Catalytic effect of monovalent cations on the amine oxidation by cofactor TTQ of quinoprotein amine dehydrogenases

Article abstract of DOI:10.1039/a908914d

Oxidation of benzylamine by a model compound of cofactor TTQ (tryptophan tryptophylquinone) of quinoprotein amine dehydrogenases is made possible by coordination of a monovalent cation such as Li⁺ in anhydrous MeCN.

Full text of DOI:10.1039/a908914d

Catalytic effect of monovalent cations on the amine oxidation by cofactor TTQ
of quinoprotein amine dehydrogenases
a
b
b
Shinobu Itoh,* Masato Taniguchi and Shunichi Fukuzumi*
a
Department of Chemistry, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku,
Osaka 558-8585, Japan. E-mail: shinobu@sci.osaka-cu.ac.jp
Department of Material and Life Science, Graduate School of Engineering, Osaka University; CREST, JAPAN
Science and Technology Corporation, 2-1 Yamada-oka, Suikta, Osaka 565-0871, Japan
b
Received (in Cambridge, UK) 9th November 1999, Accepted 19th January 2000
Oxidation of benzylamine by a model compound of cofactor
TTQ (tryptophan tryptophylquinone) of quinoprotein amine
dehydrogenases is made possible by coordination of a
from the slope. A similar spectral change was obtained in the
titration of 1 with NaClO although the binding constant was
4
3
–1
12,13
smaller (KML = 5.5 dm mol , lmax = 454 nm).
It should
+
monovalent cation such as Li in anhydrous MeCN.
be noted that the spectral change obtained in this system is fairly
close to that observed in the titration of MADH with
monovalent cations.4 This result suggests that the monovalent
cation binds to the quinone moiety of 1 at a similar position to
that proposed in the enzymatic systems.14
,8
Bacterial methylamine dehydrogenase (MADH) and aromatic
amine dehydrogenase (AADH) comprise a new class of
enzymes which contain a heterocyclic o-quinone cofactor, TTQ
A - A0
A• - A
A - A0
A• - A0
+
=
KML([M ] -
[Q]0)
0
(1)
Although no reaction takes place between benzylamine and
quinone 1 in an aprotic solvent such as anhydrous MeCN, 1 was
+
converted into the reduced form of TTQ in the presence of Li .
–
5
23
Thus, the reaction of quinone 1 (5.0 3 10 mol dm ) and
2
2
–3
benzylamine (1.5 3 10 mol dm ) in the presence of LiClO
4
2
3
(
0.15 mol dm ) in anhydrous MeCN at 25 °C under anaerobic
(
tryptophan tryptophylquinone), at their active sites in the light
conditions resulted in a drastic spectral change where the
1–3
subunits.
+
absorption band at 461 nm due to the Li complex of the
Monovalent cations have recently been shown to influence
not only the UV–VIS spectrum of the enzyme but also the redox
reactivity of TTQ in the amine oxidation and the subsequent
electron transfer to biological electron acceptor proteins such as
amicyanin.4 The enzymes have two different cation binding
sites, one of which is located near the quinone carbonyl oxygen
quinone decreases, accompanied by an increase in a new band
at 323 nm due to the reduced TTQ in the aminophenol form
15
(
Fig. 2). As reported in the previous model reactions between
16
the TTQ model compound and benzylamine in MeOH, the
reaction consists of three distinct steps, where the first one [Fig.
–9
2
(a)] corresponds to addition of the amine to the quinone (k
the second one [Fig. 2(b)] is due to the spontaneous (k ) and the
amine-catalyzed (k A) rearrangement from the substrate imine to
the product imine intermediates, and the third one [Fig. 2(c)]
is ascribed to the imine exchange reaction (k ) to generate
the aminophenol and N-benzylidenebenzylamine
PhCH
1
),
4
O(6) of the cofactor. Thus, the cationic species incorporated
2
into this binding pocket have been suggested to interact with the
quinone moiety directly, affecting the electronic structure of the
cofactor as well as the reactivity both in the amine oxidation
reaction and the subsequent electron transfer process.5–9
However, little is known about the binding model of the cationic
species toward TTQ or how it affects the electronic structure as
well as the reactivity of the TTQ cofactor. In this study, we have
investigated the interaction of a TTQ model compound10 with
monovalent cations to demonstrate that an alkaline metal ion
such as Li+ binds to the quinone, leading to a significant
enhancement of the amine oxidation reaction in anhydrous
organic media.
2
3
15
(
2
NNCHPh), as summarized in Scheme 1. Each process
has its own isosbestic point, at 418, 383 and 372 nm,
respectively, demonstrating the accuracy of the stepwise
mechanism shown in Scheme 1. Since the difference in the rate
4
Addition of a certain amount of LiClO to an anhydrous
MeCN solution containing TTQ model compound 1 (5.0 3
2
5
–3
22
23
1
0
mol dm ) and benzylamine (1.0 3 10 mol dm
caused a rapid spectral change in the UV–VIS region. Fig. 1
shows the spectral change of the titration of 1 with LiClO
)
4
22
2
3
(
0–0.15 mol dm ) in the presence of benzylamine (1.0 3 10
2
3 11
mol dm ). The characteristic absorption band at 408 nm due
to the quinone itself decreases, accompanied by an increase in a
new absorption band at 461 nm, with increasing the concentra-
tion of added LiClO
4
. The binding constant KML for 1+1
complex formation between the quinone (Q) and the metal ion
+
(
M ) can be expressed by eqn. (1), where A and A are the
0
∞
+
initial and final absorptions of the titration, and [M ] and [Q]
denote the concentration of the added metal ion and the initial
quinone concentration, respectively. The plot of (A 2 A )/(A
)] gives a
straight line passing through the origin, as shown in the inset in
0
0
0
∞
4
Fig. 1 Spectral change observed upon addition of LiClO (0–0.15 mol
+
2
A) vs. ([M ]
0
2 a[Q]
0
) [a = (A 2 A
0
)/(A
∞
2 A
0
23
25
23
dm ) to an MeCN solution of 1 (5.0 3 10 mol dm ) in the presence of
benzylamine (1.0 3 1022 mol dm23) at 25 °C. Inset: plot of (A 2 A )/(A
2 A) vs. [LiClO
0
∞
3
21
Fig. 1, from which a KML value of 15.6 dm mol was obtained
]
4 0
2 a[1]
0
{a = (A 2 A
0
)/(A
∞
0
2 A )}.
DOI: 10.1039/a908914d
Chem. Commun., 2000, 329–330
This journal is © The Royal Society of Chemistry 2000
329

Fig. 2 Spectral change for the reaction of 1 (5.0 3 102 mol dm23) with benzylamine (1.5 3 10 mol dm–3) in the presence of LiClO
in anhydrous MeCN at 25 °C under anaerobic conditions. (a) The first stage (0–7200 s), 800 s interval. Inset: plot of kobs(1) vs. [PhCH NH
stage (9600–24000 s), 2400 s interval. Inset: plot of kobs(2) vs. [PhCH NH ]. (c) The third stage (24000–49600 s), 3200 s interval. Inset: plot of kobs(3) vs.
PhCH NH ].
5
22
(0.15 mol dm23
]. (b) The second
)
4
2
2
2
2
[
2
2
Scheme 1
4 V. Kuusk and W. S. McIntire, J. Biol. Chem., 1994, 269, 26136.
of each step was not large enough to determine the rate
independently, the pseudo-first order rate constants for the three
steps (kobs(1), kobs(2) and kobs(3)) were determined simultaneously
by computer simulation of the time course of the absorption
change using a non-linear curve-fitting program (Mac curve fit)
as reported in the previous study.16 From the dependence of kobs
in each step on the amine concentration the rate constants were
5
6
A. C. F. Gorren and J. A. Duine, Biochemistry, 1994, 33, 12202.
A. C. F. Gorren, S. de Vries and J. A. Duine, Biochemistry, 1995, 34,
9
748.
7
8
A. C. F. Gorren, P. Moenne-Loccoz, G. Backes, S. de Vries, J. Sanders-
Loehr and J. A. Duine, Biochemistry, 1995, 34, 12926.
P. Moenne-Loccoz, N. Nakamura, S. Itoh, S. Fukuzumi, A. C. F.
Gorren, J. A. Duine and J. Sanders-Loehr, Biochemistry, 1996, 35,
4713.
2
2
21 21
25 21
determined as k
1
2
= 5.9 3 10
M
, and k
s
, k
2
= 8.7 3 10
s ,
2
21 21
22
21
s21, as
k
2
A = 2.6 3 10
M
s
3
= 1.4 3 10
M
9 G. R. Bishop and V. L. Davidson, Biochemistry, 1997, 36, 13586.
10 S. Itoh, M. Ogino, S. Haranou, T. Terasaka, T. Ando, M. Komatsu, Y.
Ohshiro, S. Fukuzumi, K. Kano, K. Takagi and T. Ikeda, J. Am. Chem.
Soc., 1995, 117, 1485.
shown in the insets in Fig. 2. It should also be emphasized that
the oxidation of benzylamine by the Li complex of 1 proceeds
catalytically, and molecular oxygen is used as an electron
acceptor to regenerate the iminoquinone form from the reduced
TTQ. Thus, benzylamine (0.10 mol dm ) was converted into
+
1
4
1 Each spectrum in Fig. 1 was taken just after the addition of LiClO , since
the redox reaction between 1 and benzylamine gradually proceeded over
a prolonged reaction time.
2
3
N-benzylidenebenzylamine quantitatively when it was treated
1
4 4 4 4
2 Because of the low solubility of NH ClO , KClO , CsClO and CsOTf
2
3
23
with a catalytic amount of 1 (1.0 3 10 mol dm ; 1 mol %)
in anhydrous MeCN, interaction between the quinone and those
monovalent cations could not be examined accurately.
2
3
in the presence of LiClO
4
(1.0 mol dm ) under aerobic
conditions for 24 h.
13 In the absence of benzylamine, the spectral changes (bathochromic
shifts) and the KML values are much smaller than those measured in the
In summary, a monovalent cation such as Li+ has been
demonstrated for the first time to bind to TTQ at its quinone
moiety and makes it possible for the catalytic amine oxidation
+
3
21
presence of the amine: for Li , lmax = 430 nm; KML = 3.6 dm mol
;
+
3
21
for Na , lmax = 425 nm; KML = 2.3 dm mol . Benzylamine may
+
abstract the pyrrole proton of 1 to enhance the Li binding.
+
to occur efficiently in anhydrous MeCN. The Li binding may
accelerate the addition step of the amine (k ) and enhances the
1
stability of the intermediates, leading to the efficient catalytic
oxidation of benzylamine. These results gave us an important
insight into the catalytic mechanism of the monovalent cations
in MADH- and AADH-catalysed reactions.
+
1
4 In spite of our great efforts, a single crystal of the Li complex of 1
1
suitable for X-ray analysis has yet to be obtained. The H NMR
23
23
3
spectrum of 1 (1 3 10 mol dm ) in CD CN in the presence of
23
LiClO
(Dd
4
(1 mol dm ) showed downfield shifts for 1-H, 2-H and 5-H
0.39, 0.16 and 0.05 ppm, respectively), suggesting the
coordination of Li at the quinone moiety of 1.
=
+
The present study was financially supported in part by a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Science, Culture, and Sports of Japan.
15 Although the reaction intermediates (substrate imine and product imine)
and the aminophenol product of TTQ have been successfully isolated in
the previous reaction in MeOH (ref. 16), isolation and detailed
characterization of those species in the present system were hampered
+
by Li existing in large excess. The spectral change of the present
Notes and references
reaction is, however, fairly close to that of the reaction of 1 and
benzylamine in MeOH, (ref. 16), strongly supporting the idea that the
intermediates and reduced product are the same in both systems.
1
2
3
W. S. McIntire, D. E. Wemmer, A. Chistoserdov and M. E. Lidstrom,
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L. Chen, F. S. Mathews, V. L. Davidson, E. G. Huizinga, F. M. D.
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Chistoserdov, V. L. Davidson and S. L. Edwards, J. Bacteriol., 1994,
1
6 S. Itoh, N. Takada, S. Haranou, T. Ando, M. Komatsu, Y. Ohshiro and
S. Fukuzumi, J. Org. Chem., 1996, 61, 8967.
1
76, 2922.
Communication a908914d
330
Chem. Commun., 2000, 329–330