Oxidation of benzyl alcohol with Cu(II) and Zn(II) complexes of the phenoxyl radical as a model of the reaction of galactose oxidase

Article abstract of DOI:10.1002/(SICI)1521-3773(19990917)38:18<2774::AID-ANIE2774>3.0.CO;2-E

Profound insights into the catalytic mechanism of galactose oxidase (GO) are offered by new models of the active form of the metalloenzyme. The important role of the Cu(II) center in the oxidation of benzyl alcohol to benzaldehyde by the Cu(II)-phenoxyl radical complex of ligand 1 has been revealed by comparison with the reactivity of the corresponding Zn(II)- phenoxyl radical complex; py = 2-pyridyl.

Full text of DOI:10.1002/(SICI)1521-3773(19990917)38:18<2774::AID-ANIE2774>3.0.CO;2-E

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residue (Tyr272), which is directly coordinated to a Cu2‡ ion
at the equatorial position, is covalently bound to the sulfur
atom of the adjacent Cys228, constituting a new organic
cofactor (Scheme 1).[2] The active form of GO has recently
been suggested to be the CuII ± phenoxyl radical of this
organic cofactor, which is converted into the two-electron
reduced species [CuI(phenol)] in the oxidation of alcohols to
aldehydes.[3±6]
[13] K. H. Schündehütte in Houben-Weyl, Methoden Org. Chem. (Hou-
ben-Weyl) 4th ed. 1965 ± , Vol. 10/3, p. 240.
[14] J. Wachtveitl, T. Nägele, B. Puell, W. Zinth, M. Krüger, S. Rudolph-
Böhner, D. Oesterhelt, L. Moroder, J. Photochem. Photobiol. A 1997,
105, 283 ± 288.
[15] T. Nägele, R. Hoche, W. Zinth, J. Wachtveitl, Chem. Phys. Lett. 1997,
272, 489 ± 495.
[16] L. Moroder, D. Besse, H.-J. Musiol, S. Rudolph-Böhner, F. Siedler,
Biopolym. Pept. Sci. 1996, 40, 207 ± 234.
[17] J. Kuriyan, T. S. R. Krishna, L. Wong, B. Guenther, A. Pahler, C. H.
Williams, Jr., P. Moldel, Nature 1991, 352, 172 ± 174.
[18] J. Wachtveitl, T. Nägele, A. J. Wurzer, M. Schenk, L. Moroder in
Springer Series in Chemical Physics, Vol. 63 (Eds.: T. Elsaesser, J. G.
Fujimoto, D. A. Wiersma, W. Zinth), Springer, Stuttgart, 1998,
pp. 609 ± 611.
[19] K. Wüthrich, NMR of Proteins and Nucleic Acids, Wiley, New York,
1986.
[20] A. Bax, D. G. Davis, J. Magn. Reson. 1985, 65, 355 ± 360.
[21] J Jeener, B. H. Meier, P. Bachman, R. R. Ernst, J. Chem. Phys. 1979,
71, 4546 ± 4553.
[22] M. Rance, O. W. Sorensen, G. Bodenhausen, G. Wagner, R. R. Ernst,
K. Wüthrich, Biochem. Biophys. Res. Commun. 1983, 117, 479 ± 485.
Scheme 1. Representation of the coordination at the CuII center of
galactose oxidase (left; the unlabeled O atom is from an acetate ion or
water), the organic cofactor (center), and the protonated form of the ligand
used in the model complexes 1 (right; py ˆ 2-pyridyl).
Extensive efforts have been made recently to develop
synthetic model complexes that can mimic the catalytic
function and the spectroscopic characteristics of the CuII ±
phenoxyl radical species of the native enzymes.[7±10] Wieghardt
et al. have succeeded in developing a very unique functional
model of GO using 2,2'-thiobis(4,6-di-tert-butylphenol) (LH2)
as the ligand; this complex can oxidize alcohols under aerobic
conditions.[7b] In this system the active species has been shown
Oxidation of Benzyl Alcohol with CuII and ZnII
Complexes of the Phenoxyl Radical as a Model
of the Reaction of Galactose Oxidase**
Shinobu Itoh,* Masayasu Taki, Shigehisa Takayama,
Shigenori Nagatomo, Teizo Kitagawa, Norio Sakurada,
Ryuichi Arakawa, and Shunichi Fukuzumi*
.
to be the dimeric phenoxyl radical complex [CuI2I(L )2]2‡, in
Redox interactions between a transition metal ion and a
redox-active amino acid side chain such as the phenol group
of tyrosine have been recognized to play a crucial role in
several biologically important processes.[1] Recently, a tyrosyl
radical directly coordinated to a CuII center has been
discovered in the active site of galactose oxidase (GO,
EC 1.1.3.9), which catalyzes the oxidation of d-galactose and
primary alcohols to the corresponding aldehydes, coupled to
the reduction of O2 to H2O2 [Eq. (1)].[1, 2] The crystal structure
of GO at 1.7-Š resolution clearly shows that the tyrosine
which only the radicals act as oxidation sites and no redox
reaction occurs at the CuII site. Now the important question
remains as to the essential role of the CuII site.
We report herein two synthetic model complexes whose
structures and spectroscopic features are similar to those of
the active form of GO. One is a CuII ± phenoxyl radical
complex that can accept two electrons, and the other is a
ZnII ± phenoxyl radical complex that can accept only one
electron. To our surprise, both model complexes can oxidize
benzyl alcohol to yield the same product, benzaldehyde. The
important difference between the two model complexes is
found to lie in the different kinetic formulation for the
oxidation of the alcohol. These features of our model
complexes provide valuable insights into the catalytic mech-
anism of GO.
RCH2OH ‡ O2 ! RCHO ‡ H2O2
(1)
[*] Dr. S. Itoh, Prof. S. Fukuzumi, M. Taki, S. Takayama
Department of Material and Life Science
Graduate School of Engineering, Osaka University
2 ± 1 Yamada-oka, Suita, Osaka 565-0871 (Japan)
Fax: (‡81)6-6879-7370
Addition of (NH4)2[CeIV(NO3)6] (ceric ammonium nitrate,
CAN) to a solution of the dimeric copper(ii) ± phenolate
complex [CuI2I(1)2](PF6)2 (2.5 Â 10 4 m in CH3CN)[11] caused an
immediate decrease in the absorption band at 522 nm due to
the dimeric copper(ii) ± phenolate complex (ligand-to-metal
charge-transfer (LMCT) transition from the phenolate to the
Cu2‡ ion), accompanied by the concomitant increase in the
E-mail: fukuzumi@chem.eng.osaka-u.ac.jp
Dr. S. Nagatomo, Prof. T. Kitagawa
Institute for Molecular Science
Myodaiji, Okazaki 444-8585 (Japan)
N. Sakurada, Prof. R. Arakawa
Department of Applied Chemistry
Faculty of Engineering, Kansai University
3-3-35 Yamate-cho, Suita, Osaka 564-8680 (Japan)
1
new absorption bands at 415 nm (e ˆ 1790m 1 cm ) and
1
867 nm (e ˆ 550m 1 cm ) at 258C (Figure 1). A similar
[**] This study was financially supported in part by a Grant-in-Aid for
Scientific Research on Priority Area (Molecular Biometallics:
10129218; Electrochemistry of Ordered Interface: 10131242) and a
Grant-in-Aid for General Scientific Research (08458177) from the
Ministry of Education, Science, Culture, and Sports of Japan.
spectrum was obtained in the oxidation of [ZnII(1)-
[11]
(CH3CN)]PF6
by CAN under the same experimental
conditions (418 nm (e ˆ 1250m 1 cm ) and 887 nm (e ˆ
1
510m 1 cm ) in CH3CN at 258C). The absorption bands in
1
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silent. Furthermore, ligand 1-H was recovered almost quanti-
tatively after workup with aqueous NH3, in contrast to the
case of self-decomposition.[15] Quantitative formation of
benzaldehyde was confirmed by GC-MS.[16] The rate of the
oxidation of benzyl alcohol to benzaldehyde obeys pseudo-
first-order kinetics, and the plot of the pseudo-first-order rate
constant kobs(Cu) versus the concentration of benzyl alcohol
gives a straight line (Figure 2a). The second-order rate
Figure 1. UV/Vis spectra of [CuI2I(1)2](PF6)2 (2.5 Â 10 4 m in CH3CN,
.
‡
dotted line) and [CuII(1 )(NO3)] (solid line) generated from the former
by adding CAN (5.0 Â 10 4 m) at 258C. Inset: Time course of the absorption
change at 867 nm due to the formation and decomposition of
.
‡
[CuII(1 )(NO3)] .
the resulting spectra are very similar to those of the phenoxyl
radical species of 2-methylthio-p-cresol (lmax ˆ 400 and
830 nm)[12, 13] and of the active form of GO (445 and ca.
800 nm).[3] This indicates that the characteristic absorption
bands obtained by the CAN oxidation are due to the phenoxyl
.
radical species (1 ) of the ligand. Such an absorption band in
the long-wavelength region (above 800 nm), a characteristic
of the active form of GO, has never been observed for other
synthetic CuII± and ZnII ± phenoxyl radical complexes report-
ed so far except one recent example described by Halcrow
et al.[10]
Further evidence in support of the formation of
.
‡
[MII(1 )(NO3)] (M ˆ Cu or Zn) was obtained from the
1
resonance Raman spectra (nÄ ˆ 1512, 1589 cm for M ˆ Cu;
1
nÄ ˆ 1517, 1595 cm for M ˆ Zn), the electrospray ionization
mass spectra (ESI-MS),[14] and the ESR spectra of the
solutions resulting from the CAN oxidation of the corre-
sponding phenolate complexes (ESR-silent at 1968C in
CH3CN for M ˆ Cu; phenoxyl radical at g ˆ 2.0052 at 808C
in CH3CH2CN for M ˆ Zn). The isotropic hyperfine coupling
Figure 2. a) Plot of (kobs(Cu) kdec(Cu)) versus the concentration of benzyl
.
‡
alcohol for the oxidation of benzyl alcohol by [CuII(1 )(NO3)] in CH3CN
at 258C. Inset: Pseudo-first-order plot for the oxidation; y ˆ ln(A A1).
b) Plot of (kobs(Zn) kdec(Zn)) versus the concentration of benzyl alcohol for
.
‡
the oxidation of benzyl alcohol by [ZnII(1 )(NO3)] in CH3CN at 258C.
constants
(a2-SMe ˆ 3.72 G,
a3-H ˆ 1.15 G,
a5-H ˆ 0.87 G,
.
.
aCH(benzyl) ˆ 2.43 G) of [ZnII(1 )(NO3)] determined by com-
puter simulation indicate that a significant amount of the spin
density is delocalized into the methylthio group, as in the case
of metal-free phenoxyl radicals.[12] As a result, the structures
and spectroscopic characteristics have been found to be
almost the same for the CuII± and the ZnII ± phenoxyl radical
complexes.
‡
Inset: Second-order plot for the oxidation; y ˆ (A0 A) [Zn(1 )] (A
/
0
4
A1) Â 10
.
constant k(Cu) was obtained from the slope as 4.79 Â
1
10 3 m 1 s . A large kinetic deuterium isotope effect (kH
/
(Cu)
kD ˆ 6.8) was obtained when PhCH2OH was replaced by
(Cu)
PhCD2OH, indicating that the abstraction of the benzylic
hydrogen atom is involved in the rate-determining step.
The same product, benzaldehyde, was also obtained in the
The CuII ± phenoxyl radical complex is relatively stable at
ambient temperature, but gradually decomposes to obey first-
.
order kinetics (kdec(Cu) ˆ 1.62  10 3 s , see inset in Fig-
reaction of benzyl alcohol and [ZnII(1 )(NO3)] .[16] In contrast
1
‡
.
ure 1).[15] Addition of a large excess of benzyl alcohol to a
to the case of [CuII(1 )(NO3)] , however, the reaction of the
zinc ± phenoxyl radical complex with benzyl alcohol obeys
second-order kinetics with respect to the decay of the
absorption at 887 nm, and the second-order rate constant
kobs(Zn) shows the second-order dependence on the alcohol
concentration (Figure 2b).[17] Furthermore, the yield of benz-
aldehyde was nearly half that of the copper case.
‡
.
solution of [CuII(1 )(NO3)] in CH3CN accelerates the decay
‡
rate of the phenoxyl radical complex. Formation of [CuI(1-
‡
H)] as a final product was evident, since a) there were no
characteristic absorption bands due to the LMCT and ligand
field excitation (d ± d) of the CuII complex in the final
spectrum of the reaction, and b) the final product was ESR-
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complex solution) at 258C. The rate constants (kobs) for the redox reaction
were also determined by following the decrease in the absorption due to the
phenoxyl radical. The yield of benzaldehyde was determined by GC-MS
with mesitylene as an internal standard.
These results clearly show that the oxidation of benzyl
.
‡
alcohol by [CuII(1 )(NO3)] proceeds by means of coordina-
tion of benzyl alcohol in the monomeric form by formally a
‡
2e /2H mechanism to give benzaldehyde and [CuI(1-H)]
quantitatively (Scheme 2a (type I)). On the other hand, no
redox reaction is expected at the ZnII site. In such a case, two
Received: February 1, 1999
Revised version: April 13, 1999 [Z12979IE]
German version: Angew. Chem. 1999, 111, 2944 ± 2946
Keywords: alcohols ´ copper ´ galactose oxidase ´ kinetics ´
phenoxyl complexes
[1] J. Stubbe, W. A. van der Donk, Chem. Rev. 1998, 98, 705 ± 762, and
references therein.
[2] a) N. Ito, S. E. V. Phillips, C. Stevens, Z. B. Ogel, M. J. McPherson,
J. N. Keen, K. D. S. Yadav, P. F. Knowles, Nature 1991, 350, 87 ± 90;
b) N. Ito, S. E. V. Phillips, K. D. S. Yadav, P. F. Knowles, J. Mol. Biol.
1994, 238, 794 ± 814.
[3] M. M. Whittaker, D. P. Ballou, J. W. Whittaker, Biochemistry, 1998, 37,
8426 ± 8436, and references therein.
[4] R. M. Wachter, M. P. Montague-Smith, B. P. Branchaud, J. Am. Chem.
Soc. 1997, 119, 7742 ± 7749, and references therein.
[5] a) K. Clark, J. E. Penner-Hahn, M. M. Whittaker, J. W. Whittaker, J.
Am. Chem. Soc. 1990, 112, 6433 ± 6434; b) K. Clark, J. E. Penner-
Hahn, M. M. Whittaker, J. W. Whittaker, Biochemistry 1994, 33,
12553 ± 12557.
[6] Such a copper± phenoxyl radical motif has been also found as the
catalytic form of glyoxal oxidase from Phanerochaete chrysosporium
and in the prokaryotic FbfB protein: a) P. J. Kersten, Proc. Natl. Acad.
Sci. USA 1990, 87, 2936 ± 2940; b) M. M. Whittaker, P. J. Kersten, N.
Nakamura, J. Sanders-Loehr, E. S. Schweizer, J. W. Whittaker, J. Biol.
Chem. 1996, 271, 681 ± 687; c) P. Bork, R. F. Doolittle, J. Mol. Biol.
1994, 236, 1277 ± 1282.
[7] a) A. Sokolowski, J. Müller, T. Weyhermüller, R. Schnepf, P.
Hildebrandt, K. Hildengrand, E. Bothe, K. Wieghardt, J. Am. Chem.
Soc. 1997, 119, 8889 ± 8900; b) P. Chaudhuri, M. Hess, U. Florke, K.
Wieghardt, Angew. Chem. 1998, 110, 2340 ± 2343; Angew. Chem. Int.
Ed. 1998, 37, 2217 ± 2220.
Scheme 2. Type I (a) and type II mechanisms (b) for the oxidation of
benzyl alcohol by the copper and zinc model complexes, respectively. (The
complexes are given without their charges.)
[8] a) J. A. Halfen, V. G. Young, Jr., W. B. Tolman, Angew. Chem. 1996,
108, 1832 ± 1835; Angew. Chem. Int. Ed. Engl. 1996, 35, 1687 ± 1690;
b) J. A. Halfen, B. A. Jazdzewski, S. Mahapatra, L. M. Berreau, E. C.
Wilkinson, L. Que, Jr., W. B. Tolman, J. Am. Chem. Soc. 1997, 119,
8217 ± 8227.
[9] a) Y. Wang, T. D. P. Stack, J. Am. Chem. Soc. 1996, 118, 13097 ± 13098;
b) Y. Wang, J. L. DuBois, B. Hedman, K. O. Hodgson, T. D. P. Stack,
Science 1998, 279, 537 ± 540.
[10] M. A. Halcrow, L. M. L. Chia, X. Liu, E. J. L. McInnes, L. J. Yellow-
lees, F. E. Mabbs, J. E. Davies, Chem. Commun. 1998, 2465 ± 2466.
[11] Details of the synthetic procedures for the ligand and the CuII and ZnII
complexes as well as the crystal structures and their physicochemical
characteristics will be reported elsewhere. The structure and phys-
icochemical data of a related CuII ± phenolate complex have been
reported in ref. [12].
[12] S. Itoh, S. Takayama, R. Arakawa, F. Furuta, M. Komatsu, A. Ishida, S.
Takamuku, S. Fukuzumi, Inorg. Chem. 1997, 36, 1407 ± 1416.
[13] M. M. Whittaker, Y.-Y. Chuang, J. W. Whittaker, J. Am. Chem. Soc.
1993, 115, 10029 ± 10035.
electrons can only be accepted by phenoxyl radical sites in a
dimeric form (Scheme 2b (type II)). Thus, the reaction obeys
.
second-order kinetics in the case of [ZnII(1 )(NO3)] . The
latter mechanism of the zinc complex is very close to that of
the system of Wieghardt et al. mentioned previously.[7b] The
enzymatic reaction has so far been considered to proceed by
the type I mechanism. Thus, the different kinetic formulations
‡
.
.
‡
‡
of [CuII(1 )(NO3)] and [ZnII(1 )(NO3)] disclosed in this
study clearly demonstrate the importance of the redox cycle
between CuI and CuII as well as the interconversion between
the phenol and phenoxyl radical states for the efficient two-
electron oxidation of alcohols at the mononuclear copper site
of GO, which is fixed in the protein matrix.
Experimental Section
[14] Coordination of NO3 which comes from CAN was confirmed by ESI-
MS.
[15] Self-decomposition of the phenoxyl radical species gave a complicated
mixture of products. An ESI mass spectrum of the resulting mixture
indicated the formation of some S-oxygenated and N-dealkylated
products of the ligand.
Kinetic studies and product analysis. The phenoxyl radical species of the
CuII and ZnII complexes were generated in situ by adding an equimolar
amount of (NH4)2[CeIV(NO3)6] (CAN, 5.0 Â 10 4 m) to a deaerated solution
of [MI2I (1)2](PF6)2 (M ˆ Cu or Zn,[11] 2.5  10 4 m in CH3CN) (M ˆ Cu or
Zn)[11] in a UV cell (1-cm path length, sealed tightly with a silicon cap) at
258C. The rate constants for the self-decomposition reaction (kdec) of the
phenoxyl radical complexes were determined by following the decrease in
the absorption band due to the phenoxyl radical.
[16] Benzaldehyde was the only product detected by GC-MS, and no C C
coupling products such as hydrobenzoin [PhCH(OH)CH(OH)Ph]
were observed.
.
‡
The oxidation of benzyl alcohol by the phenoxyl radical species was
initiated by adding an excess amount of benzyl alcohol to the CH3CN
solution, after the absorption of the phenoxyl radical species had reached a
maximum (a few minutes after the addition of CAN to the phenolate
[17] Self-decomposition of [ZnII(1 )(NO3)] also obeys second-order
kinetics.
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