDTA, photoreactivity of the dimers in
phosphate buffer was also investigated in the absence of EDTA.
In a separate pulse radiolysis study, we confirmed that phosphate
buffer is a weak reductant toward several radicals.13 It is
therefore likely that FAD may undergo photoreduction by
phosphate buffer even in the absence of EDTA to produce
FADH-.14 As shown in Table 2, while the efficiencies for
decomposition of 1a,b were decreased to significant extent, the
yields of 2a,b relative to 3a,b became higher upon excluding
hydrogen-donating EDTA. It is also remarkable that the
restoration efficiency of 2a from 1a[meso] in the absence of
EDTA is much greater than those from 1a[rac] 1b[meso], and
1b[rac], and is comparable to those in the reductive γ-radiolysis
(see also Table 1).
The above results are rationalized by a mechanism involving
the primary intermediates of electron-dimer adducts (4a,b) that
undergo splitting at the C5-C5′ linkages into 5,6-dihydrothymin-
5-yl radicals (5a,b) and C5-anions (6a,b), as outlined in Scheme
4. The formation of the 5-yl radicals 5a,b is common to both
the one-electron reductive and oxidative splittings, while the
counterparts in the latter splitting are C5-cations (7a,b).8 It has
been established that â-oxoalkyl radicals possess oxidizing
property for several aromatic amines; e.g., 6-hydroxy-5,6-
dihydrothymin-5-yl radical oxidizes TMPD [E°(TMPD•+/
TMPD) ) 0.16 V vs NHE]11 readily to form 6-hydroxy-5,6-
dihydrothymine C5-anion and TMPD radical cation (TMPD•+).12
In accord with such an oxidizing property, the 5,6-dihy-
drothymin-5-yl radicals 5a,b could one-electron oxidize 5,6-
dihydrothymine dimers 1a,b to regenerate 5a,b along with the
formation of C5-cations 7a,b that produce thymines 2a,b via
protonation, as reported recently.8 Separate experiments with
deoxygenated solution of 1a[meso] containing TMPD (0.6 mM)
For comparing the reduction potentials of the four C5-C5′-
linked isomeric dimers investigated, a fluorescence quenching
kinetic study was carried out using tris(2,2′-bipyridyl)ruthe-
nium(II) complex as a fluorescent electron donor. Recently, the
reduction potentials of pyrimidine bases were successfully

Splitting of C5-C5′-Linked Dihydrothymine Dimers
J. Phys. Chem. A, Vol. 104, No. 13, 2000 2889
Figure 1. Reductive splitting of 1a[rac] (0.5 mM) in the γ-radiolysis
of deoxygenated phosphate buffer containing 2-methyl-2-propanol (50
mM): (0) decomposition of 1a[rac]; formation of ([) 1-methylthymine
2a, (b) 5,6-dihydro-1-methylthymine 3a, and (O) isomeric dimers
1a[meso].
Figure 2. Rehm-Weller plot of the fluorescence quenching rate
constant (kq) in aqueous solution as a function of the reduction potential
(Ered) of quencher. The solid curve is obtained from a best fit of
experimental data to the Rehm-Weller empirical expression (see eqs
3-7).
2+
TABLE 3: Ru(bpy)3 Fluorescence Quenching Rate
2
1/2
Constants and Reduction Potential (vs SCE) of Various
Quenchers in Aqueous Solution
∆Get
∆Get
2
λ
4
∆Gqet )
+
+
(4)
(5)
(6)
(
[
)
]
( )
2
2
quencher
Ered/V
kq /109 dm3 mol-1 s-1
q2
rꢀ
p-benzoquinone
dimethylbenzoquinone
nitrofurantoin
3,5-dinitrobenzoic acid
anthraquinone-2-sulfonate
methyl viologen
-0.16a
-0.32a
-0.50a
-0.58a
-0.63a
-0.69a
-0.77a
-1.01b
-1.04b
-1.39b
6.32
4.56
11.4
7.16
6.73
5.13
4.58
0.323
1.40
0.0379
∆G ) 96.5 E/ox ) Ered
-
(
)
et
k-diff
1
)
kdiff
Keq
5-nitrouracil
1-methylnicotinamide
terephthaldialdehyde
1,4-dimethylpyridinium
where λ refers to solvent reorganization energy, -q2/rꢀ is the
Coulomb energy of the incipient ion pair formed after electron
transfer, and ka ()κelνn) is the preexponential term of the
expression for the rate constant of electron transfer.
a Reference 10. b Reference 19. The reduction potentials vs NHE
are converted to the values vs SCE.
It is evident in Figure 2 that kq approaches the diffusion
control limit (kdiff ) 6.5 × 109 dm3 mol-1 s-1 in aqueous
solution) as the reduction potential becomes increasingly positive
(Figure 2). The best fit of the Rehm-Weller relationship in
Figure 2 was obtained with kinetics parameters of kaKeq ) 5.0
× 1012 dm3 mol-1 s-1 and λ ) 117 kJ mol-1. The rate constants
of quenching by 1a,b[meso] and 1a,b[rac] were also determined
by a Stern-Volmer analysis (Figure 3), from which the
corresponding redox potentials were estimated based on the best
fitted Rehm-Weller relationship. Thus, almost the same kq
values for isomers of 1a (kq(1a[meso]) ) 5.48 × 108 dm3 mol-1
s-1, kq(1a[rac]) ) 6.66 × 108 dm3 mol-1 s-1) and 1b
(kq(1b[meso]) ) 2.50 × 108 dm3 mol-1 s-1, kq(1a[rac]) ) 2.58
× 108 dm3 mol-1 s-1) were obtained, respectively, leading to
the reduction potentials of Ered(1a) ≈ -1.15 and Ered(1b) ≈
-1.25 V vs SCE independent of isomeric structures. These Ered
values are slightly positive compared to those of thymine (Ered
) -1.34 V vs SCE) and thymidine (Ered ) -1.33 V) in aqueous
solution.19 It follows that the difference in restoration efficiency
between the isomeric dimers cannot be explained in terms of
the reduction potentials.
Laser Flash Photolysis Study. Laser flash photolyses with
the fourth harmonic at 266 nm of aqueous solutions of the
stereoisomeric dimers 1a,b (1.0 mM) containing N,N-dimethyl-
aniline (DMA; 0.5 mM) as a reducing sensitizer were attempted
to detect transient absorptions originating from the splitting
reaction. Previous laser flash photolysis study has shown that
DMA possessing an excited-state oxidation potential of -3.3
V is a good electron donor for splitting of the cyclobutane
photodimers by a single-electron-transfer mechanism.4 In this
study, upon laser photolysis of DMA in deoxygenated aqueous
solution, hydrated electrons and radical cations of DMA
estimated from the rate constants of electron-transfer fluores-
cence quenching of several electron donors with varying
oxidation potentials.15 In general, electron transfer from an
excited-state fluorescent molecule (electron donor D*) to a
quencher molecule (electron acceptor A) proceeds as follows:
D and A diffuse to encounter at a distance where electron-
transfer becomes favorable, and thereafter an ion-pair state (D•+
A•-) forms to undergo dissociation into free radical ions D•+
and A•-.
D + A 9
hV8
ket
kdis
D* + A {kk } (D‚‚‚A)* {k } (D•+ A•-) 8 D•+ + A•-
diff
-diff
-et
Upon 450 nm excitation of Ru(bpy)32+ (10 µM; E*ox ) -1.29
V11,16) in aqueous solution, the characteristic fluorescence was
observed with a maximum wavelength of 600 nm. The
fluorescence quenching rate constants (kq) of [Ru(bpy)32+]* by
a series of electron-accepting quenchers (see Table 3) were
determined by a Stern-Volmer analysis.17 Figure 2 shows a
plot of kq against the reduction potentials of quenchers, in which
the Rehm-Weller relationship (eqs 3-6)18 was fitted to the
experimental data.
kdiff
kq )
(3)
k-diff
kaKeq
∆Gqet
1 +
exp
{
(
)}
RT

2890 J. Phys. Chem. A, Vol. 104, No. 13, 2000
Ito et al.
TABLE 4: Rate Constants for One-Electron Reductions of
1a,b and 5a,b with Hydrated Electrons (eaq-) and DMA,
Respectively
k/dm3 mol-1 s-1
substrate
eaq- + 1
DMA + 5
1a[meso]
1a[rac]
1b[meso]
1b[rac]
4.4 × 109
3.7 × 109
2.6 × 109
3.0 × 109
3.1 × 109
4.8 × 109
7.1 × 109
6.7 × 109
SCHEME 3
Figure 3. Stern-Volmer plots of the fluorescence quenching of
*Ru(bpy)32+ by (b) 1a[meso] and (O) 1a[rac]. Relative intensity (I0/I)
of the emission at 600 nm was measured in the presence of 10 µM
2+
Ru(bpy)3 in deoxygenated phosphate buffer solution (10 mM, pH
7).
Figure 4. Time courses of the absorbancies at 460 nm in the 266 nm
laser photolysis of DMA (0.5 mM) in phosphate buffer containing
1b[rac] (1.0 mM): in the (O) presence and (b) absence of 1b[rac].
Figure 5. Dependence of kobs of differential absorption growth at 460
nm on the concentration of DMA.
-
Inset: Kinetic plot of the absorption at 650 nm due to eaq in the
presence of DMA.
with a first-order decay profile for DMA•+ generated by
photoionization (Figure 4). It is therefore most likely that the
oxidizing 5-yl radicals 5a,b, which should be generated by the
C5-C5′ bond splitting of the primary intermediate radical anions
of 1a,b (reaction 9), are spontaneously reduced by DMA to form
the corresponding C5-anions and DMA•+ (reaction 10), as in
the reaction of 5a,b with TMPD (Scheme 3).12
(DMA•+) were generated with the characteristic transient
absorptions at λmax ) 460 and 650 nm, respectively.20,21
Therefore, the hydrated electron generated by the direct pho-
toionization of DMA (reactions 7 and 8) may be responsible
for reductive splitting of the C5-C5′-linked dimers, rather than
the photoexcited DMA (DMA*).
-
DMA + hν f DMA•+ + eaq
T-T + eaq- f T-T•-
(7)
(8)
T-T•- f T- + T•
(9)
T• + DMA (TMPD) f T- + DMA•+ (TMPD•+) (10)
Transient absorption spectra of DMA•+ (λmax ) 460 nm) and
hydrated electron (λmax ) 650 nm) were detected immediately
after the laser flash photolysis of deoxygenated aqueous
solutions of 1a,b and DMA. As shown in Figure 4, the
absorption at 650 nm due to hydrated electrons decayed
following pseudo-first-order kinetics (k ) 2.6-4.4 × 109 dm3
mol-1 s-1, [1a,b] ) 1.0 mM), possibly as a consequence of the
diffusion-controlled reaction with 1a,b (Table 4). Previous pulse
radiolysis and laser flash photolysis studies have demonstrated
that N-substituted 5,6-dihydrothymin-5-yl radicals 5a,b show
broad absorption spectra at around 400 nm (5a: λmax ) 400
nm;8 5b: λmax ) 430 nm22), but such transient absorption spectra
of 5a,b were not observed in this study. Alternatively, a slow
buildup component of the transient absorption of DMA•+ was
observed about 5 µs after the laser flash excitation, concomitant
In accord with the secondary one-electron oxidation of DMA
in the ground state, the slow buildup component of DMA•+
could be obtained by subtracting the contribution from the decay
of primarily photogenerated DMA•+, as occurred within 5 µs
after the laser flash, from the apparent overall absorption at 460
nm in the presence of the dimers. Thus, the slow buildup of
DMA•+ was of the pseudo-first-order kinetics, and the corre-
sponding formal rate constant was in proportion to the concen-
tration of DMA (Figure 5). The slope of the linear plot in Figure
5 gives the intrinsic rate constants for reaction 10, as listed in
Table 4. The rate constants thus evaluated are almost in a
diffusion-controlled rate limit and are in good accordance with
the literature value for the one-electron oxidation of TMPD by
5,6-dihydro-6-hydroxythymin-5-yl radical (k ) 1.3 × 109 dm3
mol-1 s-1) as derived from pulse radiolysis.12

Splitting of C5-C5′-Linked Dihydrothymine Dimers
J. Phys. Chem. A, Vol. 104, No. 13, 2000 2891
SCHEME 4
SCHEME 5
to a pair of C5-anion 6a,b and C5-cation (7a,b) is involved in
the reductive splitting pathway (Scheme 4, path 2), which is a
common reaction with the oxidative splitting mechanism.8
Flavin-Photosensitized Splitting of C5-C5′-Linked Dihy-
drothymine Dimers. Because of the existence of multiple
protonation-reduction states of the flavin chromophore, the
photoreactivation mechanism of photodimers has not been fully
understood. The most likely mechanism involves initiation by
exoergic one-electron transfer from the excited state of FADH-
in DNA photolyase to the photodimers (∆Get ) -125 ( 30 kJ
mol-1 24
)
with rate constants of ket ) (5.5-6.5) × 109 s-1 25
.
The Rehm-Weller analysis on the flavin-photosensitized split-
ting of C5-C5′-linked dimers gave reasonable reduction
potentials Ered ) -1.15-1.25 V, which are slightly more
positive than those of the monomers 2a,b. Taking into account
the reduction (E(FADH•/FADH-) ) -0.364 V at pH 7.0),26
and the excitation energy (E00(*FADH-/FADH-) ) 2.58 V)27
of FADH-, the free energy for the electron transfer from
*FADH- to the dimers 1a,b is calculated from eq 5 as ∆Get )
-79.4 kJ mol-1, which shows exothermicity of the reaction.
Discussion
Reductive Splitting Mechanism of C5-C5′-Linked Dihy-
drothymine Dimers. In light of the proposed mechanisms for
the photorepair of cyclobutane pyrimidine dimers3 and the
oxidative splitting of the C5-C5′-linked dihydrothymine dimers,8
possible pathways for the reductive splitting of C5-C5′-linked
dihydrothymine dimers are proposed as illustrated in Schemes
4 and 5. Under the present experimental conditions of photo-
chemical and radiation-chemical generation of hydrated elec-
trons, the splitting is likely to be initiated by one-electron
reduction of the C5-C5′-linked dimers into the dimer radical
anions 4a,b, although direct observation of the corresponding
transient absorption spectra was unsuccessful in the present
nanosecond laser flash photolysis. In view of the earlier results
that the formation of radical anion intermediate of cyclobutane
pyrimidine photodimer (Pyr<>Pyr•-) could not be confirmed
clearly by nano- and picosecond laser flash photolysis, such
radical anion intermediates 4a,b may have much shorter
lifetimes.4,23 Subsequent C5-C5′-bond cleavage of 4a,b pro-
duces the C5-anions (6a,b) and the 5,6-dihydrothymin-5-yl
radicals 5a,b, as confirmed by the redox reaction with reducing
aromatic amines (DMA and TMPD). The isomerization of C5-
C5′-linked dimers supports the formation of 5a,b (Scheme 4,
path 3). Concomitant formation of 2a,b in the absence of
aromatic amines suggests that disproportionation of 5a,b leading
The remarkably high efficiency of thymine restoration in the
photoreduction of 1a[meso] by *FADH-, compared with those
of the other dimers (1a[rac], 1b[meso], and 1b[rac]), could not
be explained by the difference in the initial electron transfer
efficiency. As a possibility, this feature may be related to the
X-ray crystal structures of 1a[meso] that are distinct from those
of 1a[rac] 1b[meso], and 1b[rac].7b For better understanding,
the frame structures of 1a[meso] and 1a[rac] are illustrated in
Figure 6.7b In a crystal, 1a[meso] has a stacked conformation
by which the two pyrimidine rings face each other, while the
pyrimidine rings of 1a[rac] are separated to reduce the mutual
overlapping. In view of these structures, 1a[rac] may allow
successive two-electron reduction by FADH- (Scheme 5, path
4), whereas 1a[meso] would favor one-electron reduction, rather
than two-electron reduction, because of the generation of an
electrostatic repulsion between the one-electron-reduced dihy-
dropyrimidine rings. The intermediates (4′a,b) derived from
successive two-electron reduction are expected to afford 2 equiv
of C5-anions 6a,b, thus resulting in exclusive formation of 3a,b
by protonation at C5. To characterize the conformation of 1a,b
in solution, NMR spectra were measured in dimethyl sulfoxide-
d6 at room temperature (Figure 7). In accord with the results of
X-ray crystallography, the NOE difference spectra between H6

2892 J. Phys. Chem. A, Vol. 104, No. 13, 2000
Ito et al.
Figure 6. Frame structures of (a) 1a[meso] and (b) 1a[rac] obtained
by X-ray crystallography (see ref 7b).
protons and N1-methyl protons of the two pyrimidine rings
increased about 16% for 1a[meso] and 6% for 1a[rac],
respectively. This result suggests that 1a[meso] exhibits a
“closed-shell” conformation in solution in contrast to the
“opened-shell” conformation of 1a[rac]. Furthermore, the NOE
difference spectra for 1b[meso] and 1b[rac] in solution increased
to a much lesser extent and there was no substantial difference
between these stereoisomers (data not shown), although the
dihydropyrimidine rings of 1b[meso] and 1b[rac] are stacked
in a similar manner as those of 1a[meso] in the crystal.7b Hence,
due to the conformational preference in solution that may relieve
the intramolecular electrostatic repulsion between the pyrimi-
dine-electron adducts when formed, 1b[meso] and 1b[rac]
could undergo successive two-electron reduction similar to
1a[rac]. In this context, previous EPR spectroscopic studies have
demonstrated that the radical anion of trans-syn-1,3-dimethyl-
uracil dimer, pyrimidine rings of which are likely to be separated
to each other,2a is relatively long-lived in contrast to the radical
anions of the stacked conformations of cis-syn-thymine and
uracil dimers that are unstable to split into monomer anions
spontaneously.28
Another possibility is that the “closed-shell” conformation
of 1a[meso] may produce a relatively stable complex state with
*FADH-, thereby the 5-yl radical intermediate 5a formed
undergoing readily back electron transfer (BET) (path 5 in
Scheme 5). However, such a back electron transfer reaction is
less likely in the present case because of the oxidizing properties
of pyrimidine 5-yl radicals. This is clearly in contrast to the
photoinduced splitting mechanism of photodimers by which
thymine monomers are regenerated by BET from the monomer
radical anion to the FAD chromophore.
Figure 7. 400 MHz 1H NMR spectra (lower trace) and NOE difference
spectra (upper trace) of (a) 1a[meso] and (b) 1a[rac] in dimethyl
sulfoxide-d6 at 303 K. Arrows show irradiated H6 peaks.
ing thymine monomers 2a,b. The efficiency for the reductive
restoration into 2a,b was significantly lower than those in the
previously reported oxidative splitting reaction. According to
the proposed reductive splitting mechanism, electron adducts
of the dimers undergo the C5-C5′-bond cleavage to produce
the 5,6-dihydrothymin-5-yl radicals 5a,b and the C5-anions 6a,b.
Disproportionation of 5a,b is the most likely reaction pathway
for regeneration of the monomers 2a,b. The relatively high
efficiency of restoration into 2a in the photoreduction of
1a[meso] by *FADH- suggested that the stacked pyrimidine
rings of 1a[meso] does not favor a successive two-electron
reduction as in the other dimers (1a[rac], 1b[meso], and 1b[rac])
with a common conformation of separated pyrimidine rings.
This is in accord with the conformational studies by NOESY
and X-ray crystallography.
Acknowledgment. This work was supported by the Grant-
in-Aids for JSPS Fellows (No. 00086200) and for Scientific
Research (C) (No. 09640632) from the Ministry of Education,
Science, Sports, and Culture of Japan. We are grateful to Mr.
H. Fujita for performing nuclear magnetic resonance spectral
analysis.
References and Notes
Finally, the reductive splittings of 1b[meso] and 1b[rac] seem
to be slightly more effective than those of 1a[meso] and 1a[rac]
(see Table 1), although the reduction potentials of 1a are more
positive than those of 1b. It is likely that N-methyl substitution
may increase a strain in the dimers to enhance the exothermicity
of the splitting reaction. In fact, the enthalpy for splitting of
uracil and thymine cyclobutane photodimers tended to become
more negative by the methyl-substitution.29 Accordingly, the
instability would make the radical anions of 1b undergo the
C5-C5′-bond cleavage somewhat readily.
(1) For reviews, see: (a) von Sonntag, C. The Chemical Basis of
Radiation Biology; Taylor and Francis: London, 1987. (b) Fuciarelli, A.
F.; Zimbrick, J. D. Radiation Damage in DNA: Structure/Function Relation-
ship at Early Times; Battelle Press: Columbus, OH, 1995. (c) Kuwabara,
M. Radiat. Phys. Chem. 1991, 37, 691-704. (d) Steenken, S. Free Radical
Res. Commun. 1992, 16, 349-379.
(2) (a) Cadet, J.; Vigny, P. In Bioorganic Photochemistry: Photo-
chemistry and the Nucleic Acids; Morrison, H., Ed.; John Wiley & Sons:
New York, 1990; pp 1-272. (b) Taylor, J.-S. Acc. Chem. Res. 1994, 27,
76-82. (c) Go¨rner, H. J. Photochem. Photobiol. B: Biol. 1994, 26, 117-
139. (d) Pfeifer, G. P. Photochem. Photobiol. 1997, 65, 270-283.
(3) (a) Sancar, G. B.; Smith, F. W.; Reid, R.; Payne, G.; Levy, M.;
Sancar, A. J. Biol. Chem. 1987, 262, 478-485. (b) Payne, G.; Sancar, A.
Biochemistry 1990, 29, 7715-7727. (c) Kim, S.-T.; Heelis, P. F.; Okamura,
T.; Hirata, Y.; Mataga, N.; Sancar, A. Biochemistry 1991, 30, 11262-11270.
(d) Burdi, D.; Begley, T. P. J. Am. Chem. Soc. 1991, 113, 7768-7770. (e)
Kim, S.-T.; Heelis, P. F.; Sancar, A. Biochemistry 1992, 31, 11244-11248.
(f) Malhotra, K.; Kim, S.-T.; Walsh, C.; Sancar, A. J. Biol. Chem. 1992,
267, 15406-15411. (g) Sancar, A. Biochemistry 1994, 33, 2-9. (h) Kim,
S.-T.; Sancar, A. Photochem. Photobiol. 1993, 57, 895-904. (i) Begley,
Conclusion
We have demonstrated that reductive splitting of C5-C5′-
linked dihydrothymine dimers 1a,b by hydrated electrons and
the photoexcited reduced form of flavin (*FADH-) affords the
5,6-dihydrothymine derivatives 3a,b along with the correspond-

Splitting of C5-C5′-Linked Dihydrothymine Dimers
J. Phys. Chem. A, Vol. 104, No. 13, 2000 2893
T. P. Acc. Chem. Res. 1994, 27, 394-401. (j) Carell, T.; Epple, R. Eur. J.
Org. Chem. 1998, 1245-1258. (k) Park, H.-W.; Kim, S.-T.; Sancar, A.;
Deisenhofer, J. Science 1995, 268, 1866-1872. (l) Tamada, T.; Kitadokoro,
K.; Higuchi, Y.; Inaka, K.; Tasui, A.; de Ruiter, P. E.; Eker, P. M.; Miki,
K. Nature Struct. Biol. 1997, 11, 887-891. (m) van de Berg, B. J.; Sancar,
G. B. J. Biol. Chem. 1998, 273, 20276-20284. (n) Ramaiah, D.; Kan, Y.;
Koch, T.; Ørum, H.; Schuster G. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
12902-12905. (o) Butenandt, J.; Burgdort, L. T.; Carell, T. Angew. Chem.,
Int. Ed. Engl. 1999, 38, 708-7011.
addition, photoreduction of FADH• by pyrimidine photodimer has not been
observed (Heelis, P. F.; Payne, G.; Sancar, A. Biochemistry 1987, 26, 6,
4634-4640).
(15) (a) Yeh, S.-R.; Falvey, D. E. J. Am. Chem. Soc. 1992, 114, 7313-
7314. (b) Scannell, M. P.; Prakash, G.; Falvey, D. E. J. Phys. Chem. A
1997, 101, 4332-4337. (c) Scannell, M. P.; Fenick, D. J.; Yeh, S.-R.;
Falvey, D. E. J. Am. Chem. Soc. 1997, 119, 1971-1977.
(16) Excited-state oxidation potential E*ox was calculated using E*ox
)
2+
Eox - E00 with the oxidation potential of Ru(bpy)3 (Eox) and zero
excitation energy (E00) evaluated from λ0-0 ()530 nm) in aqueous solution.
(17) The dynamic quenching rate constants kq were determined by the
Stern-Volmer equation: I0/I ) 1 + kqτ0[Q], where I0 and I are the intensity
of the fluorescence at 600 nm when excited at 450 nm in the absence and
presence of a quencher, respectively. The lifetime τ0 of *Ru(bpy)32+ in the
absence of a quencher is known to be 0.62 µs (Kalyanasundaram, K. Coord.
Chem. ReV. 1982, 46, 159-244).
(4) Yeh, S.-R.; Falvey, D. E. J. Am. Chem. Soc. 1991, 113, 8557-
8558.
(5) Pouwels, P. J. W.; Hartman, R. F.; Rose, S. D.; Kaptein, R.
Photochem. Photobiol. 1995, 61, 575-583.
(6) (a) Todo, T.; Takemori, H.; Ryo, H.; Ihara, M.; Matsunaga, T.;
Nikaido, O.; Sato, K.; Nomura, T. Nature 1993, 361, 371-374. (b) Kim,
J.-K.; Patel, D.; Choi, B.-S. Photochem. Photobiol. 1995, 62, 44-50. (c)
Todo, T.; Ryo, H.; Yamamoto, K.; Toh, H.; Inui, T.; Ayaki, H.; Nomura,
T.; Ikenaga, M. Science 1996, 272, 109-112. (d) Kim, S.-T.; Malhotra,
K.; Smith, C. A.; Taylor, J.-S.; Sancar, A. J. Biol. Chem. 1994, 269, 8535-
8540. (e) Zhao, X.; Liu, J.; Hsu, D. S.; Zhao, S.; Taylor, J.-S.; Sancar, A.
J. Biol. Chem. 1997, 272, 32580-32590.
(18) Rehm, D.; Weller, A. Isr. J. Chem. 1970, 8, 259-271.
(19) Steenken, S.; Telo, J. P.; Novais, H. M.; Candeias, L. P. J. Am.
Chem. Soc. 1992, 114, 4701-4709.
(20) Habersbergerova´, A.; Janovsk′y, I.; Tepl′y, J. Radiation Res. ReV.
1968, 1, 109-181.
(7) (a) Nishimoto, S.; Ide, H.; Nakamichi, K.; Kagiya, T. J. Am. Chem.
Soc. 1983, 105, 6740-6741. (b) Ito, T.; Shinohara, H.; Hatta, H.; Nishimoto,
S. J. Org. Chem. 1999, 64, 5100-5108.
(21) Land, E. J.; Porter, G. Trans Faraday Soc. 1963, 59, 2027-2037.
(22) Deeble, D. J.; Das, S.; von Sonntag, C. J. Phys. Chem. 1985, 89,
5784-5788.
(8) Ito, T.; Shinohara, H.; Hatta, H.; Fujita, S.; Nishimoto, S. J. Phys.
Chem. A. 1999, 103, 8413-8420.
(23) (a) Okamura, T.; Sancar, A.; Heelis, P. F.; Begley, T. P.; Hirata,
Y.; Mataga, N. J. Am. Chem. Soc. 1991, 113, 3143-3145. (b) Kim, S.-T.;
Volk, M.; Rousseau, G.; Heelis, P. F.; Sancar, A.; Michel-Beyerle, M.-E.
J. Am. Chem. Soc. 1994, 116, 3115-3116.
(9) The number of molecules produced or changed per 1 J of radiation
energy absorbed by the reaction system.
(10) Wardman, P. J. Phys. Chem. Ref. Data 1989, 18, 1637-1755.
(11) Kavarnos, G. J.; Turro, N. J. Chem. ReV. 1986, 86, 401-449.
(12) (a) Steenken, S.; Neta, P. J. Phys. Chem. 1979, 83, 1134-1137.
(b) Fujita, S.; Steenken, S. J. Am. Chem. Soc. 1981, 103, 2540-2545.
(13) In the course of steady-state γ-radiolysis and pulse radiolysis studies
on the behavior of deprotonated adenosine C8 radicals (Ado(-H)•) in
phosphate buffer, it was suggested that phosphate ions enhance one-electron
reduction of Ado(-H)• with a rate constant k ) 103-104 dm3 mol-1 s-1
(Nishimoto, S.; Kuno, S.; Fujita, S. unpublished results).
(14) In the absence of EDTA, 3a,b are major products (see Table 2)
whereas oxidative splitting of 1a,b by anthraquinone-2-sulfonate (AQS)
sensitization affords almost equivalent amounts of 2a,b and 3a,b (ref 8).
This indicates the reduction process is predominant in the present case. In
(24) (a) Heelis, P. F.; Kim, S.-T.; Okamura, T.; Sancar, A. J. Photochem.
Photobiol. B: Biol. 1993, 17, 219-228. (b) Heelis, P. F.; Deeble, D. J.;
Kim, S.-T.; Sancar, A. Int. J. Radiat. Biol. 1992, 62, 137-143.
(25) (a) Kim, S.-T.; Heelis, P. F.; Okamura, T.; Hirata, Y.; Mataga, N.;
Sancar, A. Biochemistry 1991, 30, 11262-11270. (b) Kim, S.-T.; Sancar,
A.; Essenmacher, C.; Babcock, G. T. J. Am. Chem. Soc. 1992, 114, 4442-
4443.
(26) Anderson, R. F. Biochim. Biophys. Acta 1983, 722, 158-162.
(27) Ramsey, A. J.; Jorns, M. S. Biochemistry 1992, 31, 8437-8441.
(28) Pezeshk, A.; Podmore, I. D.; Heelis, P. F.; Symons, M. C. R. J.
Phys. Chem. 1996, 100, 19714-19718.
(29) Heelis, P. F.; Hartman, R. F.; Rose, S. D. J. Photochem. Photobiol.
A: Chem. 1996, 95, 89-98.