Modeling of the hydrophobic microenvironment of water-soluble molybdoenzymes in an aqueous micellar solution

Article abstract of DOI:10.1039/c5dt01112d

A toluene-soluble molybdenum(vi) complex containing a bulky hydrophobic substituent, (Et₄N)₂[Mo^VIO₂{1,2-S₂-3,6-(RCONH)₂C₆H₂}₂] (R = (4-^tBuC₆H₄)₃C), was dissolved in the hydrophobic core of a micelle in an aqueous medium and catalyzed the biomimetic reduction of an amine N-oxide by an NADH analog. The kinetic isotope effect of solvent water clearly indicates that water molecules are essential for catalysis and are involved in the rate-determining step.

Full text of DOI:10.1039/c5dt01112d

Dalton
Transactions
View Article Online
View Journal | View Issue
PAPER
Modeling of the hydrophobic microenvironment
of water-soluble molybdoenzymes in an aqueous
micellar solution†
Cite this: Dalton Trans., 2015, 44,
12618
Yuki Hasenaka, Taka-aki Okamura* and Kiyotaka Onitsuka
A toluene-soluble molybdenum(VI) complex containing a bulky hydrophobic substituent, (Et
4
N) -
2
VI
t
Received 20th March 2015,
Accepted 28th May 2015
[Mo O {1,2-S -3,6-(RCONH) C H } ] (R = (4- BuC H ) C), was dissolved in the hydrophobic core of a
2
2
2
6
2 2
6 4 3
micelle in an aqueous medium and catalyzed the biomimetic reduction of an amine N-oxide by an NADH
analog. The kinetic isotope effect of solvent water clearly indicates that water molecules are essential for
catalysis and are involved in the rate-determining step.
DOI: 10.1039/c5dt01112d
www.rsc.org/dalton
Introduction
Trimethylamine N-oxide reductase (TMAOR), a member of the
DMSO reductase family, is a molybdoenzyme with two dithio-
lene ligands, called the pterin cofactor. This enzyme catalyzes
oxygen-atom-transfer (OAT) reactions via the Mo(IV) and Mo(VI)
1
–4
oxidation states (Fig. 1).
The molybdenum(IV) center reduc-
tively eliminates an oxygen atom from amine N-oxides to form
VI
a Mo vO bond, and is regenerated via protonation and
reduction accompanied by the production of water. In the
anaerobic respiratory chain, nicotinamide adenine dinucleo-
tide (NADH) acts as an electron donor to reduce amine
3
,5
N-oxides, the terminal electron acceptors.
In the case of
biotin sulfoxide reductase (BSOR), the direct hydride transfer
from NADH phosphate to the active site has been proposed by
6
–8
kinetic analyses.
The active site of TMAOR is located at the base of a large
funnel-shaped depression, and the ligands are not exposed to
1
,9
the surface (Fig. 1).
The hydrophobic pocket formed by
aromatic residues at the base of the depression is conserved in
1
,7
both TMAOR and BSOR.
The above results suggest the
importance of hydrophobicity in the enzyme activity.
A
number of molybdoenzyme models have been
1
0–16
synthesized,
some of which have simulated the catalytic
reduction of amine N-oxides by phosphines via coupled OAT
1
7–21
reactions.
Monooxomolybdenum(IV) complexes with
Fig. 1 (a) The structure of the active site and (b) catalytic cycle of tri-
methylamine N-oxide reductase.
benzene-1,2-dithiolate (bdt) ligands can be used to eliminate
Department of Macromolecular Science, Graduate School of Science,
Osaka University, Toyonaka, Osaka 560-0043, Japan.
E-mail: tokamura@chem.sci.osaka-u.ac.jp; Fax: +81 6 6850 5474;
Tel: +81 6 6850 5451
VI
the oxygen atom of trimethylamine N-oxide to yield a Mo vO
bond; the resulting dioxomolybdenum(VI) complexes can be
reduced by benzoin to regenerate the original monooxomolyb-
†
Electronic supplementary information (ESI) available: The whole spectra of
Fig. 3, kinetic plot of the catalytic reduction of DDAO by BNAH in water-contain-
ing toluene-d . See DOI: 10.1039/c5dt01112d
2
2,23
denum(IV) complexes with the production of water.
8
12618 | Dalton Trans., 2015, 44, 12618–12622
This journal is © The Royal Society of Chemistry 2015

View Article Online
Dalton Transactions
Paper
confirmed, a solution of BNAH (30 mM, 20 μL) in THF was
added. After 20 min, H O (10 μL, 180 mM) was added, and the
2
time course of the reaction was monitored by using the
absorption maximum of BNAH.
Catalysis in toluene-d
8
To an NMR tube cooled at −78 °C were successively added a
solution of DDAO (0.80 mg, 3.5 μmol) in a mixture of toluene-
d8 (0.3 mL)/H O (0.2 μL, 11 μmol), a solution of BNAH
2
(
8
0.74 mg, 3.5 μmol) in toluene-d (0.3 mL), and a solution of 2
(
0.51 mg, 0.21 μmol) in toluene-d (0.1 mL). To start the reac-
8
tion, the contents were quickly warmed up to 27 °C with
shaking. The reaction was monitored at 27 °C by using
1
H NMR spectroscopy.
Chart 1 Designation of molybdenum complexes.
Preparation of a micellar solution of 2
To a solution of 2 (1.55 mg, 0.63 μmol) in THF (0.3 mL) was
added a 0.1 M THF solution of Triton™ X-100 (0.9 mL,
We have recently reported toluene-soluble monooxomolyb-
90 μmol). The resulting reddish-brown solution was concen-
denum(IV) and dioxomolybdenum(VI) complexes, (Et
4
N)
2
-
trated under reduced pressure, and water (1.8 mL) was added
to afford a 0.35 mM aqueous micellar solution of 2.
IV
t
[
Mo O{1,2-S -3,6-(RCONH) C H } ] (1), (R = (4- BuC H ) C)
2
2
6
2 2
6
4 3
VI
4 2 2 2 2 6 2 2
and (Et N) [Mo O {1,2-S -3,6-(RCONH) C H } ] (2) (Chart 1).
The introduction of bulky hydrophobic substituents covered
the polar molybdenum center completely with a hydrophobic
Catalysis in a micellar solution
A reaction system containing 2, DDAO, and BNAH was moni-
tored in the 280–1000 nm region. The measurements were
carried out in a 1 mm UV cell at 27 °C. After the thermal equi-
librium, a 90 mM aqueous solution of DDAO (10 μL, 0.9 μmol)
and a 30 mM THF solution of BNAH (30 μL, 0.9 μmol) were
added to a 0.35 mM aqueous micellar solution of 2 (0.26 mL,
3
barrier, and the approach of Me NO to the active center was
2
4
found to be more efficient in hydrophobic media. If the com-
plexes are solubilized in an aqueous medium, the biomimetic
catalysis using protons of water is expected to be achieved.
We present here the biomimetic catalytic reduction of an
amine N-oxide by an NADH analog in an aqueous micellar
solution. There are a few reports on models promoting the
OAT reaction in aqueous media; however, the reports are
limited to the use of a mixture of polar solvents and water or
0
.09 μmol), and the cell contents were quickly mixed by
shaking. The absorption at 355 nm (BNAH) was monitored at
7 °C.
2
25,26
wet organic solvents.
Our trial in this paper is to simulate
Physical measurements
heterogeneous environments around the active sites of
enzymes. The model complex in the hydrophobic core of a
micelle mimics the active site in the hydrophobic pocket. The
outside of a micelle resembles the surroundings of water-
soluble enzymes although it does not represent a pure
aqueous solution.
1
H NMR spectra were recorded using a JEOL ECA-500 spectro-
meter in toluene-d8 at 27 °C. UV-visible absorption spectra
were recorded using a SHIMADZU UV-3100PC spectrometer.
Results
Stoichiometric and catalytic reactions in nonpolar solvent
Experimental
Because of the limitations of the solubility of the reagent in
organic solvents, we designed a model system of a catalytic
reaction using dodecyldimethylamine N-oxide (DDAO) and an
NADH analog, 1-benzyl-1,4-dihydronicotinamide (BNAH). At
first, a stoichiometric reaction in organic solvents was exam-
ined using UV-vis spectroscopy (Scheme 1, Fig. 2). When 2
equivalents of DDAO were added to a toluene solution of
complex 1 (0.1 mM), a rapid OAT reaction occurred in a
All procedures were performed under an argon atmosphere by
using the Schlenk technique. All organic solvents were dried
and distilled under argon before use. Reagents were obtained
commercially and used without further purification. (Et N) -
4
2
VI
t
24
[
Mo O
2 2 6 4 3 2 6 2 2
(1,2-S -3,6-{(4- BuC H ) CCONH} C H ) ] (2) was pre-
pared by reported methods.
3
manner similar to the reaction of Me NO (Fig. 2a, from orange
to green line). Subsequently, 2 equivalents of BNAH were
added, but no reaction occurred (Fig. 2a, red line). The charac-
Stoichiometric reaction between 1 and DDAO and the
subsequent reduction by BNAH in toluene/THF
24
The procedure was similar to that described in the previous teristic absorption of BNAH at 355 nm remained unchanged
2
4
paper for Me
3
NO in toluene, except for the use of DDAO for 20 min. The absorption obviously decreased upon the
(30 mM, 20 μL) in THF. After the complete formation of 2 was addition of excess of water, showing the consumption of
This journal is © The Royal Society of Chemistry 2015
Dalton Trans., 2015, 44, 12618–12622 | 12619

View Article Online
Paper
Dalton Transactions
1
8
Fig. 3 H NMR spectral change in water-containing toluene-d during
Scheme 1 The OAT reaction of DDAO and regeneration of 1 from 2 by
BNAH.
the catalytic reduction of DDAO by BNAH with 2 mol% of 2 at 27 °C
after (a) 0.5 h, (b) 96 h, (c) 373 h, and (d) 887 h. The asterisk (*) denotes
toluene.
The catalytic reduction of DDAO by BNAH in water-contain-
1
ing toluene-d
8
was then monitored using H NMR spec-
troscopy (Fig. 3, Fig. S1†). The consumption of DDAO and
BNAH followed by the production of DDA was observed. The
−
6 −1
pseudo-first-order reaction rate constant kobs (1.9 × 10
s ) in
the presence of 2 mol% of complex 2 was approximately 20
times higher than that observed in the absence of the catalyst
7.9 × 10−
s ), and the turnover number (TON) of the cataly-
8
−1
(
sis achieved with the complete consumption of DDAO was 46
although the reaction apparently occurred without a catalyst
(
Fig. S2†).
Catalysis in an aqueous micellar solution
In order to dissolve the hydrophobic molecules in an aqueous
medium, we used micelles. In the process of searching for a
surfactant to solubilize the complex, Triton™ X-100, a liquid
2
7,28
non-ionic surfactant,
could successfully form a transparent
micellar solution of complex 2. Because complex 2 was insolu-
ble in water, but readily soluble in toluene and Triton™ X-100,
the complex must be located in the hydrophobic core of the
micelles (Fig. 4). The catalytic reduction of DDAO by BNAH in
an aqueous micellar solution was monitored using UV-vis
spectroscopy (Fig. 5). The pseudo-first-order rate constant of
the catalytic reaction (kobs = 36 × 10−
6
s
−1
) was about 10 times
higher than that of the reaction without the complex (3.8 ×
−
6
−1
10
s
). When D
2 2
O was used instead of H O, the reaction
Fig. 2 (a) UV-vis spectral change and (b) time-course of the reaction of
slowed down (KIE = k /k = 6.5), which indicates that solvent
1
with DDAO and BNAH in toluene/THF. Red lines were added for clarity.
H
D
water molecules (or protons) are involved in the rate-determin-
ing step and are crucial for catalysis. A weak and broad absorp-
tion band was observed at 770 nm during the catalytic reaction
BNAH (Fig. 2a, black lines, Fig. 2b). These results indicate that (Fig. 5), which suggests the presence of a monooxomolyb-
water is essential for the reduction of complex 2 in the catalytic denum(V) species2
cycle.
a side reaction.
3,29,30
generated by one-electron reduction as
12620 | Dalton Trans., 2015, 44, 12618–12622
This journal is © The Royal Society of Chemistry 2015

View Article Online
Dalton Transactions
Paper
Fig. 4 Schematic drawing of the complex in an aqueous micellar solu-
tion (orange oval: hydrophobic group, blue stick: hydrophilic group).
Scheme 2 Proposed mechanism of the catalysis.
step. Moreover, the catalytic OAT reaction could be directly
1
observed using H NMR spectroscopy in water-containing
toluene-d8.
The non-ionic surfactant, Triton™ X-100, efficiently covered
the complex containing bulky hydrophobic groups and created
a heterolytic microenvironment in water as shown in Fig. 4. As
a result of kinetic experiments, the reaction mechanism of the
catalysis is considered as shown in Scheme 2. The large kinetic
isotope effect (KIE) of the solvent indicated that the protona-
tion of the dioxomolybdenum(VI) complex by water was
involved in the rate-determining step. Furthermore, the obser-
vation of the molybdenum(V) species suggested the presence
of a one-electron reduction process, which is similar to the
proposed catalytic cycle of TMAOR, where the one-electron
reduction associates with the protonation of the terminal oxo
1
ligand (Fig. 1).
Conclusions
We constructed a water-soluble molybdoenzyme model con-
taining a hydrophobic microenvironment in an aqueous micel-
lar solution. The model catalyzes the biomimetic reduction of
an amine N-oxide by an NADH analog in aqueous media. The
hydrophobic microenvironment likely promotes the uptake of
substrates from the outer aqueous media into the polar reac-
tive site. The present catalytic system, which reduces the sub-
strate followed by the production of water, is one of the most
well designed enzyme models.
Fig. 5 (a) UV-vis spectral change and (b) kinetic plot of the catalytic
reduction of DDAO by BNAH in an aqueous micellar solution.
Discussion
Dioxomolybdenum(VI) complex 2 catalyzes the oxygen-atom-
transfer (OAT) reaction of an amine N-oxide in water using an
NADH analog, BNAH, in both organic and aqueous media.
Acknowledgements
The stoichiometric reaction in non-aqueous media clearly This work was supported by JSPS KAKENHI Grant Number
showed the essential role of water molecules in the reduction 26410072.
This journal is © The Royal Society of Chemistry 2015
Dalton Trans., 2015, 44, 12618–12622 | 12621

View Article Online
Paper
Dalton Transactions
1
5 P. Basu and S. J. N. Burgmayer, Coord. Chem. Rev., 2011,
Notes and references
255, 1016–1038.
1
R. Hille, J. Hall and P. Basu, Chem. Rev., 2014, 114, 3963– 16 P. Basu, V. N. Nemykin and R. S. Sengar, Inorg. Chem.,
038. 2003, 42, 7489–7501.
C. Iobbi-Nivol, R. Haser, V. Méjean and M. Czjzek, 17 B. E. Schultz, R. Hille and R. H. Holm, J. Am. Chem. Soc.,
Trimethylamine N-Oxide Reductase, in Encyclopedia of 1995, 117, 827–828.
Inorganic and Bioinorganic Chemistry, John Wiley & Sons, 18 J. M. Berg and R. H. Holm, J. Am. Chem. Soc., 1985, 107,
Ltd, 2011. 925–932.
S. L. McCrindle, U. Kappler and A. G. McEwan, Microbial 19 E. W. Harlan, J. M. Berg and R. H. Holm, J. Am. Chem. Soc.,
Dimethylsulfoxide and Trimethylamine-N-Oxide Respir- 1986, 108, 6992–7000.
ation, in Adv. Microb. Physiol., ed. K. P. Robert, Academic 20 A. Döring, C. Fischer and C. Schulzke, Z. Anorg. Allg. Chem.,
Press, 2005, vol. 50, pp. 147–198. 2013, 639, 1552–1558.
M. Czjzek, J.-P. Dos Santos, J. Pommier, G. Giordano, 21 C. J. Whiteoak, G. J. P. Britovsek, V. C. Gibson and
V. Méjean and R. Haser, J. Mol. Biol., 1998, 284, 435–447. A. J. P. White, Dalton Trans., 2009, 2337–2344.
J. C. Cox, M. T. Madigan, J. L. Favinger and H. Gest, Arch. 22 N. Ueyama, H. Oku, M. Kondo, T. Okamura, N. Yoshinaga
4
2
3
4
5
6
7
8
Biochem. Biophys., 1980, 204, 10–17.
V. V. Pollock and M. J. Barber, J. Biol. Chem., 1997, 272,
and A. Nakamura, Inorg. Chem., 1996, 35, 643–
650.
3
355–3362.
K. J. Nelson and K. V. Rajagopalan, Biochemistry, 2004, 43,
1226–11237.
23 N. Ueyama, N. Yoshinaga, T. Okamura, H. Zaima and
A. Nakamura, J. Mol. Catal., 1991, 64, 247–256.
24 Y. Hasenaka, T. Okamura, M. Tatsumi, N. Inazumi and
K. Onitsuka, Dalton Trans., 2014, 43, 15491–
15502.
1
C. A. Temple, G. N. George, J. C. Hilton, M. J. George,
R. C. Prince, M. J. Barber and K. V. Rajagopalan, Biochemi-
stry, 2000, 39, 4046–4052.
25 S. K. Das, P. K. Chaudhury, D. Biswas and S. Sarkar, J. Am.
Chem. Soc., 1994, 116, 9061–9070.
9
S. Mondal and P. Basu, Inorg. Chem., 2001, 40, 192–193.
1
1
1
1
1
0 R. H. Holm, E. I. Solomon, A. Majumdar and 26 H. Sugimoto, M. Tarumizu, H. Miyake and H. Tsukube,
A. Tenderholt, Coord. Chem. Rev., 2011, 255, 993–1015. Eur. J. Inorg. Chem., 2006, 4494–4497.
1 F. J. Hine, A. J. Taylor and C. D. Garner, Coord. Chem. Rev., 27 M. Nakata, N. Ueyama, M. Fuji, A. Nakamura, K. Wada and
2
010, 254, 1570–1579.
2 V. W. L. Ng, M. K. Taylor and C. G. Young, Inorg. Chem.,
012, 51, 3202–3211.
3 A. Majumdar and S. Sarkar, Coord. Chem. Rev., 2011, 255,
039–1054.
4 H. Sugimoto, S. Tatemoto, K. Suyama, H. Miyake,
H. Matsubara, Biochim. Biophys. Acta, 1984, 788, 306–
312.
2
28 T. Okamura, K. Taniuchi, N. Ueyama and A. Nakamura,
Polym. J., 1999, 31, 651–657.
1
29 S. Boyde, S. R. Ellis, C. D. Garner and W. Clegg, J. Chem.
Soc., Chem. Commun., 1986, 1541–1543.
R. P. Mtei, S. Itoh and M. L. Kirk, Inorg. Chem., 2010, 49, 30 Y. Hasenaka, T. Okamura and K. Onitsuka, Dalton Trans.,
368–5370. 2015, 44, 6260–6267.
5
12622 | Dalton Trans., 2015, 44, 12618–12622
This journal is © The Royal Society of Chemistry 2015