Electron transfer reactions of methionine peptides with photochemically generated ruthenium(III)-polypyridyl complexes

Article abstract of DOI:10.1016/j.jphotochem.2014.09.003

The dynamics of the oxidation of five methionine carrying peptides with six Ru(III)-polypyridyl complexes in aqueous acetonitrile medium have been followed by spectrophotometric technique. The electron transfer (ET) reaction of [Ru(NN)₃]³⁺

Full text of DOI:10.1016/j.jphotochem.2014.09.003

Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 70–78
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Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Electron transfer reactions of methionine peptides with
photochemically generated ruthenium(III)–polypyridyl complexes
Dharmaraj Thiruppathi a, Periyakaruppan Karuppasamya, Muniyandi Ganesan a,
,
*
Veluchamy Kamaraj Sivasubramanian a,b, Thangamuthu Rajendran a,c
,
d,**
Seenivasan Rajagopal
a
Post Graduate and Research Department of Chemistry, Vivekananda College, Thiruvedakam West, Madurai 625234, India
Department of Chemistry, K.L.N College of Information Technology, Pottapalayam, Sivagangai 630611, India
Department of Chemistry, PSNA College of Engineering and Technology, Dindigul 624622, India
b
c
d
School of Chemistry, Madurai Kamaraj University, Madurai 625021, India
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 10 June 2014
Received in revised form 20 August 2014
Accepted 1 September 2014
Available online 10 September 2014
The dynamics of the oxidation of five methionine carrying peptides with six Ru(III)–polypyridyl
complexes in aqueous acetonitrile medium have been followed by spectrophotometric technique. The
electron transfer (ET) reaction of [Ru(NN)3]3+ complex (NN = polypyridyl ligand) with peptide containing
methionine is sensitive to the change in the structure of ligand of Ru(III) complex and N-terminal
component of the peptide Met-Gly Met-Ala Met-Ser Met-Val and Met-Leu. The reaction follows the
overall second-order kinetics, first order each in the oxidant and the substrate. Based on the substituent
and solvent effects, an outer-sphere electron transfer from the sulfur center of the peptide to Ru(III) has
been proposed as the rate controlling step of the reaction. The ET nature of the reaction is confirmed from
the recorded transient absorption spectrum of peptide containing methionine sulfur radical cation by
laser flash photolysis technique. Marcus theory is successfully applied to the ET reaction.
ã 2014 Elsevier B.V. All rights reserved.
Keywords:
Peptide containing methionine
Ruthenium(III)–polypyridyl complex
Kinetic study
Electron transfer reaction
Marcus theory
1. Introduction
Parkinson’s diseases in addition to biological aging processes [7–9]
and age-related human cataract formation [10].
Protein-based radicals play an important role in the most
elaborate biosyntheses in nature, including photosynthesis and
respiration [1,2]. Among the different amino acid residues
generally found in proteins, methionine (Met) is one of the two
amino acids that contain a sulfur atom (the other amino acid is
cysteine) and it is most readily oxidized via electron transfer from
its sulfur center. Met residues in peptides and proteins are
primarily susceptible to oxidation by numerous reactive oxygen
species (ROS) [3,4]. In fact, depending on the nature of the
oxidizing species, Met can undergo two-electron transfer oxidation
or oxygen transfer reaction (with oxidants HOCl, H2O2, ROOH, and
ONOOꢀ/ONOOH) to give sulfoxide or one-electron oxidation (with
RSꢁ, HOꢁ, ROꢁ, CO3ꢁꢀ, N3ꢁ, and ROOꢁ) to give sulfur radical cations
[5,6]. One electron oxidation of Met in peptides is likely to play an
important role during the pathological conditions of oxidative
stress and neurodegenerative diseases such as Alzheimer’s and
The sulfur radical cation formed due to one electron oxidation
of methionine can be stabilized by neighboring groups containing
electron-rich heteroatoms present in peptides and proteins
through the formation of a two-center three-electron bond with
an electron pair donor, :X (=O, N, S) [11]. Such an intermediate
species is represented as S;X. Three types of such two-center
three electron bonds S;O, S;N and S;S in Met derivatives have
been observed in pulse-radiolysis and flash photolysis experiments
in aqueous solution [12–14].
The recent study [15] on the ꢁOH-induced oxidation of
cyclic dipeptide c-(L-Met-L-Met) highlighted the formation of
sulfur radical cations (>Sꢁ+) stabilized both by intramolecular
sulfur–nitrogen bonding with amide nitrogen atoms in the
peptide bonds and by intramolecular sulfur–sulfur two-center,
three-electron bonding with the unoxidized sulfur atom in the
side chain of the other methionine. The authors monitored the
formation of such intermediates using time-resolved optical
spectroscopy and conductivity. Theoretical and experimental
evidence for the formation of three-electron bonds have been
well documented [16].
* Corresponding author. Tel.: +91 4543 258234; fax: +91 4543 258358.
** Corresponding author. Tel.: +91 452 2458246; fax: +91 452 2459139.
E-mail addresses: mganesan58@ymail.com (M. Ganesan),
By using time-resolved optical and conductivity methods,
Bobrowski and co-workers [17] also studied the reaction of HOꢁ
rajagopalseenivasan@yahoo.com (S. Rajagopal).
http://dx.doi.org/10.1016/j.jphotochem.2014.09.003
1010-6030/ã 2014 Elsevier B.V. All rights reserved.

D. Thiruppathi et al. / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 70–78
71
radical with two other cyclic dipeptides, cyclo-Gly-Met and
cyclo- -Met- -Met to generate the sulfur radical cation which is
stabilized through the formation of the stable intramolecular S;N
bonded five-membered radial observed in c-( -Met- -Met). In
ESI-MS spectral techniques (spectral data are given in the
supporting information).
D
L
D
L
2.3. Photochemical oxidation of [Ru(NN)3]2+ to [Ru(NN)3]3+
addition to this, the stabilization through intermolecular sulfur-
sulfur three-electron bonding with the unoxidized sulfur atom on
separate cyclic dipeptide molecules was also observed [17].
Interestingly there is no evidence for the intramolecular stabiliza-
tion by the unoxidized sulfur atom in the neighboring
The steady-state photolysis of [Ru(NN)3]2+ (2 ꢃ10ꢀ5 M) in 2.3 M
HClO4, in the presence of molecular oxygen, upon irradiation for
20 min using a 500 W tungsten-halogen lamp led to the formation
of the corresponding Ru(III) complex. The infrared (IR) and
ultraviolet (UV) radiations were cut-off by passing the light beam
through a 30 cm quartz cell filled with water. It was observed that
the color of the solution readily changed from orange–yellow to
green during irradiation. The formation of [Ru(NN)3]3+ complex
was confirmed by recording the absorption spectrum of the
irradiated solution. The absorption peak at 450 due to [Ru(bpy)3]2+
disappeared quickly on irradiation for 20 min, resulting in the
formation of [Ru(bpy)3]3+ having peaks around 420–430 and
650–670 nm, matching with the reported wavelength of maximum
absorption of Ru(III) in acidic aqueous solution [24,30]. These
peaks are assigned to charge transfer transitions from the bipyridyl
methionine in cyclo-
reported intramolecular (S;S)+ radical cations established in the
c-( -Met- -Met) isomer under the same concentrations and pH
D-Met-L-Met in contrast to the previously
L
L
conditions [15] This type of contrasting behavior is accounted for
by the structural changes in the two isomers as seen through
molecular-modeling simulations [17].
Morozova et al. [18] through chemically induced dynamic
nuclear polarization (CIDNP) technique studied the photooxida-
tion of two sulfur containing peptides, glycylmethionine (Gly-Met)
and methionylglycine (Met-Gly) with triplet 4-carboxybenzophe-
none (CBP). There are two possible mechanisms for this oxidation
reaction: (i) electron transfer from the sulfur atom and (ii)
electron/proton transfer from the amino group. The later process
takes place only in the Met-Gly not for Gly-Met to form the five-
membered cyclic radical with a three electron bond between the S
and N atoms. In fact theoretical calculations also predicted that the
cyclic sulfur–nitrogen two-center, three-electron bonded inter-
mediate with the N-terminal amino group is probably the most
stable structure of the species formed from the one-electron
oxidation of methionine in aqueous solution [19,20].
p
ligands to the electron-deficient metal (t2 g) [31]. The absence of a
peak at 450 nm in the absorption spectrum of Ru3+ confirms the
complete conversion of Ru2+ to Ru3+. The absorption spectra of
[Ru(bpy)3]2+ and [Ru(bpy)3]3+ are shown in Fig. 1. The percentage
conversion of Ru2+ to Ru3+ determined from the absorption
intensity at 650–670 nm is ꢄ95% in all cases (vide infra). The
molar extinction coefficient (
680 Mꢀ1 cmꢀ1
e
) of [Ru(bpy)3]3+ at 650–670 nm is
.
To confirm the formation of peptide containing methionine
sulfur radical cation as intermediate of the reaction, we have
followed the reaction by nanosecond laser flash photolysis
technique from which we are able to get the transient absorption
spectrum supporting the formation of the intermolecular
sulfur–sulfur dimeric three-electron-bonded radical cation Met
(S;S)+ during the course of reaction. These radical cations were
detected using nanosecond flash photolysis technique. The
interesting results of the spectral and kinetic studies on the
reaction of five N-terminal methionyl residues with six ruthenium
(III) complexes [Ru(NN)3]3+ are presented in this paper. Though
several chemical and electrochemical methods are available for the
generation of Ru(III) from Ru(II) complexes, in the present study Ru
(III) complexes have been generated using visible light irradiation
of [Ru(NN)3]2+ in the presence of molecular oxygen [21–25].
2.4. Kinetic measurements
The absorption spectra of [Ru(NN)3]2+ and [Ru(NN)3]3+ com-
plexes were obtained using a JASCO model 530 UV–vis spectro-
photometer. The Ru(III) complexes prepared from the
photochemical oxidation of Ru(II) complexes were used for the
kinetic study. As the conversion from Ru(II) to Ru(III) is more than
95% (details given in the discussion section), the Ru(III) ion
generated in 2.3 M HClO4 was used directly for the oxidation
2. Experimental
2.1. Chemicals
The ligands, 2,20-bipyridine (bpy), 4,40-dimethyl-2,20-bipyri-
dine (dmbpy), 4,40-di-tert-butyl-2,20-bipyridine (dtbpy), 1,10-
phenanthroline
(dpphen) and 4,7-diphenyl-1,10- phenanthroline-disulfonate
(BPS) and RuCl3 3H2O were obtained from Sigma–Aldrich and
methionyl-glycine-(Met-Gly), methionyl-alanine-(Met-Ala),
(phen),
4,7-diphenyl-1,10-phenanthroline
ꢂ
methionyl-serine- (Met-Ser), methionine-valine-(Met-Val) and
methionyl-leucine-(Met-Leu) (purity > 99%) from Sigma–Aldrich
and used without further purification. HPLC grade CH3CN was
obtained from Merck.
2.2. Synthesis and characterization of ruthenium(II)–polypyridyl
complexes ([Ru(NN)3]2+
Fig.1. (a) The absorption spectrum of a solution of 2 ꢃ10ꢀ5 M [Ru(bpy)3]2+ complex
in oxygen-saturated aqueous CH3CN (1:1, v/v) solution in the presence of 2.3 M
HClO4 and (b) The absorption spectrum of [Ru(bpy)3]3+ obtained from
the irradiation of 2 ꢃ10ꢀ5 M [Ru(bpy)3]2+ in oxygen saturated aqueous CH3CN
(1:1, v/v) solution in the presence of 2.3 M HClO4.
The [Ru(NN)3]2+complexes (where NN = bpy, dmbpy, dtbpy,
phen, dpphen and BPS) were synthesized by known procedures
[25–29] and characterized by UV–vis, FT-IR, 1H NMR, 13C NMR and

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D. Thiruppathi et al. / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 70–78
potential value of [Ru(NN)3]3+ complexes and the oxidation
potential value of peptide containing methionine are in good
agreement with the reported values [24,33].
2.6. Laser flash photolysis experiment
The formation and decay of the short lived intermediates due to
the reaction between [Ru(NN)3]3+ complex and peptide containing
methionine is followed using the transient absorption spectral
study with a laser flash photolysis technique employing an Applied
Photophysics SP-Quanta Ray GCR-2(10) Nd:YAG laser generating
355 nm pulses (ꢄ8 ns pulse width) [34]. The transient absorption
at preselected wavelengths was monitored using a Czerny–Turner
monochromator with a stepper motor control and a Hamamatsu
R-928 photomultiplier tube. A 250 W xenon arc lamp was used as
the monitor light source. Experiments were carried out using a
flash quartz cells (1 cm) with an optical path length of 0.5 cm for
the monitoring beam. The reaction condition used for the flash
photolysis experiment was similar to that used for the kinetic
studies. The concentrations of [Ru(dmbpy)3]3+ and Met-Gly used
for all experiments were 1 ꢃ10ꢀ3 M and 0.05 M respectively.
Typically 3–5 laser shots were averaged for each kinetic trace. All
the measurements were carried out at ambient temperature. The
solution was flowed and kinetic traces were taken at 10 nm
intervals, usually between 320 and 800 nm. The change in the
absorbance of the sample on laser irradiation was used to record
the time-resolved absorption transient spectrum. The change in
the absorbance on flash photolysis was calculated using the
expression.
Fig. 2. The absorption spectral changes for the reaction between [Ru(bpy)3]3+
(2 ꢃ10ꢀ5 M) and methionylglycine (5 ꢃ10ꢀ4 M) in oxygen saturated aqueous CH3CN
(1:1, v/v) solution in the presence of 2.3 M HClO4 at 298 K with 2 min time interval.
reaction. To initiate the reaction 1.5 ml of peptide containing
methionine was mixed with 1.5 ml Ru(III) ion in the cuvette. The
reaction between the photogenerated [Ru(NN)3]3+ ion with
peptide containing methionine was monitored by following the
increase in absorbance of [Ru(NN)3]2+
(lmax = 450 nm) at 2 min
time interval at 298 K (Fig. 2) [32]. The kinetic studies of oxidation
of all peptide containing methionine with [Ru(NN)3]3+ complexes
were carried out in aqueous CH3CN (1:1, v/v) in the presence of
2.3 M HClO4 and molecular oxygen, under pseudo first-order
condition. The pseudo first-order rate constant (k1) for each kinetic
run was evaluated from the slope of linear plot of log Abs vs time. A
set of duplicate kinetic runs proved that the rate constants were
found to be reproducible within ꢅ3%. The second-order rate
constant (k2) is evaluated from the relation k2 = k1/[substrate].
logI0
D
A ¼
ðI0 ꢀ
D
IÞ
D
I ¼ ðI ꢀ ItÞ
where
DA is the change in the absorbance at time t,I0, I and It are
the voltage after flash, the pretrigger voltage and the voltage at
particular time respectively. The time-resolved transient absorp-
tion spectrum was recorded by plotting the change in absorbance
at a particular time vs wavelength. The experiment was carried out
in the absence and presence of molecular oxygen. The presence of
oxygen was necessary to generate the Ru3+ species from Ru2+
photochemically [24,30a,35].
2.5. Electrochemical measurements
The reduction potential of [Ru(NN)3]3+ complexes in aqueous
CH3CN (1:1, v/v) medium were measured by cyclic voltammetric
technique using computer controlled Potentiostat CH Instruments
Model 680 AMP Booster. HClO4 was used as the supporting
electrolyte. The oxidation potentials of peptide containing
methionine were measured by differential pulse voltammetry
(DPV) and tetrabutylammonium perchlorate (0.1 M) was used as
the supporting electrolyte. A glassy carbon (working electrode) and
a standard (Ag/Ag+) electrode (reference electrode) were used in
the electrochemical measurements. The sample solutions were
deaerated by purging dry nitrogen gas for about 30 min before each
measurement. The oxidation potential values of five peptide
containing methionine and the reduction potential values of six
[Ru(NN)3]3+ complexes are shown in Tables 1 and 2. The reduction
2.7. Product analysis
A sample of 0.02 mM of substrate (Met-Gly) was added to a
0.002 mM solution of [Ru(dmbpy)3]3+ complex in 5 ml aqueous
CH3CN (1:1, v/v). The solution was stirred at 298 K for ꢆ20 min. The
products of the reaction were extracted with chloroform and dried
and the solvent was removed. The FT-IR spectral analysis of the
product obtained from methionine sulfoxide shows the peaks at
1036 cmꢀ1 assigned as S
O stretching frequency of sulfoxide.
¼
Table 1
Second order rate constant (k2, Mꢀ1 sꢀ1) values for the oxidation of peptide containing methionines by [Ru(NN)3]3+ in aqueous CH3CN (1:1, v/v) at 298 K.
Peptides Oxidation potential, V (Ag/Ag+)
k2, Mꢀ1 sꢀ1
[Ru(bpy)3]3+ (0.92 V) (I)
[Ru(dmbpy)3]3+ (0.75 V) (II)
[Ru(dtbpy)3]3+ (0.76 V) (III)
Observed Calculated
Gꢇ (eV)
Observed
Calculated
4.3
D
Gꢇ (eV)
Observed
Calculated
D
Gꢇ (eV)
D
Met-Gly 1.50
9.7 ꢅ 0.29
0.58
0.55
0.53
0.57
0.56
0.02 ꢅ 0.001 0.01
0.03 ꢅ 0.001 0.02
0.05 ꢅ 0.002 0.04
0.06 ꢅ 0.002 0.02
0.07 ꢅ 0.002 0.03
0.75
0.72
0.70
0.74
0.73
0.01 ꢅ 0.0001 0.01
0.74
0.71
0.69
0.73
0.72
Met-Ala
Met-Ser
Met-Val
1.47
1.45
1.49
11.2 ꢅ 0.31 13.5
14.7 ꢅ 0.38 15.3
0.02 ꢅ 0.001
0.03 ꢅ 0.001
0.02 ꢅ 0.001
0.02 ꢅ 0.001
0.02
0.04
0.01
0.01
17.6 ꢅ 0.53
20.4 ꢅ 0.62
5.7
8.4
Met-Leu 1.48
General condition: [Ru(NN)3]3+ = 2 ꢃ10ꢀ5 M, [Peptide] = 5 ꢃ10ꢀ4 M and [H+] = 2.3 M.

D. Thiruppathi et al. / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 70–78
73
Table 2
Second order rate constant (k2, Mꢀ1 sꢀ1) values for the oxidation of peptide containing methionines by [Ru(NN)3]3+ in aqueous CH3CN (1:1, v/v) at 298 K.
Peptides Oxidation potential, V (Ag/Ag+)
k2, Mꢀ1 sꢀ1
[Ru(phen)3]3+ (0.92 V) (IV)
[Ru(dpphen)3]3+ (0.89 V) (V)
[Ru(dspphen)3]3+ (0.91 V) (VI)
Observed
Calculated
D
Gꢇ (eV)
Observed
Calculated
D
Gꢇ (eV) Observed
Calculated
D
Gꢇ (eV)
Met-Gly
Met-Ala
Met-Ser
Met-Val
Met-Leu
1.50
1.47
1.45
1.49
1.48
12.6 ꢅ 0.38
16.1 ꢅ 0.43
18.4 ꢅ 0.46
22.1 ꢅ 0.66
26.5 ꢅ 0.74
6.8
15.2
23.3
6.6
0.58
0.55
0.53
0.57
0.56
10.4 ꢅ 0.31
13.8 ꢅ 0.38
15.2 ꢅ 0.45
17.6 ꢅ 0.47
1.6
6.7
9.8
3.4
4.7
0.61
0.58
0.56
0.60
0.59
15.5 ꢅ 0.46
19.6 ꢅ 0.52
5.0
9.9
0.59
0.56
0.54
0.58
0.57
23.0 ꢅ 0.64 21.1
25.7 ꢅ 0.77
29.9 ꢅ 0.89
5.7
7.2
9.7
20.8 ꢅ 0.54
General condition: [Ru(NN)3]3+ = 2 ꢃ10ꢀ5 M, [Peptide] = 5 ꢃ10ꢀ4 M and [H+] = 2.3 M.
Sulfoxide product after oxidation of methionine is only major
product formed under the present experimental conditions. This
was also confirmed from the analysis of the product with 1H NMR.
When [Ru(NN)3]2+ complexes are irradiated in the presence of
molecular oxygen, two processes, energy and electron transfer
from the excited state [Ru(NN)3]2+ to molecular oxygen, take place
to form singlet oxygen and superoxide anion radical, respectively
(Eqs. (1) and (2)). Under high acid concentration, the oxidation of
Ru2+ to Ru3+ is highly favored as indicated in Eq. (3). More than 95%
conversion of Ru2+ to Ru3+ in our present experimental condition
also supports our argumenꢁtꢀthat electron transfer from *[Ru
(NN)3]2+ to O2 to produce O2 is the predominant reaction here.
The high [H+] chosen in the present study facilitates the generation
of Ru3+ from the cage as shown in Eq. (3). Thus we propose that
under the conditions used in the present study the formation of
singlet oxygen through energy transfer is less favored compared to
1H NMR (300 MHz, D2O):
d= 2.30 (2H, m), 2.94 (2H, t), 3.71 (2H, dd),
4.10 (1H, t) and 2.62 (SOCH3, s) values are in good agreement with
reported values of methionine sulfoxide [10,18]. 1H NMR and IR
spectrum of methionine sulfoxide is given in the supporting
information (Figs. S1 and S2).
3. Results and discussion
The generation of the Ru(III) ion from the corresponding
[Ru(NN)3]2+ complex is carried out as detailed below. The steady-
state photolysis of [Ru(NN)3]2+ (2 ꢃ10ꢀ5 M) in 2.3 M HClO4, in the
presence of molecular oxygen, upon irradiation for 20 min using
a 500 W tungsten-halogen lamp led to the formation of the
corresponding [Ru(NN)3]3+ complex. The formation of [Ru(bpy)3]3+
complex was confirmed by recording the absorption spectrum of
the irradiated solution. The absorption spectra of [Ru(bpy)3]2+ and
[Ru(bpy)3]3+ are shown in Fig. 1. To learn the concentration of Ru3+
formed from the photochemical oxidation of Ru2+, we estimated
the concentration of the Ru3+ ion from the absorbance (Abs) values
electron transfer (ET) and most of the Ru2+ is converted to Ru3+
,
which is responsible for the redox reactions of peptide containing
methionine.
Chart 1 depicts the structure of ligands and abbreviation of [Ru
(NN)3]3+ complexes as well as the structure of substrates used in
the present study. The kinetics of electron transfer reaction of six
[Ru(NN)3]3+ ions with five peptide containing methionines
Met-Gly Met-Ala Met-Ser Met-Val and Met-Leu (Chart 1) has
been followed spectrophotometrically, under pseudo first order
condition by taking excess of peptide containing methionines over
[Ru(NN)3]3+ complex. The kinetics of the reaction has been carried
out by mixing the peptide containing methionine with the Ru(III)
complex immediately after the preparation of [Ru(NN)3]3+ using
photochemical oxidation method as described in the experimental
section. The progress of the reaction has been followed by
measuring the increase in the absorbance (A) of Ru(II) ion formed
at 420 and 670 nm. Since the band at 670 nm (e )
= 680 Mꢀ1 cmꢀ1
corresponds to Ru3+ with no interference from Ru2+, we considered
the concentration estimated from the Abs at 670 nm as more
reliable. This estimation shows that the chemical conversion of
Ru2+ to Ru3+ is more than 95% under the present experimental
conditions and we have used this Ru3+ solution directly for kinetic
studies.
Chart 1. Structure of ligands of [Ru(NN)3]3+ and peptide containing methionines.

74
D. Thiruppathi et al. / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 70–78
ꢀ
(450 nm) as one of the products of the reaction and a sample
kinetic run is shown in Fig. 2. The spectral changes collected in
Fig. 2 clearly show that during the course of the reaction Ru(III) ion
disappears (the peak in the region 600–700 nm) and Ru(II) ion
appears (450 nm) with isosbestic point at 542 nm. Thus the
reaction Ru(III) ! Ru(II) follows simple kinetics without involving
any complex mechanism. These experimental observations are
strongly in favor of electron transfer (ET) from Met to Ru(III) to
form Ru(II) in the rate determining step. The reaction is first-order
with respect to Ru(III) complex, the oxidant which is evident from
the linear log Abs vs time plot shown in the supporting information
(Fig. S3).
The values of pseudo first order rate constant, k1 plotted as a
function of concentration of peptide containing methionines is
shown in Fig. 3. The linear relationship between k1 and [peptide]
and constant k2 values at different [peptide] point out the first
order dependence in the substrate also. The reaction is thus overall
second order. The k2 values are obtained by dividing the pseudo
first order rate constant, k1 with substrate [peptide] concentration.
The second order rate constant values estimated from the
reaction of six [Ru(NN)3]3+ complexes as the oxidants for the one-
electron oxidation of five peptide containing methionines are
collected in Tables 1 and 2. The kinetic data collected in Table 1
show that the introduction of electron-donating groups like
methyl and tert-butyl in the 4- and 40-position of 2,20-bipyridine of
[Ru(NN)3]3+, 4,40-dimethyl-2,20-bipyridine and 4,40-tert-butyl-2,20-
bipyridine ligands, leads to decrease in the rate of oxidation by
more than two orders compared to unsubstituted 2,20-bipyridine
ligand (Table 1). The change of ligand in [Ru(NN)3]3+ changes the
—SO3 is only 0.02 eV. Thus the slightly high k2 value observed
with [Ru(dspphen)3]3+ compared to [Ru(dpphen)3]3+ may be
attributed to the change in the
When we look at the oxidation potential values of peptide
containing methionines (Tables 1 and 2) it is seen that the potential
values vary by 0.01–0.05 V with the change of the structure of the
D
Gꢇ value.
peptides. Comparison of kinetic data and
conclude that apart from the
D
Gꢇ values compels us to
D
Gꢇ value other parameters, polar
and steric effects, may change the rate constant value with the
change of structure of peptide. If the polar effect of the alkyl group
present in the peptide alone is the predominant factor in this
reaction, then the change of alkyl group should increase the rate
constant value in the order methyl (2) < isopropyl (4) < tert-butyl
(5) because
D
Gꢇ value remains almost constant for these three
peptides. Interestingly the same trend is observed on the rate
constant values for the electron transfer reaction of these three
peptides (Tables 1 and 2). The conclusion from this trend is that
steric effect plays little role on the electron transfer reaction when
the structure of peptide is changed.
In order to understand the influence of other parameters on the
rate of the reaction, the kinetics of the reaction has been followed
at different solvent (CH3CN–water) composition and the results are
collected in Table 3. Generally the increase in water content favors
the reaction when charge development takes place in the
transition state of the reaction. As the title reaction involves
electron transfer from methionine sulfur atom to Ru(III), positive
charge is developed on the sulfur center of the substrate in the
transition state. Our research group established the fact that
increase in water content increases the rate of the reaction
between organic sulfur compounds and Ru(III)–polypyridyl com-
plexes [25a]. Thus the solvent effect supports the operation of
electron transfer in the rate-controlling step.
D
Gꢇ value by 0.16 eV. Thus the main reason for the decrease in k2
values in the order [Ru(bpy)3]3+ > [Ru(dmbpy)3]3+ ꢆ [Ru(dtbpy)3]3+
may be attributed to the change in the
D
Gꢇ value. When
we compare the k2 values observed with [Ru(dmbpy)3]3+ and
To study the effect of temperature on the reaction rate, kinetics
of reaction of [Ru(NN)3]3+ with peptide containing methionines
were carried out at four different temperatures 293, 298, 303 and
[Ru(dtbpy)3]3+ the former complex has slightly more k2 value than
the latter though
D
Gꢇ values are almost equal. This may be
attributed to the steric effect of tert-butyl group present in the
complex [Ru(dtbpy)3]3+. Similar results were observed by us when
we used the excited state [Ru(NN)3]2+ complexes for the oxidation
of several phenolate ions [36]. On the other hand the introduction
of the electron-withdrawing group, disulfonate, in the ligand
1,10-phenanthroline increases the rate of reaction slightly com-
pared to the parent 1,10-phenanthroline ligand (Table 2). This is
308 K. The rate constants were found to increase with increase in
¼
temperature as presented in Table 4. The enthalpy (
D
H ) and
¼
entropy (
D
S ) of activation evaluated using the Eyring’s plot of log
¼
k2/T vs 1/T is summarized in Table 4. The negative
D
S
value
indicates the compactness of transition state. The thermodynamic
¼
¼
parameters
DS and DH values, collected in Table 4 show that
with the change of structure of peptide containing methionines as
well as the ligand in the Ru(III) complex there is little change in the
because the change in
D
Gꢇ value due to the introduction of
¼
¼
D
H as well as DS values. This little change in the thermodynamic
parameters with the change of structure of reactants also supports
the operation of electron transfer mechanism of the reaction.
3.1. Formation of Met sulfur radical cation
In this work we propose that the reaction of [Ru(NN)3]3+ with
methionylglycine produces Met-sulfur radical cation. Ruthenium
Table 3
Effect of varying solvent composition on the reaction of [Ru(NN)3]3+ with peptide
containing methionines at 298 K.
Solvent composition CH3CN:H2O (v/v)
k2, Mꢀ1 sꢀ1
[Ru(bpy)3]3+
[Ru(phen)3]3+
Met-Gly
Met-Ala
80:20
70:30
60:40
50:50
40:60
20:80
5.3 ꢅ 0.16
6.5 ꢅ 0.18
7.2 ꢅ 0.20
9.7 ꢅ 0.29
12.1 ꢅ 0.32
13.5 ꢅ 0.41
11.8 ꢅ 0.35
13.0 ꢅ 0.36
14.3 ꢅ 0.43
16.1 ꢅ 0.43
18.5 ꢅ 0.56
20.8 ꢅ 0.60
Fig. 3. Plot of k1 vs [peptide] for the oxidation of peptide containing methionines
with [Ru(bpy)3]3+ in oxygen saturated aqueous CH3CN (1:1, v/v) solution in the
presence of 2.3 M HClO4 at 298 K. (The points 1–5 refer to the peptide containing
methionine given in Table 1).
General condition: [Ru(NN)3]3+ = 2 ꢃ10ꢀ5 M, [Peptide] = 5 ꢃ10ꢀ4 M and [H+] = 2.3 M.

D. Thiruppathi et al. / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 70–78
75
Table 4
Second order rate constant (k2) values and activation parameters for the oxidation of peptide containing methionines by [Ru(bpy)3]3+ in aqueous CH3CN (1:1, v/v) at four
different temperatures.
¼
¼
DS
J Kꢀ1 molꢀ1
Peptides
k2, Mꢀ1 sꢀ1
DH
k J molꢀ1
ꢀ
293 K
298 K
303 K
313 K
Met-Gly
Met-Ala
Met-Ser
Met-Val
Met-Leu
7.3 ꢅ 0.2
8.1 ꢅ 0.2
9.7 ꢅ 0.3
11.2 ꢅ 0.3
14.7 ꢅ 0.4
17.6 ꢅ 0.5
21.7 ꢅ 0.6
13.6 ꢅ 0.4
15.5 ꢅ 0.4
20.3 ꢅ 0.5
24.4 ꢅ 0.7
27.9 ꢅ 0.8
18.5 ꢅ 0.5
20.7 ꢅ 0.6
26.5 ꢅ 0.8
30.9 ꢅ 0.9
36.5 ꢅ 1.0
35.6 ꢅ 1.1
35.1 ꢅ 1.0
34.7 ꢅ 0.99
35.6 ꢅ 1.0
36.4 ꢅ 1.1
197.1 ꢅ 5.9
197.1 ꢅ 5.4
197.1 ꢅ 5.3
197.1 ꢅ 5.7
197.1 ꢅ 5.8
11.3 ꢅ 0.3
13.1 ꢅ 0.4
15.2 ꢅ 0.1
General condition: [Ru(NN)3]3+ = 2 ꢃ10ꢀ5 M, [Peptide] = 5 ꢃ10ꢀ4 M and [H+] = 2.3 M.
(III) complexes (I–VI), [Ru(NN)3]3+, are one-electron oxidants and
the proposal of ET mechanism can be confirmed if the short lived
transient radical, formed during the course of the reaction, is
identified by nanosecond laser flash photolysis technique (Fig. 4).
The experiment was designed as follows: the reaction mixture
consisting of [Ru(dmbpy)3]2+ and methionylglycine taken in
aqueous CH3CN (1:1, v/v), was purged with molecular oxygen
for 20 min in the presence of 2.3 M HClO4. This reaction mixture
was utilized for the flash photolysis study. When this reaction
mixture was irradiated with flash of light using the flash photolysis
set up it resulted in the appearance of the absorption peaks
corresponding to various transients depending as a function of
time. The irradiation of [Ru(dmbpy)3]2+ in the presence of O2 leads
to the formation of Ru(III) species which is formed due to the
oxidative quenching of *[Ru(NN)3]2+ with molecular oxygen
particularly at high [H+] as per reactions shown in Eqs. (1)–(3)
(Scheme 1). Thus the transients formed are due to the reaction
between [Ru(dmbpy)3]3+ and methionylglycine as shown in Fig. 4.
Fig. 4 shows the formation of three new species and decay of
two species. The peak at 370 nm corresponds to the dmbpy radical
anion on the basis of its similarity with the spectrum of 2,20-
bipyridyl anion radical and 420 nm assigned to the absorption
maximum of Ru(III) ion. The bleaching around 450 nm is due to the
emission of the excited state of Ru(II) ion. Interestingly a new peak
is appeared around 480 nm at 1 ms, which is assigned to the well-
characterized two-center, three-electron-bonded (S;S)+ dimeric
radical cation of methionylglycine (Met-Gly) [17,18]. But this short-
lived species almost completely disappeared at 2 ms after the laser
pulse (Fig. 4). Bobrowski et al. [40–42] previously reported the
formation of dimeric radical cation with different kind of
methionine peptides at 480 nm. The point that we want to
emphasize from these results is that during the course of this
reaction methionine sulfur radical cation Sꢁ+ and dimeric radical
cation (S;S)+ are formed as the intermediates of the reaction. It is
important to quote that at low pH 0–1 the formation of S;S
species is more favorable compared to S;N and S;O species in the
oxidation of peptide containing methionines [40].
When we irradiated a mixture of [Ru(dmbpy)3]2+ and methio-
nylglycine (Met-Gly) in the absence of O2 with 2.3 M HClO4 no
evidence for the formation of Ru3+ and methionine sulfur radical
cation is observed (Fig. 5). This clearly shows that the excited state
[Ru(NN)3]2+ has no reaction with methionylglycine (Met-Gly).
When we look at the spectral changes indicated in Fig. 5 in the
region 330–800 nm the formation of one intermediate species and
bleaching of two species can be noticed. A sharp absorption band at
370 nm is attributed to the formation of dmbpy radical anion on
the basis of its similarity with the spectrum of 2,20-bipyridyl anion
radical. The bleaching around 450 nm is due to the loss of ground
loss of ground state absorption, dp–p* (MLCT) transition, [37–39]
while the disappearance of a peak at 650 nm corresponds to the
state absorption, dp–p* (MLCT) transition, [37–39] while the
disappearance of 650 nm correspond to the emission of the excited
state of Ru(II) ion. In the present experiment it is pertinent to
mention that a lack of absorption band at 480 nm infers that sulfur-
sulfur dimeric radical cation is not formed even at longer time
scales followed in the experiment.
We have also carried out flash photolysis study of the
deoxygenated solution of [Ru(bpy)3]2+ in the absence of
methionine and the absorption spectral changes are shown in
the supporting information (Fig. S4). The spectral changes
collected in the supporting information show that during this
photolysis study both the formation of excited state Ru2+ and
decay of *Ru2+ are observed. The important point we want to
emphasize from the flash photolysis study is that no transient
with absorption maximum at 480 nm is observed in the absence
of oxygen.
Fig. 4. Spectra of transients formed due to the reaction between the photochemi-
cally generated [Ru(dmbpy)3]3+ with 0.05 M methionylglycine in an oxygen-
saturated aqueous CH3CN (1:1, v/v) solution in the presence of 2.3 M HClO4 obtained
at different time delays, from100 ns to 2.0
m
s, by laser flash photolysis experiment.
Scheme 1. Reaction of excited state [Ru(NN)3]2+ with molecular oxygen.

76
D. Thiruppathi et al. / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 70–78
Fig. 5. Transient difference spectra of the [Ru(dmbpy)3]2+ complex with 0.05 M
methionylglycine in a deoxygenated aqueous CH3CN (1:1, v/v) solution in the
presence of 2.3 M HClO4 obtained at different time delays, from 100 ns to 1.0 ms, by
laser flash photolysis experiment.
3.2. Mechanism of [Ru(NN)3]3+ oxidation of methionine peptides
The one electron transfer from sulfur atom of methionyl peptide
to [Ru(NN)3]3+ is proposed as the rate controlling step of the
reaction (Eq. (4). The sulfur-center radical cation (>Sꢁ+) and
[Ru(NN)3]2+ are formed as the products of this step. The initially
formed sulfur radical cation can react with either water molecule
present in the solvent medium to form the hydroxyl sulfuranyl
radical (Eq. (5)) or another molecule of methionyl peptide to form
the dimer (Eq. (6)). Since the present reaction has been carried out
at high [H+] (2.3 M) the following steps are suggested to account for
the overall reaction and transients detected. (i) in the presence of
higher thioether concentration, monomeric sulfur radical cation
combines with a unoxidized Met residue according to equilibrium
(Eq. (6)) to form the intermolecular two-center, three-electron-
bonded dimeric radical cation (S;S)+ – species which has an
absorption maximum at 480 nm confirmed by laser flash
photolysis study. (ii) [Ru(NN)3]3+ abstracts an electron from the
hydroxysulfuranyl radical (ꢁS—OH) in a fast step to produce the
protonated sulfoxide (Eq. (7)). (iii) Finally the deprotonation of
hydroxysulfuranyl cation yields sulfoxide as the major product of
the reaction (Eq. (8)) is confirmed by FT-IR and 1H NMR techniques.
The electron transfer mechanism of the reaction is supported from
a satisfactory correlation between the experimentally observed
second-order rate constants and the values calculated by Marcus
semiclassical theory also support the proposed mechanism. This
mechanism is similar to the one proposed by us for the [Ru(NN)3]3+
and [Fe(NN)3]3+ oxidation of organic sulfides and sulfoxides
[25,43,44] (Scheme 2).
Scheme 2. Mechanism for the oxidation of methionine peptides by [Ru(NN)3]3+
.
ꢀ
ꢁ
4
p2
e
ꢀsSm
m!
a
X
2
1=2
ket
¼
jHDAj ð4plkTÞ
h
m¼0
"
#
2
ꢀð
l
þ
D
Gꢇ þ mhvÞ
ꢃ exp
(9)
ð4l
kTÞ
In Eq. (9), HDA is the electronic coupling matrix element, the
reorganization energy is composed of solvational l0 and
vibrational li contributions with s = li/h is the high-energy
l
n, n
vibrational frequency associated with the acceptor and m is the
density of product vibrational levels. The terms h and k are Planck’s
and Boltzmann’s constants, respectively. The free-energy change
(D
Gꢇ) of electron transfer can be calculated from Eq. (10).
D
Gꢇ ¼ E
ꢀ E
(10)
ðA=AꢀÞ
D=Dþ
ð
Þ
+
where E(
D/D
A
) is the oxidation potential of electron donor and E(
A/
ꢀ
), the reduction potential of acceptor. The reorganization energy
(l) is the sum of two contributions, l= l0 + li, where li represents
3.3. Application of Marcus semiclassical theory to the electron transfer
reaction of [Ru(NN)3]3+ with methionine peptides.
the activation of the vibrational modes of the reactants and l0
represents the changes in the solvent structure around the
reactants, which strongly dependent on the solution medium.
According to the Marcus theory of electron transfer, the l0 value is
given by Eq. (11) [48].
After establishing the electron transfer nature of the reaction,
we applied the semiclassical theory of electron transfer (Eq. (9))
[45–47] to the above redox reaction. The rate of ET from a donor to
an acceptor molecule in a solvent is controlled by free energy
ꢄ
ꢂ
ꢃꢅ
l0 ¼ e2=4pe0 ð1=2rD þ 1=2rA ꢀ 1=dÞ 1=Dop ꢀ 1=Ds
Here e is the transferred electronic charge, e0 the permittivity of
free space, Dop and Ds the optical and static dielectric constants,
(11)
change of the reaction (D l) and the
Gꢇ), the reorganization energy (
electron transfer distance between the donor and the acceptor
(Eq. (9)).

D. Thiruppathi et al. / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 70–78
77
Acknowledgements
M. Ganesan thanks University Grant Commission (UGC), New
Delhi, India, for sanctioning a Major research project (F. No. 35-140/
2008) (SR). D. Thiruppathi thanks UGC, New Delhi, India, for a
Junior Research Fellowship. M.G. and D.T. thank the Management,
Principal and Head of the Department of Chemistry of Vivekananda
College, Thiruvedakam West, Madurai, India, for providing
laboratory facilities. The authors also thank Prof. P. Ramamurthy
and Dr. C. Selvaraju, National Centre for Ultrafast Processes,
University of Madras, Chennai, India, for their help in recording
transient spectra using laser flash photolysis technique.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.jpho-
tochem.2014.09.003.
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