Gold electrodes wired for coupling with the deeply buried active site of arthrobacter globiformis amine oxidase

Article abstract of DOI:10.1021/ja029538q

Diethylaniline-terminated oligo(phenyl-ethynyl)-thiol (DEA-OPE-SH) wires on Au-bead electrodes facilitate electron tunneling to and from the deeply buried topaquinone (TPQ) cofactor in Arthrobacter globiformis amine oxidase (AGAO). Reversible cyclic volta

Full text of DOI:10.1021/ja029538q

Published on Web 05/22/2003
Gold Electrodes Wired for Coupling with the Deeply Buried Active Site of
Arthrobacter globiformis Amine Oxidase
Corinna R. Hess,† Gregory A. Juda,‡ David M. Dooley,‡ Ricky N. Amii,§ Michael G. Hill,*,§
Jay R. Winkler,† and Harry B. Gray*,†
Beckman Institute, California Institute of Technology, Pasadena, California 91125, Department of Chemistry,
Montana State UniVersity, Bozeman, Montana 59717, and Department of Chemistry, Occidental College,
Los Angeles, California 90041
Received December 1, 2002; E-mail: mgh@oxy.edu; hbgray@caltech.edu
Deeply buried enzyme active sites are difficult to study electro-
chemically, as the tunneling of electrons to internal redox centers
often is too slow to be observed even when redox mediators are
employed.1,2 The amine oxidases are a case in point: AOs catalyze
the conversion of amines to aldehydes using copper and an organic
cofactor, topaquinone (2,4,6-trihydroxyphenylalanine quinone, TPQ),
as redox centers.3-6 Accurate determination of the cofactor reduction
potentials is badly needed, as it is likely that electron transfers are
key steps in the AO catalytic mechanism.7 In the enzyme from
Arthrobacter globiformis (AGAO), the active center is only
accessible to substrates through a hydrophobic channel that is ∼20
Å deep.8 Because electronic coupling mediated by polypeptide or
water at this distance is expected to be very weak,9,10 the AGAO-
electrode kinetics would be sluggish at best.
To enhance electron tunneling to and from the AGAO active
site,11-29 we have synthesized a diethylaniline-terminated oligo-
(phenyl-ethynyl)-thiol (DEA-OPE-SH) wire to bind in the substrate
channel, thereby allowing TPQ to be coupled more strongly to an
electrode surface.
The synthesis, which involves a series of Pd cross-coupling
reactions, allows for modification of the headgroup and length of
the molecule, to match specific requirements of the protein.30
Because competitive AGAO inhibition studies have shown that
diethylaniline is a strong inhibitor of phenethylamine turnover,31
the DEA end of the wire will act as the protein-specific functional-
ity. With the opposite (thiol) end adsorbed on a Au surface, electron
tunneling through the bridge of repeating phenyl-ethynyl units to
the active site of wire-bound AGAO should be rapid.32,33
Figure 1. Cyclic voltammogram (black line) and background-subtracted
voltammogram (red line) for AGAO on Au-bead electrodes modified with
DEA-OPE-SH in 10 mM KPi, pH 7 (scan rate 100 mV/s). The background-
subtracted voltammogram is not to scale. Inset: peak current versus scan
rate.
broadening of the peaks. The linear dependence of peak current
on scan rate (Figure 1, inset) is in accord with expectation for a
protein-surface conjugate.
Gold-bead electrodes34 were soaked in millimolar solutions (1:1
CH2Cl2:CH3OH) of DEA-OPE-SH for ∼24 h to functionalize the
surface. Reductive stripping analyses of the resulting films indicated
∼70% coverage of wires on the gold surface.35 The modified
electrodes were subsequently incubated with AGAO for 24-48 h
to allow binding to the adsorbed wires. Cyclic voltammetry36 using
electrodes prepared in this manner showed a reversible reduction
at -140 mV versus SCE in phosphate buffer, pH 7 (Figure 1),
whereas electrodes modified with thiol wire alone gave no response
in this potential range. Background-subtracted voltammograms
recorded at slow scan rates (<20 mV/s) revealed anodic and
cathodic widths from ∼55 to 70 mV, with peak splittings ranging
from ∼30 to 50 mV.37 The wave shapes remained essentially
unchanged at scan rates up to 1 V/s, although there was a slight
The observed reduction potential, which is close to that reported
for quinone model complexes,38 varies linearly with pH; the slope
of ∼ -60 mV/pH indicates a 2e-, 2H+ TPQ reduction to the
hydroxyquinol. A 2e-, 3H+ reduction of related quinones occurs
between pH 4.5 and 8 in the absence of protein.38 Apparently, the
nearby Cu(II) center stabilizes the anionic form of the product
quinol, resulting in a lower pKa for the 4-hydroxy group in the
enzyme.
While AGAO coverages varied somewhat from electrode-to-
electrode, integration of the charge under the voltammetric peaks
† Beckman Institute.
‡ Montana State University.
§ Occidental College.
9
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J. AM. CHEM. SOC. 2003, 125, 7156-7157
10.1021/ja029538q CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
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Figure 2. DEA-OPE-SH modeled into the substrate channel of AGAO.
Channel residues are purple, TPQ is red, and the Cu site is blue.
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gave maximum surface concentrations on the order of ∼1 pmol/
cm2. On the basis of crystallographic parameters,8 this corresponds
to ∼25% monolayer coverage by the enzyme. Gradual loss of
electrochemical signals resulted from prolonged exposure of the
AGAO-modified electrodes to buffer solutions, possibly due to slow
dissociation of the protein from the wire-modified surface. Substrate
inhibition experiments have demonstrated that wires similar to
DEA-OPE-SH bind tightly to AGAO, with estimated dissociation
constants of ∼10 µM.31 Addition of micromolar phenethylamine
solutions completely quenches the electrochemical response in the
cell, providing further evidence that the enzyme specifically binds
to the electrode by insertion of the adsorbed thiol wire into the
substrate channel. As phenethylamine displaces DEA-OPE-SH from
this channel, the enzyme is decoupled from the electrode.
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AGAO is electroinactive at underivatized gold surfaces, high-
lighting the importance of wire interactions with the protein in
establishing electronic coupling with the active site (Figure 2).
Studies of electron tunneling through phenyl-alkynyl bridges in self-
assembled monolayers suggest that the distance decay constant is
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(30) Full synthetic details are in the Supporting Information.
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(34) The electrodes were prepared by heating a gold wire (0.5 mm diameter)
slowly in a hydrogen flame until a gold drop formed at the end of the
wire. AFM measurements revealed that the gold-bead surface was mainly
Au(111), with flat terraces extending 50-200 µm. The electrodes were
etched in boiling sulfuric acid for 30 min, and then electrochemically
cycled in 1 M H2SO4 between 1.5 and -0.3 V versus Ag/AgCl for 30
min (scan rate 20 mV/s); they were then sonicated for several minutes
prior to wire modification.
substantially lower (0.4-0.6 Å-1 32,33
than that for tunneling through
)
peptides (1.1 Å-1) or water (1.7 Å-1).9 Assuming a normal protein
reorganization energy (0.8 eV),39 we estimate ko > 4 × 104 s-1
(∆G° ) 0) for tunneling through the 22-Å wire; the corresponding
rate through polypeptide would be ∼3 s-1, and that through water
would be <10-4 s-1. Importantly, the CVs obtained at scan rates
up to 1 V/s place a lower limit of 103 s-1 for tunneling to the TPQ,
confirming that the DEA-wire is the coupling element at this
distance.
Acknowledgment. This work is dedicated to the memory of
Eraldo Antonini (per il ventesimo anniversario della sua morte, 19
Marzo 2003), a giant in metallobiochemistry. We thank R. Tanimura
and K. Niki for assistance in the preparation of Au-bead electrodes
and for helpful discussions. This work was supported by NIH
(C.R.H., J.R.W., H.B.G., G.A.J., D.M.D.) and the David and Lucille
Packard Foundation Initiative for Interdisciplinary Research (R.N.A.,
M.G.H.).
(35) Kakiuchi, T.; Sato, K.; Iida, M.; Hobara, D.; Imabayashi, S.; Niki, K.
Langmuir 2000, 16, 7238-7244.
(36) All electrochemical measurements were carried out using a BAS CV5-W
electrochemical analyzer. The electrochemical cell consisted of a small
volume (200-300 µL), three-electrode, two-compartment glass cell. A
saturated calomel electrode (SCE) served as the reference electrode, and
a Pt wire served as the auxiliary electrode. The reference compartment
was separated from the working solution by a modified Luggin capillary.
Measurements were made at ambient temperature in deoxygenated, 10
mM KPi, pH 6-8.
(37) The voltammetric wave widths are slightly broader than the expected
values for a two-electron couple, possibly because of inhomogeneities
within the films; the small peak separation at slow scan rates may reflect
similar inhomogeneity in electron-transfer rates. See, for example: Rowe,
G. K.; Carter, M. T.; Richardson, J. N.; Murray, R. W. Langmuir 1995,
11, 1797-1806. Alternatively, CV broadening could be due to a
breakdown in cooperativity resulting from stabilization of the semiquinone
radical in the enzyme.
Supporting Information Available: Details of protein preparation,
DEA-OPE-SH synthesis, binding studies, and electrochemical measure-
ments (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
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