Reduction of cytochrome c in its reaction with thiamine and its structural analogs

Article abstract of DOI:10.1023/A:1023361817590

The kinetics of reduction of ferricytochrome c in its reaction with thiamine and its O-substituted structural analogs in phosphate buffer at pH 7.5-7.8 were studied. The reduction rate is proportional to the concentration of thiamine or its derivatives. The dependence of the reaction rate on the oxidant concentration is characterized by negative deviations from linearity. In oxidation with ferricytochrome c, the reactivity of thiamine consdierably exceeds the reactivity of thiamine diphosphate and thiamine monophosphate, and in oxidation with ferricyanide the reaction rate increases in the order thiamine monophosphate < thiamine < thiamine diphosphate. With O-(2-norbornoyl)thiamine, O-(2-adamantyl)acetylthiamine, O-benzoylthiamine, O-(4-nitrobenzoyl)thiamine, or O-(5-nitro-2-chlorobenzoyl)thiamine, the rate of ferricytochrome c reduction is higher than with thiamine. Presumably, the electron transfer to the heme group of the oxidant is preceded by formation of a complex of ferricytochrome c with the neutral tricyclic form of the substrate.

Full text of DOI:10.1023/A:1023361817590

Russian Journal of General Chemistry, Vol. 72, No. 11, 2002, pp. 1808 1812. Translated from Zhurnal Obshchei Khimii, Vol. 72, No. 11, 2002,
pp. 1913 1917.
Original Russian Text Copyright
2002 by Vovk, Babii, Murav’eva.
Reduction of Cytochrome c in Its Reaction with Thiamine
and Its Structural Analogs
A. I. Vovk, L. V. Babii, and I. V. Murav’eva
Institute of Bioorganic and Petroleum Chemistry, National Academy of Sciences of Ukraine, Kiev, Ukraine
Received January 25, 2001
Abstract The kinetics of reduction of ferricytochrome c in its reaction with thiamine and its O-substituted
structural analogs in phosphate buffer at pH 7.5 7.8 were studied. The reduction rate is proportional to the
concentration of thiamine or its derivatives. The dependence of the reaction rate on the oxidant concentration
is characterized by negative deviations from linearity. In oxidation with ferricytochrome c, the reactivity of
thiamine consdierably exceeds the reactivity of thiamine diphosphate and thiamine monophosphate, and in
oxidation with ferricyanide the reaction rate increases in the order thiamine monophosphate < thiamine <
thiamine diphosphate. With O-(2-norbornoyl)thiamine, O-(2-adamantyl)acetylthiamine, O-benzoylthiamine,
O-(4-nitrobenzoyl)thiamine, or O-(5-nitro-2-chlorobenzoyl)thiamine, the rate of ferricytochrome c reduction
is higher than with thiamine. Presumably, the electron transfer to the heme group of the oxidant is preceded
by formation of a complex of ferricytochrome c with the neutral tricyclic form of the substrate.
Thiamine (vitamin B1), thiamine monophosphate,
and thiamine diphosphate can be oxidized with ferri-
cyanide in the presence of a base to thiochrome and
its derivatives [1, 2]. Thiamine transforms into thio-
chrome and oxodihydrothiochrome in reaction with
Cu(II) ions and ascorbic acid [3] and also with H2O2,
oleic acid, and peroxidase [4]. Reduction of ferricya-
nide with thiamine diphosphate can be a side reaction
in the determination of the activity of the pyruvate de-
carboxylase component of the pyruvate dehydrogenase
complex [5]. Oxidative transformations of thiamine
and its structural analogs attract attention as model
processes characterizing the stability of vitamin B1
and thiamine phosphate against possible xenobiotics
and physiological electron acceptors.
its structural analogs II VIII differing in the substit-
uent at the 5-position of the thiazolium ring were cal-
culated from the rate of ferrocytochrome c formation,
monitored spectrophotometrically. The optical density
at 550 nm, characterizing its accumulation [8], varied
with time. The initial rate of formation of ferrocyto-
chrome c in the course of oxidation of thiamine and
its derivatives II VIII increases with pH. Figure 1 il-
lustrates the kinetics of reduction of ferricytochrome c
in reaction with vitamin B1 at pH 7.5 and 7.8. In go-
ing from 3-(4-amino-2-methyl-5-pyrimidinyl)methyl
analogs of thiamine to 5-benzoyl-4-methyl-5-substi-
tuted thiazolium salts, the yield of reduced cyto-
chrome c appreciably decreases. For example, in the
5
reaction of ferricytochrome c (3 10 M) with
3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chlo-
4
Cytochrome c is a component of mitochondrial
electron-transport chain; it can fulfil the regulator
functions [6]. Cytochrome c is used as oxidant in
biomimetic systems [7]. We found that, at pH 7.5,
ferricytochrome c is reduced with vitamin B1. Our
goal was to elucidate the kinetic features of oxidative
transformations of thiamine, its O-acyl-substituted
analogs, and thiamine monophosphate and thiamine
diphosphate in the presence of cytochrome c and a
base.
ride (4 10 M) in phosphate buffer (pH 7.8), the
amount of the forming ferrocytochrome c is as low
as 10% as compared to the control experiments with
vitamin B1.
+
N
Me
N
Cl HCl
OR
Me
N
S
NH2
I VIII
The consumption of thiamine, thiamine monophos-
phate, and thiamine diphosphate in the reaction with
a base and ferricytochrome c and the formation of
the corresponding thiochromes were established by
HPLC. The initial reaction rates with thiamine I and
R = H (I), PO3H2 (II), P2O6H3 (III), NbCO (IV),
AdCH2CO (V), C6H5CO (VI), p-NO2C6H4CO (VII),
2-Cl,5-NO2C6H3CO (VIII). Nb is 2-norbornyl, Ad
is 2-adamantyl.
1070-3632/02/7211-1808$27.00 2002 MAIK Nauka/Interperiodica

REDUCTION OF CYTOCHROME c IN ITS REACTION WITH THIAMINE
1809
reaction rate increases in going from thiamine mono-
phosphate and thiamine diphosphate to thiamine.
However, in oxidation of thiamine and thiamine phos-
phates with ferricyanide, the pattern is different. In
this case, the reaction rate increases in the order thia-
mine monophosphate < thiamine < thiamine diphos-
phate (Fig. 3). Thus, the reactivity of thiamine diphos-
phate depends on the nature of the oxidant more
strongly than the reactivity of thiamine and thiamine
monophosphate. At the same time, the rate of reduc-
tion of both ferricyanide [9] and ferricytochrome c
increases on replacement of thiamine by O-(2-ada-
mantyl)acetylthiamine V or its structural analogs IV
and VI VIII. The rates of oxidation of O-(4-nitroben-
zoyl)thiamine VII and O-benzoylthiamine VI are com-
parable, whereas O-(5-nitro-2-chlorobenzoyl)thiamine
VIII is less reactive.
5
6
4
2
0
4
3
2
1
2
4
c
104, M
Fig. 1. Observed rate of reduction of ferricytochrome c
as a function of concentration of (1) thiamine mono-
phosphate, (2) thiamine diphosphate, (3, 4) thiamine,
and (5) O-(2-adamantyl)acetylthiamine. Cytochrome c
The mechanism of formation of thiochrome from
thiamine should involve transfer of two electrons and
also proton transfer with the participation of a base.
As seen from Fig. 2a, at a relatively low concentration
of ferricytochrome c, the reaction is approximately
first-order with respect to the oxidant. Apparently, the
second molecule of ferricytochrome c reacts after the
stage limiting the overall process of oxidation of thi-
amine or its structural analog. The low rate of ferricy-
tochrome oxidation in the reaction with the 3-benzyl
analog of thiamine suggests that the products of the
thiazole ring opening [10] are substrates of the redox
reaction to a considerably lesser extent. If it is pre-
sumed that the neutral tricyclic form of vitamin B1
(S ) [9] is an intermediate in the transformation of thi-
amine into thiochrome under the action of a base, then
one-electron transfer from S to the heme group of
ferricytochrome c will yield radical cation product P .
5
concentration 3 10 M; pH (3, 5) 7.5 and (1, 2, 4) 7.8.
The rate of reduction of ferricytochrome is directly
proportional to the concentration of thiamine I or its
derivative II VII (Fig. 1). The plot of the observed
rate constant vs. ferricytochrome c concentration is
characterized by negative deviations from linearity
(Fig. 2a). Similar nonlinear kinetic relationships be-
tween the observed rate constant and the oxidant con-
centration are observed with IV VI also. The hyper-
bolic curves obtained with all the substrates are line-
azied in the reciprocal coordinates 1/c vs. 1/[ferricyto-
chrome c] (Fig. 2b).
Figures 1 and 2 show that the rate of ferricyto-
chrome c reduction depends on the substituent in the
5-position of the thiazolium salt. In particular, the
(b)
2
20
(a)
1
5
4
2
1
16
12
8
3
3
4
0
4
2
1
5
2
4
0
0.5
1
1.5
2
[CytC] 105, M
5
(1/[CytC]) 10 , M
Fig. 2. Observed rate of reduction of ferricytochrome c as a function of its initial concentration: (a) direct and (b) reciprocal
4
coordinates. Substrate concentration 4 10 M, pH 7.8. (1) Thiamine monophosphate, (2) thiamine diphosphate, (3) thiamine,
(4) O-(4-nitrobenzoyl)thiamine, and (5) O-(5-nitro-2-chlorobenzoyl)thiamine.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 72 No. 11 2002

1810
VOVK et al.
The effect of molecular oxygen as one-electron accep-
tor in the subsequent base-catalyzed transformation of
radical cation P into thiochrome (P) should not alter
the reaction stoichiometry, since the superoxide radi-
cal anion is efficiently oxidized with ferricytochrome
c [8].
+
N
H+
Me
Me
N
N
N
Me
Me
OR
Me
OR
Me
S
N
H
S
N
N
NH2
H
S
S
+
N
N
CytC
N
N
+
N
H+,
e
Me
OR
Me
S
N
OR
N
H
P
S
N
H
H
Me
N
N
H+
Me
N
OR
S
N
P
The negative deviations from the linear dependence
of the observed rate of ferrocytochrome c formation
on the initial concentration of ferricytochrome c
(Fig. 2a) suggest the possibility of complexation
between the reactants. The kinetic scheme of the pro-
cess can be presented as follows:
Taking into account the material balance with re-
spect to the total concentration of the neutral tricyclic
form, free species, and the species bound in a complex
with ferricytochrome c, we can write the equation for
the reaction rate as follows:
k1k2K[Cytc][S]
v =
.
K1[Cytc]
k [H+] + k1K[Cytc]
K
fast
k2
1
S
S
[S Cytc]
P
P.
[H+]
Here, [S] is the concentration of thiamine or its
structural analog; k1 and k are the rate constants
1
of reversible complexation of the ligand with cyto-
chrome c (direct and reverse processes, respectively).
The kinetic equation was deduced assuming that the
concentration of ferricytochrome c is considerably
higher than the concentration of intermediate S occur-
ring in equilibrium (constant K) with the ionic sub-
3
5
4
3
strate species. At k [H+] >> k1K[Cytc], the rate is
1
equal to k2K1K[Cytc][S]/[H+], and at k [H+] << k1
2
1
K[Cytc] the rate is determined by the rate of decom-
position of the complex of intermediate S with cyto-
chrome c, with the constant k2. The constant k2 can
be determined from the reciprocal length of the por-
tion intercepted on the ordinate by the plot of 1/v
vs. 1/[Cytc]. The slope of the straight lines in the
reciprocal coordinates, equal to [H+]/k2K1K[Cytc][S],
characterizes the product of the equilibrium constants
in the stages of base-catalyzed formation of intermedi-
ate S and its complex with the protein. The calculated
constants k2 and the relative values of KK1 are listed
below. It is seen that the substrate structure affects
both KK1 and k2. The constant k2 increases in going
2
1
1
0
1
2
3
4
[K3Fe(CN)6] 104, M
Fig. 3. Observed rate of oxidation of (1) thiamine mono-
phosphate, (2) thiamine, and (3) thiamine diphosphate as
a function of ferricyanide concentration. Concentration
5
of K [Fe(CN) ] 2 10 M, pH 7.8.
3
6
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 72 No. 11 2002

REDUCTION OF CYTOCHROME c IN ITS REACTION WITH THIAMINE
1811
from thiamine to O-(5-nitro-2-chlorobenzoyl)thiamine
VIII and to derivatives IV VII and decreases in going
to thiamine monophosphate and thiamine diphosphate.
The similar relationship between the structure and
reactivity of vitamin B1, thiamine phosphates II and
III, and derivatives V VIII is also manifested in the
relative values of KK1, which increase in going from
thiamine monophosphate to O-(4-nitrobenzoyl)thia-
mine VII and O-benzoylthiamine VI.
[13]. The results of this work show that, in interaction
of vitamin B1 with a protein, the acid base and redox
stages can be conjugated and revealed structural fea-
tures of the ligand determining the specificity of such
interaction in the model system.
EXPERIMENTAL
In our work, we used thiamine chloride hydrochlo-
ride (Sigma), thiamine monophosphate chloride hy-
drochloride trihydrate (Serva), thiamine diphosphate
tetrahydrate (Fluka), K3Fe(CN)6, K4Fe(CN)6, KCl
(analytically pure grade), and cytochrome c from
horse heart (Serva). Compounds IV VIII were pre-
pared by reaction of 4-amino-2-methyl-5-chlorometh-
ylpyrimidine hydrochloride with appropriate 4-meth-
yl-5-substituted thiazoles as described in [9, 14, 15].
The electronic absorption spectra were recorded on a
Specord M-40 spectrophotometer. In HPLC analysis
of the reaction mixture, we used an LCD-2553 spec-
trophotometric detector, a 3.3 150-mm Separon
SGX-C18 column (5 m), and a CI-100A integrator
(Laboratorni Pristroje, Czechia). The mobile phase
was 10% aqueous methanol containing 0.025 M tetra-
butylammonium bromide and 0.23 M sodium dihydro-
gen phosphate. Detection was performed at 254 nm.
1
Substrate
k2 106, s
KK1 (rel.)
I
II
1.5 0.2
0.6 0.3
0.5 0.1
3.1 0.5
3.3 0.3
2.6 0.4
2.9 0.3
2.0 0.2
1
0.5
0.9
1.2
2.6
3.4
3.3
2.1
III
IV
V
VI
VII
VIII
Thus, vitamin B1 undergoes oxidative transforma-
tions in reaction with cytochrome c in the presence of
a base. The electron transfer to the heme group can be
preceded by fast formation of a complex of ferricyto-
chrome c with the intermediate of acid base transfor-
mations of thiamine or its structural analog. Apparent-
ly, the efficiency of the complexation and subsequent
oxidation with the heme group increases when the
ligand contains an ester moiety. The relatively low
reactivity of thiamine diphosphate in oxidation with
ferricytochrome c, as compared to its oxidation with
ferricyanide, may be due to specific features of inter-
action of the neutral tricyclic form of the substrate
with the protein molecule.
The kinetics of reduction of ferricytochrome c and
ferricyanide with thiamine and its structural analogs
were studied in a temperature-controlled spectrophoto-
metric cell at 23.5 C in K Na phosphate buffer
(0.05 M). In control experiments without the thiazoli-
um substrate, the concentration of cytochrome c
changed by no more than 5%; this was taken into
account when calculating the reaction rate. The molar
extinction coefficient of ferrocytochrome c at 550 nm
1
was taken as 29040 l mol 1 cm [8]. The rate of
It is known that vitamin B1 is a substrate or prod-
uct of a number of enzymatic transformations control-
ling the level of thiamine phosphates. It is also bound
by proteins providing biological transport. Thiamine-
binding proteins are poorly studied. The character of
the reaction of thiamine, thiamine monophosphate,
and thiamine diphosphate with cytochrome c, re-
vealed in this work, reflects the known relationships
of the interaction of vitamin B1 and its phosphate
esters with thiamine-containing proteins [11]. For ex-
ample, the dissociation constants of complexes of the
thiamine-binding protein from buckwheat grains in-
crease in going from thiamine as ligand to thiamine
monophosphate and thiamine diphosphate. Under the
same conditions, the thiamine-binding protein effi-
ciently retains O-acetylthiamine and O-benzoylthia-
mine [11, 12]. Thiamine diphosphate does not notice-
ably compete with vitamin B1 in its interaction with
the thiamine-binding protein of rat brain synaptosomes
oxidation of thiamine and its structural analogs was
determined by accumulation of thiochrome and its
analogs, detected spectrophotometrically at 367 nm,
and by a decrease in the optical density at 420 nm,
characteristic of ferricyanide. With excess oxidant,
the reaction is first-order with respect to both substrate
and oxidant. In the pH range used (pH 7.5 7.8), the
relative content of the N1 -protonated species of I VIII
is insignificant [16].
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