Mechanistic studies on the reaction of nitrocobalamin with glutathione: Kinetic evidence for formation of an aquacobalamin intermediate

Article abstract of DOI:10.1002/ejic.201300254

The essential but also toxic gaseous signaling molecule nitric oxide is scavenged by the reduced vitamin B₁₂ complex cob(II)alamin. The resulting complex, nitroxylcobalamin [NO^--Cbl(III)], is rapidly oxidized to nitrocobalamin (NO₂Cbl) in the presence of oxygen; however, it is unlikely that nitrocobalamin is itself stable in biological systems. Kinetic studies on the reaction between NO₂Cbl and the important intracellular antioxidant, glutathione (GSH), are reported. In this study, a reaction pathway is proposed in which the β-axial ligand of NO₂Cbl is first substituted by water to give aquacobalamin (H ₂OCbl⁺), which then reacts further with GSH to form glutathionylcobalamin (GSCbl). Independent measurements of the four associated rate constants k₁, k_-1, k₂, and k_-2 support the proposed mechanism. These findings provide insight into the fundamental mechanism of ligand substitution reactions of cob(III)alamins with inorganic ligands at the β-axial site. Kinetic studies on the reaction of nitrocobalamin (NO₂Cbl) with glutathione show that glutathionylcobalamin (GSCbl) is formed via an aquacobalamin (H ₂OCbl⁺) intermediate. This reaction pathway is demonstrated by independently determining individual rate constants for each step.

Full text of DOI:10.1002/ejic.201300254

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Cobalamins
D. T. Walker, R. S. Dassanayake,
K. A. Garcia, R. Mukherjee,
N. E. Brasch* .................................... 1–6
Kinetic studies on the reaction of nitro-
cobalamin (NO2Cbl) with glutathione
show that glutathionylcobalamin (GSCbl)
is formed via an aquacobalamin
(H2OCbl+) intermediate. This reaction
pathway is demonstrated by independently
determining individual rate constants for
each step.
Mechanistic Studies on the Reaction of
Nitrocobalamin with Glutathione: Kinetic
Evidence for Formation of an Aquacobal-
amin Intermediate
Keywords: Vitamin B12 / Cob(III)alamin /
Bioinorganic chemistry / Kinetics / Reac-
tion mechanisms
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DOI:10.1002/ejic.201300254
Mechanistic Studies on the Reaction of Nitrocobalamin
with Glutathione: Kinetic Evidence for Formation of an
Aquacobalamin Intermediate
David T. Walker,[a][‡] Rohan S. Dassanayake,[a][‡]
Kamille A. Garcia,[a] Riya Mukherjee,[a] and Nicola E. Brasch*[a,b]
Keywords: Vitamin B12 / Cob(III)alamin / Bioinorganic chemistry / Kinetics / Reaction mechanisms
The essential but also toxic gaseous signaling molecule nitric
oxide is scavenged by the reduced vitamin B12 complex
cob(II)alamin. The resulting complex, nitroxylcobalamin
[NO–-Cbl(III)], is rapidly oxidized to nitrocobalamin
(NO2Cbl) in the presence of oxygen; however, it is unlikely
that nitrocobalamin is itself stable in biological systems. Ki-
netic studies on the reaction between NO2Cbl and the impor-
tant intracellular antioxidant, glutathione (GSH), are re-
ported. In this study, a reaction pathway is proposed in which
the β-axial ligand of NO2Cbl is first substituted by water to
give aquacobalamin (H2OCbl+), which then reacts further
with GSH to form glutathionylcobalamin (GSCbl). Indepen-
dent measurements of the four associated rate constants k1,
k–1, k2, and k–2 support the proposed mechanism. These find-
ings provide insight into the fundamental mechanism of li-
gand substitution reactions of cob(III)alamins with inorganic
ligands at the β-axial site.
Introduction
Two essential enzymes in mammals, l-methylmalonyl-
CoA mutase and methionine synthase, and numerous bac-
terial enzymes require the vitamin B12 derivatives (= cobal-
amins, Cbls) adenosylcobalamin (AdoCbl) and methyl-
cobalamin (MeCbl) as cofactors, Figure 1.[1] MeCbl-de-
pendent methionine synthase transfers a methyl group from
methyltetrahydrofolate to homocysteine to generate tetra-
hydrofolate and methionine, whereas AdoCbl-dependent l-
methylmalonyl-CoA mutase catalyzes the isomerization of
l-methylmalonyl-CoA to succinyl-CoA.[1] Cobalamins may
also have additional roles in biological systems, including
regulating the immune response and protecting against in-
tracellular oxidative stress.[2]
The signaling molecule nitric oxide (·NO) plays a key role
in the immune response, vasodilation, and neurotransmis-
sion.[3] However, high levels of NO can be deleterious and
can result in sepsis and septic shock,[4] which leads to organ
failure and even death. Importantly, administering cobal-
Figure 1. Structure of cobalamins showing the two axial sites (up-
per = β, lower = α) with respect to the corrin ring.
·
amins suppresses NO-induced relaxation of smooth mus-
cle,[5] ·NO-induced vasodilation[6] and ·NO-mediated inhibi-
tion of cell proliferation.[7] Cobalamins also reverse NO-
·
[a] Department of Chemistry and Biochemistry, Kent State
University,
induced neural tube defects.[8] Both mammalian B12-de-
·
pendent enzymes are inhibited by NO.[9] With the excep-
Kent, OH 44242, USA
[b] School of Biomedical Sciences, Kent State University,
Kent, OH 44242, USA
tion of glutathionylcobalamin (X = glutathione, Figure 1),
·NO does not directly react with cob(III)alamins,[10]
whereas the rate of the reaction between cob(II)alamin and
nitric oxide to form nitrosylcobalamin (NOCbl) is almost
diffusion controlled and essentially irreversible.[11] Given
E-mail: nbrasch@kent.edu
Homepage: www.brasch-group.com
[‡] Equal contributions
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201300254.
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that all cob(III)alamins are readily reduced by intracellular
reductases,[12] it is likely that cob(II)alamin reacts with ·NO
in biological systems, to form NOCbl.
In discussions of the biological relevance of NOCbl for-
mation, we have observed that authors neglect to mention
that NOCbl itself is not a stable entity.[5a,6–8,9c–9e] However,
in the presence of even minute amounts of air, orange
NOCbl rapidly oxidizes to form red nitrocobalamin,
NO2Cbl.[13] Indeed, we have used this property of NOCbl
in our laboratory numerous times to check the condition of
valves and taps used in strictly anaerobic experimental set-
ups. The intracellular fate of NO2Cbl is unclear. Possibil-
ities include reduction of NO2Cbl by intracellular re-
ductases, and/or the reaction of NO2Cbl with the strong
nucleophile and reductant glutathione (GSH), which is
present in mm concentrations in cells.[14] In this work, we
report kinetic studies on the reaction of NO2Cbl with gluta-
thione. Interestingly, our kinetic data show that GSH does
not directly react with NO2Cbl, but instead reacts with
aqua-
Figure 2. (a) UV/Vis spectra for the reaction of GSH
(5.00ϫ10–2 m) with NO2Cbl (5.0ϫ10–5 m) at pH 4.00 (25.0 °C,
0.020 m NaOAc, I = 1.0 m, NaCF3SO3). Selected spectra for the
reaction are shown every 1.00 min. (b) Plot of absorbance at
354 nm vs. time for the experiment shown in (a). Data were fitted
to
a
first-order rate equation, which gives kobs
=
(1.15Ϯ0.07)ϫ10–2 s–1.
to reach a limiting value of kobs at high GSH concentrations
(saturation kinetics). The first involves rapid equilibration
to form a NO2Cbl·GSH association complex prior to rate-
–
determining substitution of the β-axial NO2 ligand of
cobalamin, which is always present in equilibrium, albeit
typically in small amounts, in cobalamins with inorganic
ligands at the β-axial cobalamin site (Figure 1).
NO2Cbl by GSH to give GSCbl. The other alternative is a
two-step process, in which H2OCbl+, which is in equilib-
rium with NO2Cbl, reacts with GSH (Scheme 1). Given
that all Cbls with β-axial inorganic ligands exist in equilib-
rium with H2OCbl+ and that Cbls undergo β-axial ligand
exchange through a dissociative interchange mechanism,[16]
the latter possibility is more likely to occur. Kinetic studies
on the reaction between H2OCbl+/HOCbl and GSH have
been reported[17] and show that H2OCbl+ reacts rapidly
with GSH to form GSCbl under the conditions of our
study.
Results and Discussion
Kinetic data were collected for the reaction of NO2Cbl
with glutathione (GSH). Experiments were initiated by add-
ing a small aliquot of concentrated aqueous NO2Cbl solu-
tion (final concentration 5.0ϫ10–5 m) to a buffered GSH
solution (3.00 mL) at a specific pH condition [I = 1.0 m
(NaCF3SO3)]. Control experiments established that rate
constants were identical within experimental error in the
absence and presence of air; hence, all experiments were
carried out under aerobic conditions. Importantly, the dis-
solving of NO2Cbl in water rather than in buffer minimized
the decomposition of NO2Cbl to aquacobalamin
(H2OCbl+) prior to the addition of an aliquot of this solu-
tion into a buffered GSH solution.
Figure 3. Plots of kobs vs. [GSH] at pH (a) 4.00 and (b) 7.00 for
the reaction between NO2Cbl (5.00ϫ10–5 m) and glutathione
[25.0 °C, (a) 0.020 m NaOAc or (b) 0.020 m KH2PO4 , I = 1.0 m
(NaCF3SO3)]. Data in (a) were fitted to Equation (2) in the text
fixing k–2 = 7.4ϫ10–4 s–1, which gives k1 = (1.75Ϯ0.02)ϫ10–2 s–1
and K = 94.5Ϯ3.7 m–1 at pH 4.00. Data in (b) were fitted to Equa-
tion (2) fixing k–2 = 0 s–1, which gives k1 = (1.73Ϯ0.05)ϫ10–2 s–1
and K = 102.1Ϯ9.7 m–1 at pH 7.00.
Figure 2(a) shows UV/Vis spectral changes observed
upon the addition of NO2Cbl to a buffered GSH solution
(5.00ϫ10–2 m) at pH 4.00. NO2Cbl (λmax = 354, 413, and
532 nm) is converted to GSCbl (λmax = 333, 372, 428, and
534 nm[15]) with isosbestic points at 336, 367, 452, and
543 nm, which indicates that a single reaction occurs. The
corresponding plot at 354 nm vs. time is given in Fig-
ure 2(b). The data fit well to the first-order rate equation,
The rate expression corresponding to the reaction path-
way shown in Scheme 1 is shown in Equation (1).[18]
which gives an observed rate constant, kobs
=
(1.15Ϯ0.07)ϫ10–2 s–1.
–
–
kobs = {k1k2[GSH]/(k–1[NO2 ]) + k–2}/{1 + k2[GSH]/(k–1[NO2 ])}
(1)
Kinetic data were collected at pH 4.00 and 7.00, in order
to determine whether the rate of the reaction is pH depend-
ent. Plots of kobs vs. total GSH concentration are shown in
Figure 3(a) and (b). These plots indicate that the observed
= (k1K[GSH] + k–2)/(1 + K[GSH])
(2)
–
rate constant reaches a limiting value at high GSH concen- where K = k2/(k–1[NO2 ]). The rate constant, k–2, for de-
trations and that there is no pH dependence in this pH re- composition of GSCbl to H2OCbl+ and GSH was indepen-
gion. There are two plausible mechanisms by which dently determined at pH 4.00 and found to be
NO2Cbl reacts with GSH to give a plot exhibiting curvature (7.4Ϯ0.5)ϫ10–4 s–1 (Figure S1, Supporting Information).
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Scheme 1. Proposed reaction pathway for the reaction of NO2Cbl
with GSH. Note that in aqueous solution H2OCbl+ exists in equi-
librium with HOCbl [pKa(H2OCbl+) = 7.8[17]]; however, at the pH
conditions of our kinetic experiments, HOCbl formation is unim-
portant, since the values of the rate constants k–1 and k2 are the
same at pH 4.00 and 7.00.
Figure 4. Plot of kobs vs. [GSH] for the reaction between NO2Cbl
(5.0ϫ10–5 m) and GSH in the presence of 5.00ϫ10–4 m NaNO2 at
pH 7.00 (25.0 °C, 0.020 m KH2PO4, 5.00ϫ 10–4 m NaNO2, I =
1.0 m, NaCF3SO3). Data were fitted to Equation (2) in the text by
fixing k–2 = 0, which gives k1 = (1.60 Ϯ0.05)ϫ10–2 s–1 and K =
16.2Ϯ0.2 m–1.
Previous studies have shown that the observed equilibrium
constant for formation of GSCbl, Kobs(GSCbl) = k2/k–2, in-
creases by approximately one order of magnitude for each
unit change in pH.[17] Since k2 is pH independent,[17] k–2 is
therefore negligible at pH 7.00. Data in Figure 3(a) were
fitted to Equation (2) by fixing k–2 = 7.4ϫ10–4 s–1, which
gives k1 = (1.75Ϯ0.02)ϫ10–2 s–1 and K = 94.5Ϯ3.7 m–1.
Data in Figure 3(b) were fitted to the same equation by
fixing k–2 = 0 s–1, which gives k1 = (1.73Ϯ0.05)ϫ10–2 s–1
and K = 102.1Ϯ9.7 m–1. K is therefore also pH independent
in the pH 4–7 region, within experimental error.
reported by others [k–1 = 99.8ϫ102 m–1 s–1 at 25 °C, I =
2.2 m (NaNO3)].[19] By using k2 = 12.00 m–1 s–1, k–1
=
–
1.23ϫ103 m–1 s–1, and [NO2 ] = 5.00ϫ10–4 m, a value of K
≈ 20 m–1 is obtained, which is in very good agreement with
the experimental value of K obtained from the best fit of
the data (16.2Ϯ0.2 m–1) and provides strong support for the
reaction pathway proposed in Scheme 1.
Finally, the rate constant k1 for decomposition of
NO2Cbl to H2OCbl+ was also independently determined by
obtaining kinetic data for the decomposition of NO2Cbl to
H2OCbl+ upon dissolving NO2Cbl in buffer. Figure S5
(Supporting Information) shows the absorbance change
(ΔAbs = 0.013 at 350 nm) that occurs at pH 4.00. Only a
small fraction of NO2Cbl is converted to H2OCbl+; how-
ever, the absorbance change is sufficient to allow calcula-
tion of k1. The results at different pH conditions are sum-
marized in Table S1 and give a mean value for k1
Importantly, if our model is correct, then K = k2/
(k–1[NO2 ]). Note, however, that the free nitrite concentra-
–
tion is not strictly constant during the reaction, but will
vary from 0 to a maximum value of 5.0ϫ10–5 m as the reac-
tion proceeds, as NO2Cbl (5.0ϫ10–5 m) reacts with GSH to
–
give GSCbl plus NO2 . Hence, K is not strictly constant
during the reaction, as reflected in the error associated with
K. In order to provide support for our model, new data was
therefore collected at pH 7.00 under the same conditions
as the experiments summarized in Figure 3(b), except that
5.00ϫ10–4 m NaNO2 was added to each solution, so the
nitrite concentration is constant (pseudo-first order condi-
tions) during the reaction. These data are shown in Fig-
of (1.48Ϯ0.22)ϫ10–2 –1 s–1 (pH 3.5–6.0; k1 is pH
m
independent). The observed rate constant for the partial
decomposition of NO2Cbl to give H2OCbl+ is actually
–
(k1 + k–1[NO2 ]) for the pseudo-first-order reversible pro-
ure 4. Fitting of this data to Equation (2) by fixing k–2
=
=
0 s–1 gave k1
=
(1.60Ϯ0.05)ϫ10–2 s–1 and
K
cess. A separate experiment showed that an absorbance dif-
ference of 0.232 is observed upon completely converting
H2OCbl+ to NO2Cbl upon the addition of a slight excess
16.2Ϯ0.2 m–1. The data now fit considerably better to
Equation (2) as expected (the error in K is now one order of
magnitude smaller), since the nitrite concentration remains
constant during the reaction.
–
of NO2 ([Cbl]T = 5.0ϫ10–5 m). Hence, an absorbance
change of 0.013 corresponds to formation of 5.6%
–
H2OCbl+ (2.8ϫ10–6 m H2OCbl+ and 2.8ϫ10–6 m NO2 )
The rate constant k2 for the reaction of H2OCbl+ with
GSH was also independently determined at pH 4.00 and
found to be 12.00Ϯ0.25 m–1 s–1 [25.0 °C, 0.020 m NaCH3-
COO, I = 1.00 m (NaCF3SO3)] (Figure S2, Supporting In-
formation). This value is in good agreement with a value
upon dissolving NO2Cbl in H2OCbl+; that is, the maximum
–
NO2 is 2.8ϫ10–6 m. For k1 = 1.48ϫ10–2
m
–1 s–1, k–1
=
–
(1.25Ϯ0.02)ϫ103 and [NO2 ] = 2.8ϫ10–6 m k1 is about 5
times larger than k–1[NO2 ], which validates the assumption
–
that kobs ≈ k1 upon dissolving NO2Cbl in H2OCbl+. By
using our values of k1 and k–1, the equilibrium constant for
formation of NO2Cbl, K(NO2Cbl) = k–1/k1, is 8.5ϫ104 m–1,
which is in reasonable agreement with a value reported by
others under different ionic strength conditions
[K(NO2Cbl) = 2.2ϫ105 m–1, 25 °C, I = 2.2 m[20]].
reported by us under slightly different conditions [k2
=
18.5 m–1 s–1 at pH 4.50, 25.0 °C, 0.10 m NaOAc, I = 0.50 m
(KNO3)[17]], and is pH independent in the pH 4–7 range.[17]
The rate constant k–1 for the reaction between H2OCbl+
–
and NO2
was independently determined to be
(1.25Ϯ0.02)ϫ103 and (1.20Ϯ0.02)ϫ103 m–1 s–1 at pH 4.00
and 7.00, respectively (Figures S3 and S4, Supporting Infor-
mation). Hence, k–1 is also independent of the pH (pH 4–7),
as expected, as the ionization of the reactants is essentially
unchanged [pKa(HNO2) ≈ 3.2; pKa(H2OCbl+) = 7.8[17]] in
Conclusions
Kinetic studies on the reaction between NO2Cbl and
this pH region. The value of k–1 agrees well with a value GSH show that the rate of the reaction is pH independent
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Kinetic Measurements: The rates of the reaction between NO2Cbl
and glutathione (GSH) were determined under pseudo-first-order
conditions with excess GSH. Stock solutions of GSH (0.500 m) in
the presence or absence of sodium nitrite (5.00ϫ10–4 m) were pre-
pared in the appropriate buffer (0.020 m) at pH 4.00 and 7.00 and
diluted as appropriate. A small aliquot of concentrated NO2Cbl
(final concentration 5.0ϫ10–5 m) in water was added to initiate the
reaction, and the absorbance at 354 nm was recorded as a function
of time.
Kinetic data for the reaction between H2OCbl+ (5.0ϫ10–5 m) with
varying concentrations of NO2– were obtained at pH 4.00 and 7.00.
Stock solutions of NaNO2 (0.010 m) were prepared in the appropri-
ate buffer (0.020 m) and diluted as needed. Data were collected at
354 nm. Kinetic data for the reaction of H2OCbl+ (5.0ϫ10–5 m)
with varying concentrations of glutathione at pH 4.00 were col-
lected at 354 nm in acetate buffer (0.020 m).
The rate of decomposition of NO2Cbl to H2OCbl+ was determined
in the pH 3.5–6.0 range by adding solid NO2Cbl directly to the
appropriate buffer solution (0.200 m; the solution was thermostat-
ted at 25.0 °C for 10 min prior to the addition of NaNO2). The
solution was quickly filtered through a micropore filter (0.45 μm),
and data collection initiated at 354 nm.
The rate of decomposition of GSCbl to H2OCbl+ was determined
at pH 4.00. An aliquot of GSCbl (4.0ϫ10–5 m) was added to pH
4.00 acetate buffer (0.020 m; the buffer solution was thermostatted
at 25.0 °C for 10 min prior to the addition of GSCbl), and data
were collected at 354 nm.
in the pH 4–7 region. By independently determining values
of k1, k–1, k2, and k–2, we have shown that the data fits a
model involving an H2OCbl+ intermediate, which then
rapidly reacts with GSH to form GSCbl (Scheme 1). To our
knowledge, this is the first time that the reaction pathway
of β-axial inorganic ligand exchange for cob(III)alamins via
an H2OCbl+ intermediate has been unequivocally demon-
strated. This may have important consequences for free and
potentially for even protein-bound cob(III)alamins incor-
porating inorganic ligands (X-ray structures of cobalamins
bound to B12 transport proteins show that the β-axial site
can be readily accessed by solvent and small molecules[21]);
that is, the amount of each of these species may reflect the
concentrations and binding constants to aquacobalamin of
the various inorganic ligands present. As such, GSCbl
would be expected to be the major intracellular non-alkyl-
cob(III)alamin, given that intracellular GSH concentrations
are in the mm region,[14] and only CN– binds more strongly
than GSH to H2OCbl+ (KCNCbl ≈ 1014 –1[22]). Finally, at
m
0.5 mm GSH, the rate constant for the reaction between
NO2Cbl and GSH is ≈8ϫ10–3 s–1 at pH 7.0 (25 °C), which
corresponds to a half-life of about 1.4 min. Hence, forma-
tion of GSCbl is one possible reaction pathway by which
NO2Cbl decomposes in biological systems.
Experimental Section
The total ionic strength was maintained at 1.0 m by using
NaCF3SO3 for all solutions.
General: Hydroxycobalamin hydrochloride (HOCbl·HCl, 98%
stated purity by manufacturer) was purchased from Fluka. Gluta-
thione (98%), acetic acid (sodium salt, Ն 99%), sodium nitrite
(97%), and CF3SO3H (99%) were obtained from Acros Organics.
TES buffer (98%) was purchased from MP Biomedicals Inc. Potas-
sium dihydrogen phosphate was purchased from Sigma. NaCF3SO3
was prepared by neutralizing a concentrated, aqueous solution of
CF3SO3H with NaOH, by reducing it to dryness by rotary evapora-
tion, and by drying it overnight in a vacuum oven at 70.0 °C. Nitro-
cobalamin was synthesized and characterized according to a pub-
Supporting Information (see footnote on the first page of this arti-
cle): Further kinetic plots, UV/Vis spectra and rate constants are
presented.
Acknowledgments
This research was funded by the US National Science Foundation
(CHE-084839) and the US National Institute of General Medical
Sciences of the National Institutes of Health (1R15GM094707-
01A1). The content is solely the responsibility of the authors and
does not necessarily represent the official views of the National
Institutes of Health. Funding for this work was also provided by
the NSF-REU program at KSU [CHE-1004987 (D. W.) and CHE-
0649017 (K. G.)].
1
lished procedure.[15] The purity was Ն 95%, as determined by H
NMR spectroscopy.
UV/Vis spectra and kinetic data for slower reactions were recorded
on a Cary 5000 spectrophotometer equipped with a thermostatted
(25.0Ϯ0.1 °C) cell changer operating with WinUV Bio software
(version 3.00). Reactant solutions were thermostatted for 15 min
prior to measurements. Kinetic data for rapid reactions were ob-
tained at 25.0Ϯ0.1 °C by using an Applied Photophysics SX20
stopped-flow spectrophotometer equipped with a photodiode array
detector in addition to a single wavelength detector. Data were col-
lected with Pro-Data SX (version 2.1.4) and Pro-Data Viewer (ver-
sion 4.1.10) software, and a 1.0 cm pathlength cell was utilized. All
data were analyzed by using Microcal Origin version 8.0.
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pH measurements were carried out by using an Orion model 710A
pH meter equipped with a Wilmad 6030–02 pH electrode. The elec-
trode was filled with a 3 m KCl/saturated AgCl solution (pH 7.0)
and standardized with standard BDH buffer solutions at pH 6.98,
4.01, and 2.02. Solution pH was adjusted by using 50% v/v aque-
ous CF3SO3H and NaOH (≈5 m).
1H NMR spectra was recorded on a Bruker Avance 400 MHz spec-
trometer equipped with 5 mm probe. Solutions for NMR measure-
1
ments were prepared in D2O. H NMR spectra were internally ref-
erenced to TSP (δ =0 ppm).
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Received: February 21, 2013
Published Online: ᭿
Eur. J. Inorg. Chem. 0000, 0–0
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