вход по аккаунту


Direct Electron Transfer between Carbon Electrodes Immobilized Mediator and an Immobilized Viologen-accepting Pyridine Nucleotide Oxidoreductase.

код для вставкиСкачать
[2] J. Rohr. A. Zeeck in P. Prive (Ed.): Juhrbuch der Biofechnologie 1988189,
Carl Hanser Verlag, Miinchen 1988, p. 263.
[3] T. Widlanski. S . L. Bender, J. R. Knowles, J Am. Chem. SOC./ l / (1989)
2299 2300.
[4] H. Drdutz. H. Zihner,J. Rohr, A. Zeeck, J. Anrrbiot. 39(1986) 1657-1669.
[ 5 ] J. Rohr, .
Chem. Soc. Chem. Commun. 1989 492-493; J. Rohr, ibid. 1990
113- 114.
[6] J. Rohr, A. Zeeck. J. Anfrbior. 40 (1987) 459-467; J. Rohr. J. M. Beale,
H. G. Floss, ihid. 42 (1989) 1151-1157; J. Rohr, A. Zeeck. H. G. Floss,
ihrd. 41 (1988) 126-129.
[7] J. Rohr, J Anfibiot. 42 (1989) 1482-1488.
[8] M. Hino. M. Iwami, M. Okamoto. K. Yoshida, H. Harutd, M. Okuhara.
J. Hosoda, M. Koshaka, H. Aoki. H. Imanaka, J Aniibiof. 42 (1989)
3578-1583; M. Hino, T. Ando, S. Takase, Y Itoh, M. Okamoto, M.
Koshaka. H. Aoki. H. Imanaka, ibid. 42 (1989) 1584-1588; M . Hino, S.
Tdkase. Y. Itoh, I. Uchida, M. Okamoto, M. Koshaka, H.Aoki, H.
Imanaka. rhid. 42 (1989) 1589-1592.
[9] T. Okabe. B. D.Yuan, F. Isono, I. Sato, H. Fukazdwa, T. Nishimura, N .
Tanaka. J. Antrhrol. 38 (1985) 230-235; T. Okabe, K. Suzuki, H. Suzuki,
Y Inouye, S. Nakdmura, N. Tanaka, rbid. 39 (1986) 316-317.
[lo] B Krone, A. Zeeck, H. G. Floss, J Org. Chem. 42 (1982) 4721-4724.
[ l l ] W Keller-Schierlein. A. Geiger, H. Zahner, M. Brdndl, Helv. Chim. Acfu
71 (1988) 2058-2070; A. Geiger, W. Keller-Schierlein. M. Brdndl, H
ZHhner, J Anfibior. 41 (1988) 1542-1551
alents between viologens [Eq. (1); V = viologen] or Co-cage
complexes [Eq. (2); Co-diamser, cf. Fig. I] and pyridine nucle~tides.[~]
Viologen-accepting pyridine nucleotide oxidoreductases (VAPORS) are present in numerous cells.
+ NAD(P)O + He
NAD(P)H + 2V2@
2C02@-diamsar+ NAD(P)@ + Hm
NAD(P)H + 2Co3@-diarnsar(2)
The VAPOR from the thermophilic Bacillus spec.
DSM 466 was immobilized together with viologen, and then
the reactions shown in Scheme 1 were carried out. It was not
fixed to the electrode
Direct Electron Transfer between Carbon
Electrodes, Immobilized Mediator and
an Immobilized Viologen-accepting Pyridine
Nucleotide Oxidoreductase **
By Helmut Giinther, Antonios S . Paxinos, Michael Schulz,
Cees van Dijk, and Helmut Simon *
Scheme 1. Flow of electrons via immobilized viologens to an immobilized
enzyme that reduces soluble NAD.
our main concern to generate NAD(P)H in this way, but to
demonstrate the flow of electrons. The mediators employed
are shown in Figure 1. An NAD(P)H or NAD(P) consuming
reaction can be coupled on.
Dedicated to Professor Ivar Ugi
on the occasion of his 60th birthday
Electrons from an electrode constitute an inexpensive,
residue-free reagent for redox reactions whose redox potential can be adjusted over wide ranges to the desired reaction.
They could be especially valuable in combination with
specific redox enzymes. However, the direct transfer of electrons between redox active prosthetic groups of oxidoreductases and electrodes is far too slow for preparative conversions. Usually recourse to synthetic redox mediators, for
example viologens or metallo cage complexes, has to be
made in order to achieve satisfactory rates for the electron
transfer. Preparative electromicrobial and electroenzymatic
systems with dissolved mediator and dissolved enzyme"'
have been reported, as have also systems in which either the
mediator or the enzyme was immobilized on the electrode
surface.['. 31 For analytical purposes, a ferrocene derivative
was deposited on a graphite electrode and subsequently covalently bound to glucose oxidase.141 We have now tried to
bind both the mediator as well as the enzyme covalently to
a functionalized carbon surface in order to check whether
electrons flow from the cathode via the mediator, acting as
"molecular wire," to the prosthetic group of the enzyme.
For the immobilization, an enzyme discovered by us in
thermophilic bacilli was chosen which transfers redox equiv[*] Prof. Dr. H. Simon, Dr. H. Gunther, Dr. A. S. Praxinos,
DipLChem. M. Schulz
Lehrstuhl f i r Organische Chemie und Biochemie der Technischen Universitat Miinchen
LichtenbergstraBe 4, D-8046 Garching (FRG)
Dr. C van Dijk
Agrotechnical Research Institute
NL-6700 AA Wageningen (The Netherlands)
This work was supported by the European Community (Biotechnology
Action Program, Contract BAP 250D).
AnKen.. C'hrm. In[. Ed Engl. 29 (1990) No. 9
H2N -(CH2)3-~
HO oc -I C H ~ ) ~ - ~ = ~ - ( - - ( C ~ ~ ~ ) = C O O H
- diamsar
Fig. I . Mediators employed
Two routes were used for the immobilization: 1) covalent
binding of the mediator to the enzyme followed by covalent
binding of the modified enzyme to the functionalized carbon
electrode, and 2) covalent binding of the enzyme to an electrode modified with covalently bound bifunctional viologen.
DAPV was covalently bound to VAPOR from Bacillus
spec. DSM 466 (route 1) under various reaction conditions
using the carbodiimide method, and the activity of the so
Verlagsgeselkhuji mbH, 0-6940 Weinheim, 1990
0570-0833190j0909-1053 X3.50f ,2510
modified VAPOR was then checked in an optical test (Table
1). Upon covalent binding of DAPV to the enzyme with
carbodiimide, not only is the enzyme activity retained, or
even increased under certain conditions, but also the K,,,
value for the binding of NAD changes only negligibly compared to the unmodified enzyme from 0.054 mM to 0.07mM
(80 mol DAPV per mol VAPOR).
The immobilizates are, after a rapid drop to ca. 50 % of the
initial activity, very stable. The starting activity is ca. 40% of
the activity of the dissolved enzyme.
The functionalization of the glassy carbon electrode and
the covalent coating of the electrode surface with a bifunctional viologen will be described elsewhere.r61By varying the
sequence of the immobilization steps for viologen and
VAPOR, differently coated electrode surfaces can be prepared (Fig. 3). The electrodes were tested as cathodes in po-
Table 1. Enzyme activity in dependence of method of immobilization. EDC =
HCI. Solutions of DAPV in
10-100 pL of 0.1 N NaOH and of EDC in 100-500 pL of H,O were added to
5- 8 m L VAPOR in TRISiHCl buffer [TRIS = tris(hydroxymethy1)aniinomethanc] (0.02 M,pH 7.0) and adjusted to the respective pH with 0.1 N HCI, which
was kept constant during the reaction time of 6 min. Finally, unbound DAPV
and other low-molecular substances were removed by repeated ultrafiltration.
C [mM]
Spec Act.
[a] [YO]
1 .o
71 [bl
mol DAPV bound
per rnol VAPOR
1 .o
80 [cl
[a] Specific activity of the modified VAPOR compared to the native enzyme.
[b] With additional 8.6 pmol N-hydroxysuccinimlde. [c] The reaction time in
this case was 2.5 h.
Similar results are to be expected upon immobilization of
VAPOR on a carbon surface modified with viologens according to the carbodiimide method (route 2). This was also
indicated by experiments in which the stability of such immobilizates was checked. The respective viologen was first
covalently bound to carbon felt and thereafter a monomolecular layer of the enzyme was applied. A solution of
NAD and MV'@ (MV = N,N'-dimethyl-4,4'-bipyridinium)
was now pumped through the carbon felt and the NADH
formed was measured samplewise in an optical test with
L-lactate dehydrogenase (L-LDH, E.C 1.I .1.27) and pyruvate. The results for various viologens are shown in Figure
2. The non-linearity of the reaction rate is in this case mainly
determined by the progressive consumption of the starting
materials and the increasing concentration of the products.
t b
Fig. 2. NADH formation of the immobilized VAPOR as a function of time.
DAPV-DCPV, APCPV or APisoCPV was bound covalently to 50 mg carbon
felt (ca. 12 cm2, Type RVG 4000,Carbone Lorraine); a monomolecular layer
of VAPOR was then immobilized on the modified felt. For the test, a solution
of NAD (10 pmol) and MV'" (25 pmol) in potassium phosphate buffer (0.1 M,
pH 7 0) was circulated through the felt at 2.7 m L min-' for about 40 min.
VerlagsgesellschafimbH, 0-6940 Weinhelm. 1990
Fig. 3. Schematic representation of the various electrode surfaces. Electrode:
glassy carbon (SIGRADUR G), area 1 cm2. a) Functionalized surface.
b) DAPV layer bound via the carboxy groups of (a). c) Monomolecular VAPOR layer: the carboxy groups on an electrode of type (a) were activated by
2 pmol N-cyclohexyl-N'-[2-(N-methylmorpholino)ethyl]carbodiimide. p-toluenesulfonate (CMC) in 100 pL 0.1 M potassium phosphate buffer pH 4.5 for 2 h
and the so modified electrode treated with 1 U VAPOR in 100 pL 0.05 M TRIS/
HCI buffer pH 7.0.d) Multimolecular layer of VAPOR: as (c), but treated
simultaneously with 1 pmol CMC in 50 pL 0.1 M potassium phosphate buffer
pH 5.5 and the enzyme. e) Monomolecular layer of VAPOR, doped with
DAPV: as (c), then both the carboxy groups still available at the electrode
surface as well as the enzyme were re-activated and DAPV immobilized
thereon. f) Monomolecular layer of VAPOR, doped with DECV: as (c), then
DECV was activated with EDC in the molar ratio 1 :1.2 and transferred onto
the enzyme layer. After 7 min this solution was treated with an additional
2 pmol of DAPV. g) Monomolecular layer of VAPOR, immobilized on a
DAPV-DECV layer: as (b), then DECV was treated with EDC analogously to
(f) and, after renewed activation ofthe resulting DAPV-DECV layer with EDC,
VAPOR was added. h) Multimolecular layer of VAPOR on DAPV: as (b), then
1 U VAPOR in 100 pL 0.05M TRIS/HCl buffer pH 7.0 and 1 pmol CMC in
50 pL 0.1 M potassium phosphate buffer pH 5.5 added simultaneously. i) Multimolecular layer of VAPOR and DAPV, cross-linked by DAPV: as (b). then
1 U VAPOR in 100 pL 0.05 M TRISiHCl buffer pH 7.0,1 pmol EDC, and
1 pmol DAPV each in 50 pL 0.1 M potassium phosphate buffer pH 5.5added
tentiostatically-controlled, electrochemical cells as shown in
Scheme 1 . All experiments were carried out with strict exclusion of oxygen; anolyte and catholyte were therefore separated by a diaphragm. The initial rate of NADH formation
was determined by the bioluminescence method ['I using a
calibration curve (Fig. 4). The following conclusions can be
drawn: 1) all electrodes without VAPOR (a, b) or without
mediator (c, d) show no NADH formation (measured values
in the region of that of the blank). 2) Monomolecular layers
of VAPOR on a cathode with viologen layer reduce NAD to
NADH (e-g). 3) In the case of electrodes modified with
DAPV and coated with a multilayer of enzyme (h), the enzyme Layers reachable by the substrate no longer have ample
electronic contact with the electrode. 4) If, however, DAPV
is covalently incorporated in the enzyme layer in the case of
such an electrode, a modified electrode (i) is formed which
reduces NAD to biologically active NADH). 5 ) Charge and
bonding conditions influence the rate of reaction (e-i).
0570-0833/90j0909-1054$ 3 5 0 i ,2510
Angew Chem. Int. Ed. Engl. 29 (1990) No. 9
The rate of NADH formation decreases significantly over
a period of 20 h. The coupling of a reaction rapidly consuming NADH (Scheme 1) increases the rate and stabilizes the
reaction, even over a longer period of time. The coupled
reaction was the reduction of pyruvate to L-lactate (catalyzed by L-LDH). The lactate formation at the electrode of
type (i) was 148 nmol cm-' in 20 h compared to 13.5 nmol
cm-2 NADH in 19.5 h with a comparableelectrode without
coupling of an NADH-consuming reaction. In control experiments it was established that all the lactate originates
[I] H. Simon, J. Bader, H. Giinther, S. Neumann, J. Thanos, Angen, Chem. 97
(1985) 541-555; Angew. Chem. Inr. Ed. Engl. 24 (1985) 539-553.
[2] C. van Dijk, T. van Eijs, J. W. van Leeuwen, C. Veeger, FEES Lerr. 166
(1984) 76-80.
M. Laval, Biorerhnol. Eioeng. 3 / (1988) 553 [3] C. Bourdillon, R. Lortie, .I.
589, and references cited therein.
[4] A. E. G . Cass, G. Davls, G. D. Francis, H. A. 0. Hill, W. J. Aston, I J.
Higgins, E. V. Plotkin. L. D. L. Scott, A. P. F. Turner, A n d Chem. 56
(1984) 667-671.
[5] S. Nagata, R. Feicht, W Bette, H. Gunther, H. Simon, Eur. J. Appl. Microbiol. Biorechnol. 26 (1987) 263-267.
161 A. S. Paxinos, H. Gunther, D. J. M. Schmedding, H. Simon, unpublished.
[7] J. W. Hastings, Methods Enzymol. 57(1978) 125- 134; P. E. Stanley, ihid. S7
(1978) 215-222.
181 T. Eguchi, T. Iizuka, T. Kagotani, J H. Lee. 1. Urabe, H. Okada, Eur. J.
Biorhem. 15.5 (1986) 415-421.
[9] C. H. Hamann, W. Vielstich: Elekrrorhemie I I , Eleklrodenprozesse. Angenun& Elektrochemie, Verlag Chemie, Weinheim 1981, p. 265.
Dependence of the Magnetic Superexchange
on the Cr-S-CrDistance in
Dinuclear Chromium(I1) Complexes**
By Ursula Bossek, Karl Wieghardt,* Bernhard Nuber,
and Johannes Weiss
Fig. 4. Time course of NADH formation at carbon electrodes modified as
described in Figure 3. Electrode surface: 1 cm2 (glassy carbon), potential:
- 407 mV vs. NHE, 6 mL oxygen-free catholyte (TRIS/HCI buffer, 0.1 M,
pH 7.0); 18 pmol NAD. (a)-(I) as in Fig. 3). Bioluminescence-NADH test:
250 pL potassium phosphate buffer (25 mM, pH 7.0) contained 25 pmol dithiothreitol. 1.25 pg luciferase EC mU flavin mononucleotide (FMN)
oxidoreductase EC, 0.6 pmol FMN, 10 pg TRITON X-100, 5 pmol
myristinaldehyde, 150 pmol NAD, and 50 pL sample. Apparatus:
BIOLUMAT LB 9500T, Berthold, D-7547 Wildbad (FRG).
from the NADH-dependent LDH-catalyzed reaction. A further experiment was carried out with such an electrode in
order to clarify the influence of the NADH decomposition
during the 19.5 h. First the NADH formation was measured
over 90 min according to the bioluminescence method, then,
after addition of pyruvate (20 pmol) and L-LDH (7U), the
increase in lactate was determined after a further 17 h. The
NADH formation slowed down in the first 30 min from
3.6 nmol cm-' h - ' to 1.2 nmol cm-2 h-', but the lactate
formation was 9 nmol cm-2 h - ' over 17 h. In a further
analogous experiment with the same electrode the lactate
formation was 8.4 nmol cm-' h-' after 2 days.
The higher lactate formation compared to the NADH
formation in the absence of an NADH-consuming reaction
can be explained in terms of the pyrurate/lactate diffusing
more rapidly than NAD/NADH and of an absorption of
LDH at the electrode.18'The NAD concentration in the reaction layer is also increased through the dehydrogenation of
the NADH.
According to the above results, current densities lying in
the lower range of the current densities of industrial processes can be expected on using carbon felts with larger internal
surfaces (referred to the macroscopic surface).[g1
Received: May 17. 1990 [Z 3963 IE]
German version: Angew. Chem. 102 (1990) 1075
Angels Chem. h i . Ed. Engl. 29 (1990) N o . 9
The spins of the unpaired electrons of a transition metal
ion in a dinuclear complex can be oriented intramolecularly
parallel (ferromagnetism) or antiparallel (antiferromagnetism) through the mediation of bridged, non-magnetic anions. This phenomenon is referred to as superexchange."]
Generally speaking, the extent of this spin-spin coupling is
satisfactorily described by the isotropic Heisenberg-DiracvanVleck model (HDVV model), whereby the form of the
isotropic spin-Hamilton operator is given by H = - 2JS,S2,
where J is the exchange coupling constant (in cm- I ) and S ,
and S , give the total spin of the metal ions 1 and 2, respectively; J is negative in the case of antiferromagnetic and
positive in the case of ferromagnetic spin coupling. The question of whether J is a function of the distance r between the
two paramagnetic centers in a bridged, dinuclear complex is
still a matter of controversy.['] Experimentally most convincing is such a dependence with - J = r - 1 2 in the case of
solid-state structures of the type XMF, and X,MF, (X = K,
Rb, TI; M=Mn", Co", Ni").[2bJHowever, the M . - - M distances vary only by ca. 0.020 A. Such a dependence on distance has not been observed for the intramolecular spin coupling in dinuclear complexes, since the distance between the
metal ions changes with changing type of bridge ligand, and
this results in the superexchange mechanism changing.
We have now synthesized dinuclear Cr"'(d3) complexes
containing H-bridges as common structural feature:
. . - H . . . -CRi"
R =aliphatic residue or H
The Cr.. .Cr distance varies because two octahedrally coordinated Cr"' ions are coupled intramolecularly to each
Prof. Dr. K. Wieghardt, U. Bossek
Lehrstuhl fur Anorganische Chemie I der Universitit
Postfach 10 21 48, D-4630 Bochum 1 (FRG)
Dr. B. Nuber, Prof Dr. J. Weiss
Anorganisch-chemisches Insitut der Universitdt
Im Neuenheimer Feld 270, D-6900 Heidelberg (FRG)
This work was supported by the Fonds der Chemischen Industrie
Verlagsgesellschafl mbH, 0-6940 Weinheim, 1990
$3.50+ ,2510
Без категории
Размер файла
351 Кб
electrode, pyridin, immobilized, nucleotide, accepting, direct, transfer, viologen, electro, mediators, oxidoreductase, carbon
Пожаловаться на содержимое документа