Studies on the reaction between reduced riboflavin and selenocystine

Article abstract of DOI:10.1002/kin.21430

Selenocysteine (Sec) is a crucial component of mammalian thioredoxin reductase (TrxR) where it serves as a nucleophile for disulfide bond rupture in thioredoxin (Trx). Generation of the reduced state of Sec in TrxR requires consecutive two electron transfer steps, namely: (i) from NADPH to flavin adenine dinucleotide, (ii) from reduced flavin to the disulfide bond Cys⁵⁹-S-S-Cys⁶⁴, and finally (iii) from Cys⁵⁹ and Cys⁶⁴ to the selenosulfide bond Cys⁴⁹⁷-S-Se-Sec⁴⁹⁸. In this work, we studied the reaction between reduced riboflavin (RibH2) and selenocystine (Sec-Sec), an oxidized form of Sec. The interaction between RibH2 and Sec-Sec proceeded relatively slowly in comparison with its reverse reaction, that is, reduction of riboflavin (Rib) by Sec. The rate constant for the reaction between RibH2 and Sec-Sec was (7.9?±?0.1)?×?10^?2?M^?1 s^?1 (pH 7.0, 25.0°C). The reaction between Rib and Sec proceeded via two steps, namely, a rapid reversible binding of Rib to Sec having a protonated selenol group to form a Sec-Rib complex, followed by nucleophilic attack of Sec-Rib by a second Sec molecule harboring a deprotonated selenol group. The equilibrium constant for the overall reduction process of Rib by Sec is (1.2?±?0.1)?×?10⁶?M^?1 (25.0°C). The finding that the interaction of RibH2 with oxidized selenol is reversible with its equilibrium favored toward the reverse reaction provides an additional explanation for the exceptional mechanism of the mammalian Trx/TrxR system involving transient reduction of a disulfide bond.

Full text of DOI:10.1002/kin.21430

Received: 1 May 2020
DOI: 10.1002/kin.21430
Revised: 1 September 2020
Accepted: 4 September 2020
A RT I C L E
Studies on the reaction between reduced riboflavin
and selenocystine
Ilia A. Dereven’kov1
Anna S. Makarova2
Sergei V. Makarov1
Pavel A. Molodtsov1
1 Ivanovo State University of Chemistry
and Technology, Ivanovo 153000, Russia
Abstract
2 G.A. Krestov Institute of Solution
Chemistry of Russian Academy of
Sciences, Ivanovo 153045, Russia
Selenocysteine (Sec) is a crucial component of mammalian thioredoxin reduc-
tase (TrxR) where it serves as a nucleophile for disulfide bond rupture in thiore-
doxin (Trx). Generation of the reduced state of Sec in TrxR requires consecu-
tive two electron transfer steps, namely: (i) from NADPH to flavin adenine din-
ucleotide, (ii) from reduced flavin to the disulfide bond Cys59-S-S-Cys64, and
Correspondence
IliaA. Dereven’kov, IvanovoState Uni-
versity of Chemistry andTechnology,
Sheremetevskiy str 7, Ivanovo 153000,
Russia.
finally (iii) from Cys59 and Cys64 to the selenosulfide bond Cys497-S-Se-Sec498
.
In this work, we studied the reaction between reduced riboflavin (RibH2) and
selenocystine (Sec-Sec), an oxidized form of Sec. The interaction between RibH2
and Sec-Sec proceeded relatively slowly in comparison with its reverse reaction,
that is, reduction of riboflavin (Rib) by Sec. The rate constant for the reaction
between RibH2 and Sec-Sec was (7.9 0.1) × 10−2 M−1 s−1 (pH 7.0, 25.0◦C). The
reaction between Rib and Sec proceeded via two steps, namely, a rapid reversible
binding of Rib to Sec having a protonated selenol group to form a Sec-Rib com-
plex, followed by nucleophilic attack of Sec-Rib by a second Sec molecule har-
boring a deprotonated selenol group. The equilibrium constant for the overall
reduction process of Rib by Sec is (1.2 0.1) × 106 M−1 (25.0◦C). The finding that
the interaction of RibH2 with oxidized selenol is reversible with its equilibrium
favored toward the reverse reaction provides an additional explanation for the
exceptional mechanism of the mammalian Trx/TrxR system involving transient
reduction of a disulfide bond.
Email:derevenkov@gmail.com
Fundinginformation
Council ongrants of thePresident ofthe
RussianFederationfor statesupportof
young Russianresearchers, Grant/Award
Number:МК-1083.2019.3
K E Y WO R D S
kinetics, reaction mechanism, riboflavin, selenium, selenocysteine
gen selenide, selenocyanate) forms.1,2 Its main biological
effects are predominantly associated with cycling of sele-
1
INTRODUCTION
Selenium is an ultra-microelement, which plays impor-
tant roles in vivo.1 In nature, Se is found in organic (eg,
selenocysteine [Sec], selenomethionine, methyl selenides,
selenosugars) and inorganic (eg, selenate, selenite, hydro-
noenzymes, that is, glutathione peroxidase, thioredoxin
reductase (TrxR), iodothyronine deiodinase, and others,
whose active sites contain Sec, a selenium analogue of cys-
teine (Figure 1).3,4
Int J Chem Kinet. 2020;1–8.
wileyonlinelibrary.com/journal/kin
© 2020 Wiley Periodicals LLC
1

2
DEREVEN’KOV et al.
melanogaster DmTrxR and found that flavin as well as
Cys59 and Cys64 residues exhibited poor reactivity toward
selenocystine.18
In this work, we interrogate the direct reactivity of flavin
toward Sec and of reduced flavin toward selenocystine
to gain insights into the exceptional mechanism of the
Trx/TrxR system.
2
EXPERIMENTAL
Riboflavin (Rib) (Sigma, St. Louis, MO, USA; ≥98%) and
seleno-l-cystine (Sec-Sec) (Aldrich, St. Louis, MO, USA;
95%) were used without additional purification. Oxygen-
free argon was used to deoxygenate reagent solutions.
Reduction of selenocystine to Sec was performed using
sodium borohydride.19 An excess of borohydride was
quenched by adding hydrochloric acid or phosphate buffer
solution. The reduced form of Rib (RibH2) was pre-
pared by mixing equivalent amounts of sodium dithionite
(Na2S2O4) and Rib anaerobically.20
Buffer solutions (phosphate and acetate; 0.1 M) were
used to maintain pH during the measurements. The
pH values of solutions were determined using Mul-
titest IPL-103 pH-meter (SEMICO, Novosibirsk, Rus-
sia) equipped with ESK-10601/7 electrode (Izmeritelnaya
tekhnika, Moskow, Russia) filled by 3.0 M KCl solution.
The electrode was calibrated using standard buffer solu-
tions (pH 1.65-12.45).
Ultraviolet-visible (UV-vis) spectra were recorded on a
cryothermostated ( 0.1◦C) Cary 50 UV-vis spectropho-
tometer in quartz cells under anaerobic conditions.
The measurements were performed under pseudo-first
conditions using at least 10-fold excess21,22 of Sec or Sec-
Sec over Rib species. Data were obtained from at least three
independent experiments and were analyzed using Origin-
Pro 9.1 software. Mean values and standard deviations are
derived from the Levenberg Marquardt iteration algorithm
implemented in the OriginPro 9.1 software.
F I G U R E 1 Structures of cysteine (A), selenocysteine (B), and
riboflavin (C)
Thioredoxin reductases are protein disulfide reductases,
which maintain Trx in its reduced state in vivo. TrxRs
and Trxs comprise a system involved in the defense
against oxidative stress, redox signaling, protein repair,
DNA repair, and other biological processes.5–8 In mam-
mals, TrxRs are selenoenzymes comprising one Sec residue
per protein molecule, although Sec is lacking in TrxRs
derived from other living species.9,10 Hypotheses explain-
ing the presence of the noncanonical amino acid Sec as a
catalytic residue in mammalian TrxRs include higher sta-
bility of selenoenzymes over thiol species in the presence
of strong oxidants (eg, ONOOH, hypohalites, and others),
lower unwanted reactivity of selanyl over thiyl radicals
toward the protein backbone (that in turn increases pro-
tein longevity), as well as greater reactivity of Sec toward
oxidants in comparison with cysteine.11,12
One of the most characterized Se-containing TrxRs
species is cytosolic TrxR1.13–17 In the oxidized form
of TrxR1, Sec exists as selenosulfide Cys497-Sec498
(Scheme 1). TrxR1 contains flavin coenzyme (viz., flavin
adenine dinucleotide, FAD), which transfers electrons
from NADPH to the Cys59-Cys64 disulfide bond. The
latter reaction liberates Cys59 (thiol) and produces a
charge-transfer complex between FAD and Cys64. Cys59
reacts with the Cys497-Sec498 selenosulfide bond of TrxR1
to form a Cys59-Sec498 intermediate and Cys497. Next,
Cys64 attacks the Cys59-Sec498 bond at the Cys59 site
to yield free Sec498 and a Cys59-Cys64 disulfide bond.
Sec498 is located in a flexible tail of TrxR1, which enables
conformational flexibility to react with oxidized Trx.
Reduction of oxidized Trx proceeds via attack of Sec at the
Trx disulfide bond to give a Se-S bond between the enzyme
and the substrate, which is further cleaved by Cys497.
Therefore, TrxR1 utilizes a relatively complex pathway
of electron transfer from reduced flavin to selenosulfide
where direct contact between these species is eliminated.
A previous study examined the reactivity of selenocystine
as a substrate for the truncated ortholog Trx of Drosophila
3
RESULTS AND DISCUSSION
First, we investigated possible interactions between
reduced riboflavin (RibH2) and selenocystine (Sec-Sec) by
UV-vis spectroscopy. Figure 2 shows the spectral changes
that occurred upon mixing RibH2 and an excess of Sec-
Sec. A slow reaction was characterized by an increase in
absorption at 445 nm with isosbestic points appearing at
289 and 331 nm. The final UV-vis spectrum coincides with
that of the oxidized flavin (Rib) with a slight deviation
below 350 nm, where the spectral contribution of Sec-Sec
is pronounced (Figure S1).

DEREVEN’KOV et al.
3
S C H E M E 1 Catalytic cycle of cytosolic
thioredoxin reductase 1
We next studied the kinetics of the reaction between
RibH2 and an excess of Sec-Sec at pH 7.0. A typical
kinetic curve is shown in Figure 3. The RibH2 oxida-
tion event is best described by an exponential equation
that indicates first order with respect to RibH2. This per-
mitted the investigation of initial rates (r0) with vary-
ing concentrations of Sec-Sec. Plotting of r0 versus [Sec-
Sec] exhibited a linear relationship, which indicates first
order dependence with respect to Sec-Sec (Figure 4). The
value of the rate constant between RibH2 and Sec-Sec is
kSec-Sec = (7.9 0.1) × 10−2 M−1 s−1 (pH 7.0, 25.0◦C). The
presence of a small intercept can be explained by a reverse
reaction. To test this hypothesis, we studied the reaction
between Rib and Sec.
Addition of Sec to a solution of Rib led to the decrease
in absorbance at 445 nm (Figure 5). The UV-vis spec-
trum of the product is identical to that of RibH2 prepared
using dithionite. The reaction proceeds significantly faster
F I G U R E
2
UV-vis spectra of the reaction between RibH2
(3.2 × 10−5 M) and Sec-Sec (2.5 × 10−3 M) at pH 7.0, 25.0◦C [Color
figure can be viewed at wileyonlinelibrary.com]

4
DEREVEN’KOV et al.
F I G U R E
3
Kinetic curve of the reaction between RibH2
(3.2 × 10−5 M) and Sec-Sec (2.5 × 10−3 M) at pH 7.0, 25.0◦C [Color
F I G U R E
5
UV-vis spectra of the reaction between Rib
(1.7 × 10−5 M) and Sec (1.0 × 10−3 M) at pH 7.0, 25.0◦C [Color figure
figure can be viewed at wileyonlinelibrary.com]
can be viewed at wileyonlinelibrary.com]
F I G U R E
4
Plot of initial rates (r0) of the reaction between
F I G U R E
6
Kinetic curve of the reaction between Rib
RibH2 (2.5 × 10−5 M) and Sec-Sec versus [Sec-Sec] at pH 7.0, 25.0◦C.
(1.7 × 10−5 M) and Sec (1.0 × 10−3 M) at pH 7.0, 25.0◦C. Fitting of the
data to a single exponential equation gives kobs. = (1.7 0.1) × 10−3 s−1
[Color figure can be viewed at wileyonlinelibrary.com]
The data have been fitted to a straight line to obtain values of slope
and intercept, (2.0 0.1) × 10−6 s−1 and (1.9 0.1) × 10−10 M s−1
,
respectively [Color figure can be viewed at wileyonlinelibrary.com]
compared to the oxidation of RibH2 by Sec-Sec: for exam-
ple, the reduction of Rib by Sec (1 mM) proceeds within
30 min, whereas the interaction between RibH2 and Sec-
Sec (1 mM) reaches completion only after 6 h at pH 7.0,
25.0◦C.
kobs. versus [Sec] is nonlinear (Figure 7), but it can be lin-
earized in “kobs. – [Sec]2” coordinates, which indicates sec-
ond order with respect to Sec. The same profile of con-
centration dependencies was observed at other pH as well
(Figure S2). A second-order dependence can be the result
of the involvement of two Sec molecules for the reduction
of one Rib molecule, that is, the first Sec molecule reacts
rapidly and reversibly with Rib to form a complex, which
is subsequently decomposed upon attack, albeit slower, by
a second Sec molecule.
Next, we characterized the kinetics of Rib reduction by
an excess of Sec. A typical time-course curve of the reac-
tion indicates first order with respect to Rib (Figure 6). The
observed rate constants for the reaction (kobs.) were deter-
mined at varying concentrations of Sec. The direct plot of

DEREVEN’KOV et al.
5
F I G U R E 7 Plot of observed rate constants (kobs) of the reaction
between Rib and Sec versus [Sec] at pH 7.0, 25.0◦C. The data fit to the
kobs = k′[Sec]2 equation provides k′ = (1.4 0.1) × 103 M−2 s−1 [Color
figure can be viewed at wileyonlinelibrary.com]
F I G U R E 8 Plot of rate constant for the reaction between Rib
and two Sec molecules (k′) versus pH at 25.0◦C [Color figure can be
viewed at wileyonlinelibrary.com]
where KSec is the equilibrium constant for the complexa-
tion of Sec with Rib, M−1, kSec is the rate constant for
−
the reaction of Sec–Rib complex with Sec−, M−1
s
−1, and
Ka is the ionization constant of the selenol group of Sec.
Fitting of the data to this equation (Figure 8) retrieves
the values KSec kSec = (9.7
0.1) × 104 M−2 s−1 and
−
pKa = (5.4 0.1) (25.0◦C), which coincides with the lit-
(R1; pKa)
erature value pKa = 5.4.24 Using KSec kSec = (9.7
−
Sec + Rib ⇌ Sec − Rib,
(R2; KSec
)
0.1) × 104 M−2 s−1 and the rate constant for the reverse
reaction (kSec-Sec = (7.9 0.1) × 10−2 M−1 s−1), as described
above, the resulting overall equilibrium constant is K = (1.2
0.1) × 106 M−1 (R4; 25.0◦C).
−
+
Sec − Rib + Sec (+H ) → RibH2 + Sec − Sec,
−
=
(R3; kSec
)
−
Sec + Sec + Rib ⇌ RibH2 + Sec − Sec.
(R4; K)
ꢀSec
−
ꢁ = ꢁSec
.
The dependence of k′ (k′ is taken from equation kobs.
ꢀSec−Sec
k′ [Sec]2) on pH exhibits a bell-shaped profile with a maxi-
mum at approximately pH 5.5 (Figure 8). Rib is not engaged
in acid-base equilibria within the pH range used in our
experiments,23 whereas deprotonation of the selenol group
of Sec has a pKa = 5.4 (reaction R1; 25.0◦C).24 Our experi-
mental results suggest that both the protonated and depro-
tonated Sec species are involved in the reaction pathway,
that is, Rib rapidly and reversibly forms a complex with
protonated Sec (reaction R2), which further interacts with
the selenolate species (Sec−; reaction R3). In light of these
considerations, the following equation can be proposed to
describe this reactivity:
The rate of the forward reaction depends on pH, where
KpH 7 = (1.8 0.1) × 104 M−1 can be calculated for pH 7.0,
25.0◦C. At pH 7.0, the electrode potentials of Sec-Sec/2Sec
and Rib/RibH2 couples are −0.3825 and −0.2226,27 V ver-
sus normal hydrogen electrode, which can be employed
to evaluate the equilibrium constant using the Nernst
equation (viz, KpH 7(pot.) = 2.5 × 105 M−1). This value is
substantially higher than the equilibrium constant cal-
culated from the kinetic analysis. One explanation for
this discrepancy is that the association step between Sec
and Rib (reaction R2) slightly decreases the electrode
potential of the Rib/RibH2 couple. For instance, a shift
from −0.22 to −0.26 V results in an equilibrium con-
stant value close to that determined via kinetic charac-
terization of the reaction. In any case, the analysis of
− pH+pꢁ
(
)
a
10
′
ꢀ = ꢁSec × ꢀSec
−
(
)
2.
−pꢁ
−pH
a
10
+ 10

6
DEREVEN’KOV et al.
the selenolate species to give RibH2 and Sec-Sec. Because
RibH2 reacts with Sec-Sec, the system is reversible. Yet,
the significantly slower rate of reaction between RibH2
and Sec-Sec in comparison with that of Rib with Sec sug-
gests that Sec-Sec reduction by RibH2 would be negli-
gible in a system containing equal quantities of Sec-Sec
and Sec.
The mechanism of this reaction is different from previ-
ously described Sec oxidation processes. For instance, reac-
tions of Sec with radicals (eg, a tyrosyl radical, Tyr•25,35
;
reaction R5) or with metal complexes (eg, cobalamins,
Cbl(III)36,37; reaction R6) involve one-electron transfer and
produce selanyl radical dimerizing to Sec-Sec. The interac-
tions with disulfides (R’SSR’; reactions R7,R8)38 and hypo-
halites (eg, hypothiocyanite, HOSCN19; reactions R9-R12)
proceed via a sequence of nucleophilic substitution steps.
This study revealed that reactivity with Rib is initiated
by the addition of a first Sec molecule (protonated) fol-
lowed by nucleophilic attack by a second, deprotonated
Sec.
−
∙
∙
−
Sec + Tyr → Sec + Tyr ,
(R5)
−
−
∙
Sec + H OCbl III ⇌ Sec − Cbl III → Sec + Cbl II ,
(
)
(
)
( )
2
(R6)
−
′
′
′
′
−
Sec + R SSR ⇌ Sec − SR + R S ,
(R7)
S C H E M E 2 Mechanism of riboflavin reduction by selenocys-
teine
−
′
′
−
Sec + Sec − SR ⇌ Sec − Sec + R S ,
(R8)
−
the electrode potentials shows that the equilibrium in the
system is strongly shifted toward the reduced flavin and
selenocystine.
Sec + HOSCN → Sec − SCN,
(R9)
−
−
Sec + Sec − SCN → Sec − Sec + SCN ,
(R10)
Earlier works on the reaction between flavins and
thiols28–31 are in line with the results obtained in this study
for Rib and selenolate. The reactions of flavins with mer-
captoethanol are second order with respect to thiol con-
centration and exhibit a dependence of the rate on pH.
However, fastest reactions between flavins and thiols were
therein observed under alkaline conditions. This is in con-
trast to what is observed between Rib and Sec (maximum at
ca pH 5.5) due to the higher acidity of selenols over thiols.
The authors suggested transient formation of a flavin C4a-
thiol adduct,28–31 which was also postulated to occur with
flavoenzymes.32–34 The proposed mechanism involves pro-
tonation of the N5-atom of the flavin moiety as a critical
step.34 Therefore, we posit that the first step of the interac-
tion between Rib and Sec is the formation of a Rib C4a-Sec
intermediate product protonated at the N5 atom, which is
preceded by a preassociation between the reactants to drive
selenium and hydrogen atoms into the appropriate posi-
tions about the Rib molecule (Scheme 2). Further, the Rib
C4a-Sec intermediate undergoes nucleophilic attack by
−
+
Sec − SCN + H2O → Sec − OH + SCN + H , (R11)
−
−
Sec + Sec − OH → Sec − Sec + HO .
(R12)
4
CONCLUSION
This study shows that the fully reduced form of Rib
reduces selenocystine. This reaction proceeds relatively
slowly in comparison with the corresponding reverse reac-
tion, that is, oxidation of Sec by Rib. Oxidation of Sec
by Rib proceeds via two steps, that is, (i) rapid reversible
binding of Rib to a Sec molecule having a protonated
selenol group to form a Sec-Rib complex, and (ii) subse-
quent nucleophilic attack of the Sec-Rib complex by a sec-
ond Sec molecule having a deprotonated selenol group.
Although the structures of selenocystine and selenosulfide
do not completely coincide, the results of this study might

DEREVEN’KOV et al.
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O RC I D
IliaA. Dereven’kov https://orcid.org/0000-0003-1332-
4998
Sergei V. Makarov https://orcid.org/0000-0002-1919-
3813
Anna S. Makarova https://orcid.org/0000-0003-1111-
7186
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