Redox turnover of organometallic B12 cofactors recycles vitamin C: Sulfur assisted reduction of dehydroascorbic acid by cob(II)alamin

Article abstract of DOI:10.1016/j.jorganchem.2017.01.002

This work reports the reactivity of cob(II)alamin (Cbl(II)) toward reduction of dehydroascorbic (DHA) to ascorbic acid (AA) mediated by sulfur-containing compounds such as glutathione (GSH) and thiocyanate. The reaction supported by GSH proceeded more efficiently than with SCN^?. Our findings demonstrate new aspects of interactions between vitamins B₁₂ and C. It has been accepted that simultaneous presence of these vitamins results in their decomposition (viz., irreversible modification of the corrin ring of Cbl and oxidation of AA). We have shown, however, that Cbl(II), the biologically active one-electron reduction product of methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl), is capable of recovering AA in the presence of natural sulfur-containing ligands, within a process that can occur in?vivo without glutathione spending, both in a stoichiometric and catalytic manner. Our studies highlight the redox versatility of Cbl(II) and expands the repertoire of reactions whereby redox turnover of the unique B₁₂ organometallic cofactors MeCbl and AdoCbl generates Cbl(II), which in turn recycles oxidized vitamin C.

Full text of DOI:10.1016/j.jorganchem.2017.01.002

Journal of Organometallic Chemistry xxx (2017) 1e7
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Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Redox turnover of organometallic B cofactors recycles vitamin C:
1
2
Sulfur assisted reduction of dehydroascorbic acid by cob(II)alamin
Ilia A. Dereven'kov , Luciana Hannibal b , Maximilian Dürr , Denis S. Salnikov ,
a
,
*
,
c
c
a
a
a
a
c
Thu Thuy Bui Thi , Sergei V. Makarov , Oscar I. Koifman , Ivana Ivanovi ꢀc -Burmazovi ꢀc
Institute of Macroheterocyclic Compounds, Ivanovo State University of Chemistry and Technology, Sheremetevskiy str. 7, 153000 Ivanovo, Russian
a
Federation
b
Laboratory of Clinical Biochemistry and Metabolism, Department for Pediatrics, Medical Center, University of Freiburg, Mathildenstraße 1, 79106 Freiburg,
Germany
Department of Chemistry and Pharmacy, Friedrich-Alexander University of Erlangen-Nürnberg, Egerlandstrasse 1, 91058 Erlangen, Germany
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 November 2016
Received in revised form
This work reports the reactivity of cob(II)alamin (Cbl(II)) toward reduction of dehydroascorbic (DHA) to
ascorbic acid (AA) mediated by sulfur-containing compounds such as glutathione (GSH) and thiocyanate.
The reaction supported by GSH proceeded more efficiently than with SCN . Our findings demonstrate
ꢀ
3
January 2017
new aspects of interactions between vitamins B12 and C. It has been accepted that simultaneous presence
of these vitamins results in their decomposition (viz., irreversible modification of the corrin ring of Cbl
and oxidation of AA). We have shown, however, that Cbl(II), the biologically active one-electron
reduction product of methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl), is capable of recovering AA in
the presence of natural sulfur-containing ligands, within a process that can occur in vivo without
glutathione spending, both in a stoichiometric and catalytic manner. Our studies highlight the redox
versatility of Cbl(II) and expands the repertoire of reactions whereby redox turnover of the unique B12
organometallic cofactors MeCbl and AdoCbl generates Cbl(II), which in turn recycles oxidized vitamin C.
© 2017 Elsevier B.V. All rights reserved.
Accepted 3 January 2017
Available online xxx
Keywords:
Cobalamin
Glutathione
Vitamin B12
Vitamin C
Dehydroascorbic acid
Ascorbic acid
Thiocyanate
Reaction mechanisms
1. Introduction
hemoglobin leading to the formation of methemoglobin and NO
liberation [6].
Dehydroascorbic acid (DHA; Fig. 1A and B) is the product of two-
electron oxidation of ascorbic acid (AA; vitamin C; Fig. 1C). Its
therapeutic significance attracted a lot of attention, because DHA
seems to be the pharmaceutically active form of vitamin C that is
preferentially imported by colorectal cancer cells and is responsible
for selective toxicity of vitamin C to tumor cells [1]. Upon cell up-
take it is reduced to AA, predominantly by glutathione (GSH; Fig. 1
D), thioredoxin and NADPH [2e5], which represents an important
mechanism of its biological and therapeutic actions [1]. The ques-
tion remains, whether DHA reduction is always related to GSH
consumption or if it can proceed through other pathway, e.g.
involving metal centers. The only example of DHA reduction by
metal complexes relates to the reaction with iron-nitrosyl-
Cobalamin (B12, Cbl) is an essential micronutrient required by all
cells in the body. An important fraction of the intracellular pool of
cobalamin, the only metal-containing vitamin (Fig. 1E), is its one-
electron reduced form, i.e. cob(II)alamin (Cbl(II)) [7e9], which
can be considered as a reducing agent. The vast majority of cob(II)
alamin present in biological systems derives from the one-electron
reduction of MeCbl and AdoCbl, as part of the catalytic cycle of
cognate enzymes methionine synthase and methylmalonyl-CoA
mutase [8,9]. Chemically, the reducing activity of Cbl(II) was
observed in the reactions with oxidizing free radicals, e.g. super-
ꢀ
oxide (O
2
) [10,11], nitric oxide (NO) [12e14], nitrogen dioxide (NO
2
)
[15,16] and chlorine dioxide (ClO
form complexes with GSH and SCN that are abundant compounds
2
) [17]. Both Cbl(III) and Cbl(II)
ꢀ
in nature [18e23], with concentrations of GSH reaching values of
ꢀ
1
0 mM in cells [24] and those for SCN being 0.5e6.0 mM in
mucosal liquids and ca. 10e100-fold lesser in blood [25,26]. In cells,
cobalamins are present in relatively low concentrations
*
Corresponding author.
E-mail address: derevenkov@gmail.com (I.A. Dereven'kov).
http://dx.doi.org/10.1016/j.jorganchem.2017.01.002
022-328X/© 2017 Elsevier B.V. All rights reserved.
0
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I.A. Dereven'kov et al. / Journal of Organometallic Chemistry xxx (2017) 1e7
Fig. 1. Structures of (A) monomeric, MDHA, and (B) dimeric, DDHA, forms of dehydroascorbic acid, (C) ascorbic acid, (D) ascorbyl free radical, (E) glutathione and (F) cobalamin
ꢀ
(
R ¼ H
2
O, CN ).
(
0.15e0.20 nM), but their reduced forms cob(II)alamin and cob(I)
potassium thiocyanate (Sigma-Aldrich; ꢁ 99%) were used as
e
alamin are highly reactive [27]. Glutathionylcob(III)alamin (GS -
Cbl(III)) was found in vivo [27]. Important characteristic of these
complexes is the existence of a CoeS bond, strengthening the
notion that CoeS bonded cobalamins, including the fraction of
SCN that binds through the sulfur atom partake in the complex
map of cellular cobalamin species [20,21].
received. Concentrations of Cbl were found by means of conversion
ꢀ1
ꢀ1
to dicyano form (ε367 ¼ 30,400 M cm ) [28]. Cbl(II) was prepared
using sodium borohydride according to a published procedure [29].
Excess borohydride was destroyed by adding acetone. Stock solu-
ꢀ
tions of DHA were prepared by dissolving the commercial com-
ꢂ
pound in diluted H
3
PO
4
(pH 2.3), kept at 5 C and used within 3 h.
Knowing that extra-ligands may influence redox behavior of the
metal centers we were interested to reveal the reactivity of Cbl(II)/
GSH-system toward DHA. Therefore, in this work we report kinetic
and mechanistic studies of reaction of Cbl(II) with dehydroascorbic
acid in acidic and neutral medium (at room temperature) in the
presence of glutathione. For comparison, the studies were per-
formed with thiocyanate as well, which differs from GSH in that it is
a redox inactive (under applied experimental conditions) S-
donating ligand. Interestingly, we have observed a catalytic effect of
S-containing ligands on the reduction of DHA by Cbl(II). This has
important mechanistic implications related to physiological and
therapeutic aspects of vitamin C: (1) Cbl(II) formed during normal
redox cycling of organometallic derivatives MeCbl and AdoCbl can
recycle vitamin C in a process assisted by natural sulfur-containing
species, and (2) the intracellular conversion of DHA to vitamin C
mediated by Cbl(II) does not occur at the expense of glutathione
consumption.
Concentrations of DHA were determined after its reduction to AA
([GSH] ¼ 50 mM, pH 5e6) using published extinction coefficients
[30]. Air-free argon was used to deoxygenate solutions. Phosphoric
acid, mono- and disubstituted potassium phosphate and sodium
acetate were used to adjust pH. All experiments were performed in
0.1 M buffers under anaerobic conditions.
2.2. Methods
ꢂ
UVevis spectra were recorded on thermostated (±0.1 C) Cary
50 spectrophotometer in gas-tight quartz cells with optical path-
length 1.00 cm. Stopped-flow experiments were performed on
mSFM-20 BioLogic device equipped with J&D TIDAS detector.
Cyclic voltammetry (CV) experiments were carried out using an
Autolab instrument with a PGSTAT 30 potentiostat. A conventional
three electrode arrangement was employed consisting of a glassy
carbon working electrode (Metrohm), a platinum wire as the
counter electrode and silver chloride (in 3 M NaCl) reference
ꢂ
2
. Experimental
electrode (0.222 V vs NHE at 20 C). Prior to the measurements, the
surface of the glass-carbon electrode was polished with alumina
powder. Lithium perchlorate (0.1 M) was used as the supporting
electrolyte.
2.1. Chemicals
Hydroxocobalamin hydrochloride (Fluka; ꢁ 95%), dehydro-L-
Analysis of reaction products was performed anaerobically us-
ing water as a solvent (the resulting pH of the samples was 5.3).
Starting material, adducts and products were identified by cryo-
(
þ)-ascorbic acid dimer (Sigma-Aldrich; ꢁ 80%), sodium borohy-
dride (Aldrich; ꢁ 96%), glutathione (Sigma-Aldrich; ꢁ 98%),
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I.A. Dereven'kov et al. / Journal of Organometallic Chemistry xxx (2017) 1e7
3
inside the anaerobic glovebox and data collection in the mass
spectrometry instrument. CSI-MS measurements were performed
on a UHR-TOF Bruker Daltonik (Bremen, Germany) maXis plus 5G,
an ESI-quadrupole time-of-flight (qToF) mass spectrometer capable
of resolution of at least 60,000 FWHM, which was coupled to a
Bruker Daltonik Cryospray unit. Detection was in positive ion mode,
the source voltage was 3.8 kV. The flow rates were 280 mL/h. The
ꢂ
drying gas (N
2
), to aid solvent removal, was held at þ5 C and the
ꢂ
spray gas was held at þ5 C. The machine was calibrated prior to
every experiment via direct infusion of the Agilent ESI-TOF low
concentration tuning mixture, which provided an m/z range of
singly charged peaks up to 2700 Da in both ion modes.
Determination of equilibrium constants was performed on a
thermostated Cary 50 UVevis spectrophotometer under anaerobic
conditions. Equilibrium constants were calculated using Eq. (1)
[21].
A þ A
∞
K½Lꢄ;
0
A ¼
(1)
1
þ K½Lꢄ
[
A
L] is the total (free þ bound) ligand concentration in solution, M; A,
0
, A are absorbances at the monitoring wavelength for the
∞
metallocomplex at a particular ligand concentration, for the start-
ing complex, and for the final complex, respectively; K is equilib-
ꢀ
1
rium constant at a given pH, M
.
3. Results and discussion
At first the effect of biologically relevant sulfur-containing li-
gands on the oxidation of Cbl(II) by DHA was monitored by UV/Vis
ꢀ
spectroscopy. In the absence of GSH or SCN the reaction between
Cbl(II) and DHA does not proceed in acidic and neutral medium.
However, in their presence it clearly takes place, resulting in the
e
formation of GS -Cbl(III) (appearance of absorbance maxima at
374, 532 and 556 nm [23]; Fig. 2A) and thiocyanato-Cbl(III) (in-
crease of absorbance at 356 and 537 nm [21]; Fig. 2B) adducts.
In the case of GSH, the rate of oxidation of Cbl(II) was higher
ꢀ
than in the presence of SCN (insets in Fig. 2A and B; Table 1) and
the linear dependence of the observed rate constants (kobs) on
[DHA] was observed (Fig. 3A) with a positive intercept, whose
origin is discussed below. This behavior indicates that the reaction
is first order with respect to the oxidant. The pH dependence of the
ꢀ
ꢀ3
5
Fig. 2. UV/Vis spectra for the reaction between Cbl(II) (4
ꢃ
10
M) and DHA
ꢀ
4
ꢀ3
(
5.9 ꢃ 10 M (A), 2.7 ꢃ 10 M (B)) in the presence of GSH (1 ꢃ 10 M) at pH 5.0 (A),
ꢀ ꢀ2
ꢂ
and SCN (1 ꢃ 10 M) at pH 1.55 (B), 25 C, and typical kinetic curves of these re-
actions (insets).
0
second order rate constants (k , obtained from slopes of DHA con-
centration dependencies) demonstrates substantial increase of the
reaction rate upon a pH increase from 3.6 to 7.0 (Fig. S1) and this is
related to the deprotonation of GSH (see SI). Importantly, reduction
mass spectrometry. Hydroxocobalamin (final concentration
ꢀ5
1
ꢃ 10 M) was reduced with sodium borohydride to produce
of DHA by
a
Cbl(II)/GSH mixture proceeds much faster
Cbl(II). The excess of reducing agent was eliminated by addition of
0
ꢀ1 ꢀ1
(k ¼ 1977 ± 66 M
s
at pH 7.0) than the direct reaction between
acetone. The reactions of Cbl(II) with DHA (final concentration
ꢀ1 ꢀ1
ꢀ
4
DHA and GSH (0.43 ± 0.01 M
s at pH 7.0; Fig. S2), thus, the latter
5
ꢃ 10 M) in the presence or absence of glutathione (final con-
ꢀ
4
being negligible under our experimental conditions (Table 1).
Titration of Cbl(II) with DHA in the presence of GSH (1 mM) (Fig. S3)
centration 1 ꢃ 10 M) were followed continuously for 10 min, by
cryospray-ionization MS (CSI-MS) measurements. A dead-time of
demonstrated that the ratio of [DHA]
for the complete conversion of Cbl(II) into GS -Cbl(III), suggesting
0
: [Cbl(II)]
0
¼ 1: 2 is sufficient
3
min was estimated between the rapid mixing of the reagents
e
Table 1
Rate constants of reactions related to this study.
ꢀ
ꢀ1
Reaction
Conditions
pH 7.0, 25
Rate constant, M 1
s
DHA þ GSH / AFRꢀ þ GS þ H
ꢅ
þ
ꢂ
C
0.43
1977
ca. 6000
3.1
29.8
Cbl(II) þ DHA þ GSH / GSCbl(III) þ AFR þ Hþ
ꢀ
[GSH] ¼ 1 mM, pH 7.0, 25
ꢂ
C
ꢂ
[
GSH] ꢁ 10 mM, pH 7.0, 25
C
C
ꢀ
ꢀ
ꢀ
ꢂ
Cbl(II) þ DHA þ SCN / SCNCbl(III) þ AFR
[SCN ] ¼ 0.5 M, pH 6.4, 25
þ
ꢀ
ꢀ
ꢀ
þ
ꢂ
H
2
OCbl þ HAA / Cbl(II) þ AFR þ H þ H
2
O
pH 7.0, 21
C
GSCbl(III) þ HAA / Cbl(II) þ AFRꢀ þ GSH
[GSH] ¼ 1 mM, pH 7.0, 21
ꢂ
C
not detectable
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4
I.A. Dereven'kov et al. / Journal of Organometallic Chemistry xxx (2017) 1e7
decreased the Y-intercept (Fig. S8; at [GSH] ꢁ 2.5 mM the inter-
cept is equal to zero, within the experimental error limits).
However, an increase in [GSH] accelerated the reaction (increase
in slope; Fig. S8) reaching a maximum of the second order rate
0
ꢀ1 ꢀ1
constant k at high GSH concentrations (app. 6300 ± 900 M
s
at [GSH] ꢁ 10 mM; Fig. 3B). This saturation kinetic behavior in-
dicates involvement of a pre-equilibrium in the reaction mecha-
e
nism (vide infra), with formation of a precursor adduct GS -Cbl(II)
that has been earlier confirmed by EPR [22]. Herein, we provide
further evidence for the existence of glutathionylcob(II)alamin by
application of cryo ESI-MS analysis (Fig. 4) and UV/Vis spectros-
copy (Fig. S9). The formation and properties of glutathionylcob(II)
alamin have been demonstrated by two independent groups by
EPR [22,31]. Herein, we adopted an ultra-high resolution, anaer-
obic, low-temperature mass spectrometry approach to detect
glutathionylcob(II)alamin, a species predicted to be both short-
lived and produced in low-yields in our reaction. Cryo ESI-MS
analysis has revealed that Cbl(II) (experimental mass/charge ra-
þ
þ 2þ
tio of [Cbl(II) þ H þ Na ] is 676.28, Table S1) as well as Cbl(III)
þ 2þ
(
experimental mass/charge ratio of [Cbl(III) þ Na ] is 675.77,
Table S1) (presence of Cbl(III) was not possible to avoid under
conditions of MS experiments) are capable of binding GSH (mass/
þ
þ 2þ
charge ratio of [GSCbl(II) þ 2H þ Na ] is 829.82, and the ratio
þ
þ 2þ
of [GSCbl(III) þ H þ Na ] is 829.32, Table S1) (Fig. 4; Table S1).
This is the first study to document direct structural information
(mass) demonstrating formation of glutathionylcob(II)alamin. Our
results show that coordination of GSH to Cbl(II) occurs indepen-
dently of DHA (Fig. 4A) and further suggests that this process
represents the pre-equilibrium preceding the electron transfer to
e
DHA. The generation of GS -Cbl(II) was also detected in the
presence of DHA (Fig. 4B), though in lower quantity, in agreement
with its intermediate nature in the overall reaction mechanism
(vide infra). Titration of Cbl(II) by GSH results in UV/Vis spectral
changes shown in Fig. S9. UV/Vis spectra are established imme-
diately after mixing of reactants that is typical for rapid ligand
exchange on soft Co(II)-center and absorbance at 550 nm reaches
maximum value at [GSH] > 15 mM. The value of equilibrium
e
constant for GS -Cbl(II) formation was found using Eq. (1):
ꢀ
1
ꢂ
K(GSH) ¼ 287.1 ± 17.5 M at pH 6.6, 25 C, I(NaNO ) ¼ 0.3 M.
3
Fig. 3. (A) Plots of kobs. vs. [DHA] for the reaction of oxidation of Cbl(II) by DHA in the
ꢀ
3
ꢂ
presence of GSH (1 ꢃ 10 M) at pH 5.0, 25 C. (B) Dependence of the second order rate
Cryo ESI-MS was also used to characterize reaction products. In
the mixture of Cbl(II) and DHA without GSH no reduction of DHA
could be observed (Fig. 4C). However, immediately after addition
of GSH to the Cbl(II)/DHA mixture, formation of AA was detected
0
0
constant k on [GSH] at pH 7.0, room temperature (k was determined as a slope of the
linear dependence of kobs. on [DHA]).
(
Fig. 4B). Approximately 2% of DHA was converted to AA (corre-
ꢀ
5
sponding to ca. 10
M of AA, obtained from the mixture of
that DHA accepts two electrons and is transformed to AA.
ꢀ
5
ꢀ4
ꢀ4
2
ꢃ 10 M Cbl(II) and 5 ꢃ 10 M DHA in the presence of 10
GSH), in good correlation with the expected reaction stoichiom-
etry ([Cbl(II)] /[DHA]
M
We further investigated the effect of AA on possible reverse or
side reactions. Neither Cbl(II) nor a Cbl(II)/DHA mixture reacted
with AA during the timescale of all measurements performed in
this work (Fig. S4). Although AA can reduce Cbl(III), the corre-
0
0
¼ 2). Importantly, transformation of GSH,
e.g. its oxidation to GSSG, was not detected by ultra high resolu-
tion ESI-MS. In addition, we performed oxidation of Cbl(II) by
excess of DHA in the presence of deficient quantities of GSH (as
ꢀ
1
ꢀ1
sponding rate constant is so low (29.8 ± 0.2 M
s
and pH 7.0;
Table 1 and Fig. S5), thus, considering the formation of AA in the
ꢀ5
ꢀ4
0
compared to Cbl(II)) and found that the ratio of [GSH] /
concentration range of 10 -10
M (based on the above
e
[
Cbl(II)]
0
ꢁ 1 is necessary for complete oxidation of Cbl(II) to GS -
mentioned reaction stoichiometry) it cannot contribute to the
value of the experimentally observed intercept. Furthermore, the
presence of GSH additionally decreases the rate of Cbl(III) reduc-
tion by AA (Table 1; Fig. S6). Importantly, addition of 1 mM AA
shifted the Y-intercept to zero, while the slope remained un-
changed compared to experiments carried out without addition of
AA (Fig. S7). This may suggest the involvement of a transient
radical species (e.g. complex of glutathionyl-Cbl(III) with ascorbyl
Cbl(III) (Fig. S10).
Taken together, our results support a general mechanism
whereby GSH acts as a catalyst for the reduction of DHA to vitamin
C by cob(II)alamin (Eq. (2)), GSH accelerates this process from its
non-detectable rate in its absence to the rate of up to ca.
ꢀ
1 ꢀ1
6000 M
s , while remaining unaffected.
e
e
GSH
free radical (AFR), GS -Cbl(III)-AFR , that is produced after one-
electron reduction of DHA, vide infra) in the process responsible
for the intercept, which can be scavenged by AA. Similarly, GSH
DHA þ 2CblðIIރ!AA þ 2 GS ꢀ CblðIIIÞ=CblðIIIÞ
(2)
By way of comparison, we further investigated kinetics of the
reaction in the presence of redox silent S-donor, SCN . Observed
ꢀ
e
can also scavenge AFR . And indeed an increase in [GSH]
rate constants linearly depend on [DHA] indicating the first order
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I.A. Dereven'kov et al. / Journal of Organometallic Chemistry xxx (2017) 1e7
5
Fig. 4. Cryo ESI-MS spectra of (A) mixture of Cbl(II) (2 ꢃ 10 M) with GSH (10 M), (B) reaction mixture of Cbl(II), GSH and DHA (5 ꢃ 10ꢀ4 M) and (C) mixture of Cbl(II) and DHA
ꢀ
5
ꢀ4
(
without GSH).
kinetics with respect to oxidant (Fig. S11), as observed in the case
of GSH. However, the corresponding second order rate constants
GSH systems by cyclic voltammetry (CV). Nevertheless, CV studies
of GS -Cbl(III) have been performed earlier [33], but applied
relatively low GSH concentrations showed no influence on Cbl(II)
e
0
0
ꢀ1 ꢀ1
s for 1 mM
(
k ; Table 1) are significantly lower (k ¼ 274.1 M
0
ꢀ1 ꢀ1
ꢀ
GSH and k ¼ 3.1 M
s
for 500 mM SCN at pH ¼ 6.4).
oxidation.
0
ꢀ
Importantly, as observed with GSH, k is a function of [DHA] and
Addition of SCN and GSH, respectively, shifted the position of
exhibits a saturation behavior, achieving a maximal value at
the oxidation wave of Cbl(II) (0.075 V vs. Ag/AgCl) to more negative
values. GSH exerts stronger effect than SCN , by shifting the
ꢀ
ꢀ
[SCN ] > 0.7 M (Fig. S12). This is again in agreement with the
formation of a thiocyanato-Cbl(II) precursor described earlier [21],
in a pre-equilibrium step characterized by the equilibrium con-
stant K(SCN) ¼ 14.7 ± 4.6 M (obtained by fitting the equation
oxidation wave to ꢀ0.25 V (at [GSH] ¼ 0.14 M; Fig. 5A) in com-
ꢀ
parison to ꢀ0.099 V vs. Ag/AgCl in the presence of 1 M SCN
ꢀ
1
e
(Fig. 5B). These results clearly demonstrate that GS -Cbl(II) and
0
ꢀ
ꢀ
k ¼ ksat $K(SCN)$[SCN ]/(1 þ K(SCN)$[SCN ]) to data in Fig. S12).
thiocyanato-Cbl(II) adducts are stronger reducing agents than
Cbl(II) explaining the activation effects of S-donating ligands, GSH
The obtained value corresponds well to the formation constant of
Co(II)eSCN determined previously (23.8 ± 1.1 M
ꢀ
ꢀ1
ꢂ
ꢀ
at 25 C,
and SCN , respectively. (Redox potentials relevant to this study are
0
I ¼ 1 M) [21]. The pH dependence of k is described by a sigmoidal
summarized in Table S2). Thus, the less prominent efficiency of
ꢂ
ꢀ
curve with an inflection point at pH ¼ 4.41 ± 0.03 (25 C,
SCN as compared to GSH results from its lower influence on the
ꢀ
[
SCN ] ¼ 0.5 M; Fig. S13). Such pH profile can be explained by
Co(II) redox potential.
According to the presented results an overall mechanistic pic-
ture can be visualized (Schemes 1 and S1). It is important that
transformation of DHA dimeric form (DDHA) to monomeric one
MDHA) (Fig. 1A and B) that occurs at pH ~4 and the fact that
MDHA exhibits more pronounced oxidizing properties than DDHA
32].
(
e
ꢀ
coordination of GS or SCN within the fast pre-equilibrium in-
2þ
[
duces either cleavage or labilization of the axial Co -Nax(DMBI)
bond, respectively (Fig. 1E, Schemes 1 and S1), as suggested in the
literature based on the EPR studies [21,22]. This additional activa-
tion effect of S-donating ligands permits a closer interaction be-
tween the Co(II) center and DHA to yield ascorbyl free radical
(AFR ) and thiocyanato-Cbl(III) or GS -Cbl(III), respectively
(Schemes 1 and S1). Probably, AFR remain bound in transient
LeCbl(III)eAFR complex and capable to reduce Co(III) to Co(II).
0
Based on the saturation dependence of the rate constants (k )
on [GSH] and [SCN ] (vide supra), respectively, and characteriza-
tion of the GS -Cbl(II) adduct, we can conclude that coordination
of GSH or SCN to initial five-coordinate Cbl(II) (through its vacant
eaxial site) modifies redox properties of Co -ion and activates
an electron-transfer from Co to DHA. To probe such paradigm,
we examined the redox behavior of the Cbl(II)/SCN and Cbl(II)/
ꢀ
e
ꢀ
2
þ
e
e
b
2þ
e
ꢀ
e
Please cite this article in press as: I.A. Dereven'kov, et al., Journal of Organometallic Chemistry (2017), http://dx.doi.org/10.1016/
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6
I.A. Dereven'kov et al. / Journal of Organometallic Chemistry xxx (2017) 1e7
e
scavengers (e.g., AA and GSH) and acidification of medium (AFR
e
rapidly disproportionates in acidic medium to DHA and HAA [34])
that is observed in studied system (viz., the value of intercept is
decreased upon addition of AA and GSH and acidification). Exper-
imental evidences for the formation of a Cbl(II)-DHA adduct were
not found, but it is expected to be labile and short lived due to the
rapid inner-sphere electron transfer resulting in unstable radical
species.
Another potential representation of reaction mechanism can be
performed via generation of highly reactive cob(I)alamin (Cbl(I))
and its further reaction with DHA. Indeed, generation of Cbl(I) in
the presence of thiols was proven in several Cbl-systems [35,36]. To
access a role of Cbl(I) in reduction of DHA in Cbl(II)/GSH system, we
attempted to perform the oxidation of Cbl(II) by nitrite and thio-
sulfate (efficient oxidants of Cbl(I) [37,38]) in the presence of GSH in
neutral medium and observed no detectable reactions. Latter dis-
criminates formation of Cbl(I) as a necessary step in reaction
mechanism of studied system.
Collectively, the reported process represents a stoichiometric
reduction of DHA by the S-ligated Cbl(II) adducts that results in
vitamin C and Cbl(III) (i.e. its S-ligated adducts), without con-
sumption of S-donors that serve rather as catalysts (Eq. (2)).
All biological cob(II)alamin derives from the processing of di-
etary MeCbl and AdoCbl by the glutathione-dependent dealkylase
MMACHC (CblC) [39,40], and from the continuous redox cycling
alternating cob(I)alamin, cob(II)alamin and CoeC bond formation
leading to re-formation of MeCbl and AdoCbl in the active site of
the two cobalamin-dependent enzymes, methionine synthase and
methylmalonyl-CoA mutase [8,9]. Oxidation of cob(II)alamin is
repaired by dedicated reductases, that re-couple cobalamin meta-
bolism [8,9], thus, providing a sustainable source of cob(II)alamin
that can participate in non-canonical reactions, such as the reduc-
tion of DHA to regenerate vitamin C. Herein, we presented evidence
that cob(II)alamin, an essential intermediate in biological organo-
metallic chemistry, can be side-tracked to catalytically and stoi-
chiometrically rescue oxidized vitamin C.
4. Conclusion
For the first time we have demonstrated that AA can be recov-
Fig. 5. (A) Cyclic voltammograms of Cbl(II) (0.3 mM) (1), Cbl(II) (0.3 mM) þ GSH
ered from DHA within a process involving cob(II)alamin in a stoi-
chiometric manner (Eq. (2)). This process does not expend GSH,
whose binding to the Co(II) center modulates the underlying redox
mechanism. These findings not only open up a completely new
network of the biological interplay between vitamin C, glutathione
and metal centers, but also new possibilities for therapeutic
approaches.
ꢂ
(
0.14 M) (2) and GSH (0.14 M) (3) at pH 7.0; 21 C. A poorly resolved oxidation wave
observed at ꢀ0.1 V in the red curve (2) probably relates to the catalytic oxidation of
GSH that occurs under conditions of the electrochemical experiment. (B) Cyclic vol-
ꢀ
ꢀ
tammograms of Cbl(II) þ SCN mixtures. [Cbl(II)] ¼ 0.3 mM; [SCN ] ¼ 0 (1), 0.01 (2),
ꢂ
0
.1 (3), 0.5 (4), 1.0 (5) M; pH 7.0; 21 C. Arrows indicates direction of scan. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
Scheme 1. Mechanism of the reduction of DHA by Cbl(II) in the presence of glutathione.
This reverse reaction can provide an explanation of intercepts on
concentration dependencies (Fig. 3A). In this case, an extent of
reverse reaction is expected to be decreased after addition of AFR
Acknowledgements
e
IAD and LH gratefully acknowledge financial support from
Please cite this article in press as: I.A. Dereven'kov, et al., Journal of Organometallic Chemistry (2017), http://dx.doi.org/10.1016/
j.jorganchem.2017.01.002

I.A. Dereven'kov et al. / Journal of Organometallic Chemistry xxx (2017) 1e7
7
German Academic Exchange Service (DAAD) and Russian Ministry
of Education and Science (Mikhail Lomonosov program) as well as
Russian Scientific Foundation, agreement Nꢂ
knowledges financial support by the intramural grant from the
Friedrich-Alexander University (Emerging Field Initiative: Medici-
nal Redox Inorganic Chemistry).
1081e1083.
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[
17] I.A. Dereven’kov, N.I. Shpagilev, L. Valkai, D.S. Salnikov, A.K. Horv aꢀ th,
S.V. Makarov, J. Biol. Inorg. Chem. (2016), http://dx.doi.org/10.1007/s00775-
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AA
AFR
Cbl
Ascorbic acid
Ascorbyl free radical
Cobalamin
e
[
Cryst. B59 (2003) 51e59.
DMBI
DHA
DDHA
GSH
5,6-Dimethylbenzimidazole
Dehydroascorbic acid
Dehydroascorbic acid dimer
Glutathione
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[
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MDHA Dehydroascorbic acid monomer
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848e6857.
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Please cite this article in press as: I.A. Dereven'kov, et al., Journal of Organometallic Chemistry (2017), http://dx.doi.org/10.1016/
j.jorganchem.2017.01.002