Control of xanthine oxidase activity by light

Article abstract of DOI:10.1002/1521-3773(20001103)39:21<3886::AID-ANIE3886>3.0.CO;2-A

Turning the light on and off can control the activity of xanthine oxidase (XOD) when allopurinol (1) is the substrate. Alloxanthin (2) is formed in the reaction but also acts as an inhibitor of the enzyme. Irradiation can free the active site and let the enzyme get on with its job. The alloxanthin - XOD system might have important future applications as a photoswitch/integrator in molecular-scale optobioelectronic devices.

Full text of DOI:10.1002/1521-3773(20001103)39:21<3886::AID-ANIE3886>3.0.CO;2-A

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Method 2: A solution of nBu4NBr A7.85 g, 24.36 mmol) and SOBr A8.3 mL,
2
Control of Xanthine Oxidase Activity by
Light**
105.56 mmol) in MeCN A10 mL) was added dropwise to
a stirred
suspension of Na8H[PW9O34] ´ 7 H2O A20.81 g, 8.12 mmol) and K2CO3
A8.00 g, 57.88 mmol) in MeCN A200 mL) at À708C. After 1 h, the solution
was filtered and the volatiles removed under reduced pressure. The
resulting solid was redissolved in MeCN A40 mL) and filtered. Removal of
the solvent and trituration with diethyl ether gave an orange powder
A26.13 g). IR: nÄ ˆ 1064 As), 1022 Aw), 998 Aw), 989 Aw), 974 As), 928 As), 868
Lin Ai Tai and Kuo Chu Hwang*
Photocontrol of enzyme activities has been a subject of
active research.[1±8] Natural photoenzymes are very rare. To
date, only three natural photoenzymes have been reported,
namely, DNA photolyase,[9] [6 ± 4] photoproduct lyase,[10] and
protochlorophyllide reductase.[11] With their ability of trans-
forming optical signals into chemical motion, photoenzymes
might have important future applications in molecular-scale
electronics in the areas of signal transformation, amplifica-
tion, integration, and information storage.
Am), 787 As, br), 737 Aw), 580 Aw), 519 Am) cmÀ1
.
Received: January 20, 2000
Revised: August 7, 2000 [Z14574]
[1] W. H. Knoth, J. Am. Chem. Soc. 1979, 101, 759.
[2] P. Gouzerh, A. Proust, Chem. Rev. 1998, 98, 7 7 .
Previously, photoinduced release of a light-active antigen
from an antibody was reported.[12] Photochemical inhibition
of nicotinic acetylcholine receptors by a photoisomerizable
inhibitor was also reported.[13] Enzymes can also be chemi-
cally modified by covalently bonding a ªgating moleculeº to
the neighborhood of the enzyme active site[3±5] or at the
enzyme cofactor[6±8] to control the entry of substrates.
Common gating molecules of choice are photoisomerizable
olefins and photochromic compounds. Chemical modification
of natural enzymes often results in partial loss of enzyme
activity. Moreover, the location of the gating molecule is
limited to the availability of chemically modifiable amino
acids around the active site, and is quite difficult to control at
will. Herein, we report a simple, novel xanthine oxidase
AXOD) photoenzyme system, with allopurinol as a substrate,
in which the activity can be switched on or off by light. A
photoinduced intra-enzyme electron-transfer model is pro-
posed to rationalize the photoregulation of XOD activity.
XOD is a 300 kDa homodimer protein, with each unit
containing a molybdenumAvi), two Fe/S clusters, and one
flavin adenine dinucleotide AFAD) moiety.[14] The MoVI ion at
the active site can activate a water molecule and oxidize a
[3] W. H. Knoth, P. J. Domaille, D. C. Roe, Inorg. Chem. 1983, 22, 198.
[4] Y-J Lu, R. H. Beer, Polyhedron 1996, 15, 1667.
[5] We have shown by X-ray crystallography that this compound has the
a-PW11 structure.
[6] Crystal data for AnBu4N)3-1 ´ 1.56MeCN: C51.08H112.62Br6N4.56O28PW9,
Mr ˆ 3403.9, monoclinic, space group P21/n, a ˆ 15.0129A10), b ˆ
25.831A2), c ˆ 22.8859A15) Š, b ˆ 93.452A2)8, Vˆ 8859.0A10) Š3, Z ˆ
4, 1calcd ˆ 2.552 gcmÀ3
,
MoKa radiation, l ˆ 0.71073 Š, m ˆ
14.43 mmÀ1, Tˆ 160 K; of 37732 measured reflections corrected for
absorption, 14773 were unique, Rint
ˆ 0.0668; R ˆ 0.0600 AI > 2s),
Rw ˆ 0.1345 AF 2, all data), GOF ˆ 1.147, 968 parameters, final differ-
ence map extremes ‡2.38 and À1.20 eŠÀ3. Disorder was refined for
some alkyl chains and one partially occupied acetonitrile position with
the aid of restraints. Crystallographic data Aexcluding structure
factors) for the structure reported in this paper have been deposited
with the Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC-139046. Copies of the data can be obtained free
of charge on application to CCDC, 12 Union Road, Cambridge
CB21EZ, UK Afax: A‡44)1223-336-033; e-mail: deposit@ccdc.cam.
ac.uk).
[7] S. Liu, S. N. Shaikh, J. Zubieta, Inorg. Chem. 1988, 27, 3064.
[8] A. Mazeaud, N. Ammari, F. Robert, R. Thouvenot, Angew. Chem.
1996, 108, 2089; Angew. Chem. Int. Ed. Engl. 1996, 35, 1961.
[9] F. Xin, M. T. Pope, Organometallics 1994, 13, 4881.
[10] C. R. Mayer, R. Thouvenot, J. Chem. Soc. Dalton Trans. 1998, 7 .
[11] J. Lefebvre, F. Chauveau, P. Doppelt, C. Brevard, J. Am. Chem. Soc.
1981, 103, 4589.
[12] W. McFarlane, A. M. Noble, J. M. Whitfield, J. Chem. Soc. +A) 1971,
948.
[13] P. J. Domaille, Inorg. Synth. 1990, 27, 100.
À
À
C H bond in various substrates to C OH. After gaining two
electrons from the substrate, MoIV passes two electrons
sequentially, by way of the Fe/S clusters, to the FAD cofactor
which then reduces molecular oxygen to generate superoxide
Aor hydrogen peroxide in acidic conditions).[15] As an XOD
substrate, allopurinol A1) is oxidized and converted to
alloxanthine A2) [Equation A1)], which is a potent, slow-
[14] R. J. Errington, R. L. Wingad, W. Clegg, R. A. Coxall, unpublished
results.
[15] R. J. Errington, Advanced Practical Inorganic and Metalorganic
Chemistry, Blackie Academic and Professional, London, 1997.
[*] K. C. Hwang, L. A. Tai
Department of Chemistry
National Tsing Hua University
Hsinchu ATaiwan)
Fax : A‡886)3-571-1082
E-mail: kchwang@mx.nthu.edu.tw
[**] The authors are grateful for financial support from the National
Science Council, Taiwan ANSC 88 ± 2113-M-007± 016).
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binding XOD inhibitor capable of binding to the reduced
form MoIV-XOD, but not to the oxidized MoVI-XOD.[16]
The enzymatic conversion of allopurinol into alloxanthine
can be monitored by UV/Vis absorbance at 277 nm, the
absorption maximum of alloxanthine. During the enzymatic
reaction, the increase of absorption at 277 nm slowly levels
off, due to the slow inhibition of XOD by alloxanthine Asee
Figure 1a, trace 1). Upon exposure to the output from a high-
pressure Hg lamp, the enzyme activity was recovered, as
xanthine. The formation rate becomes smaller in later dark
cycles, which is due to the higher concentration of alloxan-
thine Athe inhibitor) present during later cycles. Figure 1b
shows the variation of the alloxanthine formation rates Aor
catalysis velocity) during the dark ± light cycles. In the dark,
the catalysis velocity is approximately 2 mm hÀ1, before the
onset of the subsequent light cycle. Upon irradiation, the
catalysis velocity is increased to about 40 mm hÀ1. Irradiation
increases the enzyme activity by a factor of approximately
2000%! Upon switching the light off, the catalysis velocity
soon decays back to its initial low values. The data in Figure 1
clearly shows that the XOD activity can be regulated on or off
photochemically.
Before irradiation, most of the XOD was inhibited by
alloxanthine. To recover the enzyme activity, the inhibitor has
to be released from the active site. It seems that exposure to
light triggers the release of inhibitor Athat is, the alloxanthine)
from the XOD active site. How does light initiate the
dissociation of the alloxanthine ± XOD complex? It is known
that alloxanthine can only bind the reduced MoIV-XOD.[16]
Photoexcitation probably converts the MoIV ion into other
forms, such as MoV or MoVI. The change in the electron
density at the Mo ion then leads to the dissociation of
alloxanthine from the active site.[17] The MoVI species does not
exhibit an ESR signal, but MoV does. To examine this
possibility, alloxanthine ± XOD complexes were prepared by
mixing XOD with excess allopurinol for 10 min. The solution
was then frozen at 180 K in the presence of air, and irradiated
by the output of a high-pressure Hg lamp for 3 min. Figure 2a
shows the difference spectrum obtained by subtracting the
ESR spectrum after irradiation from the one obtained before
irradiation. The left ESR band Ag ˆ 2.0047, linewidth DHpp
ˆ
19.3 Gauss) has the same g value and linewidth as that
.
reported in the literature for FADH .[18] The two smaller ESR
bands Ag ˆ 1.9716, 1.9636; DHpp ˆ 8.5, 9.0 G, respectively)
resemble the ªalloxanthine signalº[16, 18, 19] and are designated
to the MoV species.[20] Figure 2b shows the computer-simu-
Figure 1. a) The optical density AOD) at 277 nm from a solution of
allopurinol and XOD A200 mm and 0.15 mm, respectively) as a function of
the reaction time. Trace 1: The solution was maintained in the dark.
Trace 2: The solution was put through many dark ± light cycles. In each light
cycle, the solution was irradiated for 900 s Apower density at the sample
ꢀ0.01 WcmÀ2; l ꢁ 320 nm). Inset: The OD changes Afrom top to bottom)
of the first, second, third, and fourth dark cycles from trace 2 as a function
of the reaction time. b) The enzyme catalysis rate AV) obtained from trace 2
in Aa) at different reaction times: 50 s before irradiation Aopen circle),
during irradiation Asolid triangles), and 50 s after irradiation Asolid
squares).
.
lated FADH and MoV signals.
The results in Figure 2 support the hypothesis that irradi-
ation causes electron transfer from MoIV to the other two
cofactors, Fe/S and FAD, to generate MoV, AFe/S)red, and
.
FADH intermediate states. Subsequent electron transfer of
the second electron from the Mo center to the other cofactors
and release of the alloxanthine from the active site leads to
recovery of free, active XOD. Therefore, the overall XOD
activity is recovered by light. The ESR signal of Fe/S was not
observed at the experimental conditions used ATˆ 180 K). It
has been reported that the ESR signal of Fe/S has never been
observed at temperatures above 42 K because of its very short
relaxation time and, thus, very broad ESR linewidth.[21]
We have reported a novel XOD system in which the
enzyme activity can be regulated on or off by light. This
system represents a prototype of natural biological photo-
switches/integrators. For example, as shown in trace 2 of
Figure 1a, the amount of alloxanthine product is continuously
being integrated Aor accumulated) upon stimulation by the
incoming light signals. We believe that this system might have
future applications as a photoswitch/integrator in molecular-
scale optoelectronics.
evidenced by the dramatic increase in the absorption at
277 nm Asee Figure 1a, trace 2). When the enzyme solution
was removed from the light, the XOD activity was inhibited
again by the alloxanthine Asee Figure 1a, the dark sections of
trace 2). The XOD activity can be turned on or off by many
dark ± light cycles without noticeable deterioration of the
enzyme activity. The absorbance change Athat is, the amount
of alloxanthine produced) during the ªlightº cycles is clearly
far more than that in the ªdarkº cycles, indicating the
dramatic photoeffect on increasing the XOD activity.
The inset of Figure 1a shows the alloxanthine formation
rate during the first 500 s of the first, second, third, and fourth
dark cycles Afrom top to bottom). Each trace in the inset
shows the typical slow-binding inhibition behavior of allo-
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[2] P. R. Westmark, J. P. Kelly, B. D. Smith, J. Am. Chem. Soc. 1993, 115,
3416 ± 3419.
[3] I. Willner, E. Zahavy, Angew. Chem 1994, 106, 594 ± 595; Angew.
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[4] S. Monti, G. Montagnoli, L. Nannicini, J. Am. Chem. Soc. 1977, 99,
3808 ± 3811.
[5] S. Martinek, I. V. Berezin, Photochem. Photobiol. 1979, 29, 637± 645.
[6] R. Blonder, E. Katz, I. Willner, V. Wray, A. F. Bückmann, J. Am.
Chem. Soc. 1997, 119, 11747 ± 11757.
[7] E. Zahavy, I. Willner, J. Am. Chem. Soc. 1996, 118, 12499 ± 12514.
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1995, 376, 672 ± 675.
[9] a) D. B. Sanders, O. Wiest, J. Am. Chem. Soc. 1999, 121, 5127± 5134;
b) D. Burdi, T. P. Begley, J. Am. Chem. Soc. 1991, 113, 7768 ± 7770.
[10] a) S. T. Kim, K. Malhatra, C. A. Smith, J. S. Taylor, A. Sancar, J. Biol.
Chem. 1994, 269, 8535 ± 8540; b) T. Todo, H. Takamon, H. Ryo, M.
Ihara, T. Matsunaga, O. Nikaido, K. Sato, T. Nomura, Nature 1993,
361, 371 ± 374.
[11] T. P. Begley, H. Young, J. Am. Chem. Soc. 1989, 111, 3095 ± 3096.
[12] a) M. Harada, M. Sisido, J. Hirose, M. Nakanishi, FEBS 1991, 286,
6 ± 8; b) R. Blonder, S. Levi, G. Tao, I. Ben-Dov, I. Willner, J. Am.
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[14] a) R. C. Bray, Q. Rev. Biophys. 1988, 21, 299 ± 329; b) R. Hille,
Biochem. Biophys. Acta 1994, 1184, 143 ± 169.
Figure 2. a) The ESR spectrum obtained by subtracting the spectrum after
irradiation from the spectrum obtained before irradiation for an aqueous
solution of XOD A20 mm) in the presence of excess allopurinol in air. The
enzyme solution was frozen at 180 K, and the first spectrum was recorded.
Then the frozen enzyme solution was irradiated Apower density at the
sample ꢀ0.13 WcmÀ2) for 3 min, and the second spectrum was obtained.
The microwave power was 10 mW, the modulation was 5 Gauss, and the
.
frequency was 9.5819 GHz. b) Computer-simulated spectra of the FADH
Ag ˆ 2.0047,DHpp ˆ 19.3 G) and MoV Ag ˆ 1.9716, 1.9636; DHpp ˆ 8.5, 9.0 G)
signals.
[15] R. Hille, Chem. Rev. 1996, 96, 2757 ± 2816.
[16] J. W. Williams, R. C. Bray, Biochem. J. 1981, 195, 753 ± 760.
[17] Photooxidation of alloxanthine by the excited MoIV center is also one
of the possible pathways. We isolated the alloxanthine ± XOD
complex and split it into two portions. One portion was maintained
in the dark, waiting for dissociation into alloxanthine and the free
enzyme; the other portion was photolyzed to force dissociation of the
complex. The UV spectra of these samples were then compared. No
clear difference was observed in the absorption of the dissociated
alloxanthine. In addition, irradiation of the alloxanthine ± XOD
complex did not show any detectable ESR signal corresponding to
an alloxanthine cation radical. Therefore, it seems that majority of
alloxanthine molecules leave the enzymeꢁs active site without being
oxidized. However, the current data can certainly not exclude the
possibility of photooxidation of alloxanthine as a minor process.
[18] V. Massey, H. Komai, G. Palmar, G. B. Elion, J. Biol. Chem. 1970, 245,
2837± 2844.
Experimental Section
XOD A0.08 unitmgÀ1, Sigma) and allopurinol ATCI) were used as received.
The enzyme solution was adjusted to pH 7.5 in Na HPO4 buffer A0.1m). The
2
pH value and buffer concentration are the same in all experiments. The
concentration of XOD was determined by the its absorption at 450 nm,
with the absorption coefficient e450 ˆ 37800MÀ1 cmÀ1 [22]
.
In general, allo-
purinol A2 mm) in NaOH solution A0.01n) was prepared first, then aliquots
of the solution were added to the enzyme solution to reach the required
final concentration. The enzyme catalysis rate was monitored at the
absorption maximum of the product, 277 nm for alloxanthine.
All experiments were performed in the presence of air. The light source is
the output from a high-pressure Hg lamp AOriel) fitted with a CuSO4 filter
solution Al ꢁ 320 nm). ESR spectra were taken on a Bruker EMX-12
spectrometer. A 1,1-diphenyl-2-picrylhydrazyl ADPPH) sample in a second
resonator chamber was used as the reference for the g value measurements.
The temperature was controlled by a Bruker B-VT 2000 variable temper-
ature control unit.
[19] T. R. Hawkes, G. N. George, R. C. Bray, Biochem. J. 1984, 218, 961 ±
968.
.
[20] The ESR data shown in Figure 2 resemble very closely the FADH
and MoV signals of Figure 6 in Ref. [18] except that the phase of the
ESR spectrometer is reversed.
Received: February 29, 2000 [Z14789]
[21] a) R. Hille, W. R. Hagen, W. R. Dunham, J. Biol. Chem. 1985, 260,
10569 ± 10575; b) D. J. Lowe, R. C. Bray, Biochem. J. 1978, 169, 47 1 ±
479.
[22] V. Massey, R. E. Brumby, H. Komai, G. Palmer, J. Biol. Chem. 1969,
244, 1682 ± 1691.
[1] For excellent reviews of photocontrol of enzyme activities, see: a) I.
Willner, S. Rubin, Angew. Chem. 1996, 108, 419 ± 439; Angew. Chem.
Int. Ed. Engl. 1996, 35, 367± 385; b) I. Willner, Acc. Chem. Res. 1997,
30, 347± 356; c) T. P. Begley, Acc. Chem. Res. 1994, 27, 394 ± 401, and
references therein.
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