Electrocatalytic hydrocarbon hydroxylation by ethylbenzene dehydrogenase from Aromatoleum aromaticum

Article abstract of DOI:10.1021/jp512562k

We report the electrocatalytic activity of ethylbenzene dehydrogenase (EBDH) from the β-proteobacterium Aromatoleum aromaticum. EBDH is a complex 155 kDa heterotrimeric molybdenum/iron-sulfur/heme protein which catalyzes the enantioselective hydroxylation of nonactivated ethylbenzene to (S)-1-phenylethanol without molecular oxygen as cosubstrate. Furthermore, it oxidizes a wide range of other alkyl-substituted aromatic and heterocyclic compounds to their secondary alcohols. Hydroxymethylferrocenium (FM) is used as an artificial electron acceptor for EBDH in an electrochemically driven catalytic system. Electrocatalytic activity of EBDH is demonstrated with both its native substrate ethylbenzene and the related substrate p-ethylphenol. The catalytic system has been modeled by electrochemical simulation across a range of sweep rates and concentrations of each substrate, which provides new insights into the kinetics of the EBDH catalytic mechanism.

Full text of DOI:10.1021/jp512562k

Subscriber access provided by MCGILL UNIV
Article
Electrocatalytic Hydrocarbon Hydroxylation by Ethylbenzene
Dehydrogenase from Aromatoleum Aromaticum
Palraj Kalimuthu, Johann Heider, Daniel Knack, and Paul Vincent Bernhardt
J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp512562k • Publication Date (Web): 30 Jan 2015
Downloaded from http://pubs.acs.org on February 2, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
The Journal of Physical Chemistry B is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 24
The Journal of Physical Chemistry
1
2
3
4
5
6
7
8
Electrocatalytic Hydrocarbon Hydroxylation by Ethylbenzene
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Dehydrogenase from Aromatoleum aromaticum
Palraj Kalimuthu,1 Johann Heider2, Daniel Knack2 and Paul V. Bernhardt1,*
1School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, 4072, Australia
2Laboratory for Microbial Biochemistry and Synmikro Center for Synthetic Microbiology, Philipps
University Marburg, 35043 Marburg, Germany
E-mail: p.bernhardt@uq.edu.au
Abstract
We report the electrocatalytic activity of ethylbenzene dehydrogenase (EBDH) from the β-
proteobacterium Aromatoleum aromaticum. EBDH is
a complex 155 kDa heterotrimeric
molybdenum/iron-sulfur/heme protein which catalyses the enantioselective hydroxylation of non-
activated ethylbenzene to (S)-1-phenylethanol without molecular oxygen as co-substrate. Furthermore,
it oxidizes a wide range of other alkyl-substituted aromatic and heterocyclic compounds to their
secondary alcohols. Hydroxymethylferrocenium (FM) is used as an artificial electron acceptor for EBDH
in an electrochemically driven catalytic system. Electrocatalytic activity of EBDH is demonstrated with
both its native substrate ethylbenzene and the related substrate p-ethylphenol. The catalytic system has
been modelled by electrochemical simulation across a range of sweep rates and concentrations of each
substrate, which provides new insights into the kinetics of the EBDH catalytic mechanism.
1
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 2 of 24
1
2
3
4
5
Introduction
Molybdenum is an essential element for most life forms as it is required by enzymes catalyzing
redox reactions relevant to carbon, sulfur and nitrogen metabolism.1-3 Apart from the multinuclear
enzyme nitrogenase, Mo is bound to the bidentate molybdopterin ligand cofactor (Scheme 1) where it is
involved in coupled two-electron, O-atom transfer reactions of a range of substrates.3 The relevant
metal centred oxidation state changes are either O=MoVI → MoIV (substrate oxidation) or MoIV →
O=MoVI (substrate reduction), with the oxido ligand often, but not always, exchanged with the substrate.
On the basis of their sequences and of structural differences at the active site Mo enzymes are classified
into three major families such as xanthine oxidase family, sulfite oxidase family and DMSO reductase
family.4 Dependent on the enzyme family, either one or two molybdopterins may be bound per Mo, and
the dithiolene chelating group(s) is (are) essential in tuning the redox properties of the respective active
sites.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
One of the more recently discovered Mo-enzymes is ethylbenzene dehydrogenase (EBDH)
isolated from the β-proteobacterium Aromatoleum aromaticum. which is involved in the direct
anaerobic oxidation of non-activated ethylbenzene to 1-(S)-phenylethanol.5-8 Furthermore, EBDH
catalytically converts a broad spectrum of aromatic and heterocyclic compounds with ethyl or propyl
substituents to their secondary alcohols with high enantioselectivity and thus it is potentially useful in
the generation of precursors to fine chemicals.9-11 It is the first known enzyme capable of the direct
anaerobic oxidation of non-activated hydrocarbons.8
2
ACS Paragon Plus Environment

Page 3 of 24
The Journal of Physical Chemistry
1
2
3
4
5
6
7
8
9
FS0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
FS2
Mo
FS4
FS1
heme
FS3
Figure 1. Crystal structure of EBDH (PDB code 2IVF)12 showing the seven redox active cofactors.
EBDH consists of three subunits of molecular masses of 96, 43 and 23 kDa. The largest subunit
contains the Mo active site ligated by two molybdopterin guanine dinucleotides and an Fe-S cluster (FS0,
Fig. 1). The middle subunit bears three [4Fe-4S] clusters (FS1-FS3) and a single [3Fe-4S] cluster (FS4)
while the smallest subunit contains a heme b.12 EBDH belongs to the DMSO reductase family and
exhibits high sequence similarities with other enzymes such as selenate reductase from Thauera
selenatis, dimethylsulfide dehydrogenase from Rhodovulum sulfidophilum and chlorate reductase from
Dechloromonas sp.4, 13-15 These enzymes belong to a group collectively known as the complex iron sulfur
molybdoenzymes (CISMs).16 In this case the two electrons resulting from ethylbenzene oxidation at the
Mo active site flow (one at a time) from left to right as shown in Figure 1 via the five Fe-S clusters to the
heme.
3
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 4 of 24
1
2
3
4
5
6
7
8
9
The crystal structure of EBDH also revealed a relatively large active site cavity which permitted
entry of several aromatic substrates containing a variety of substituents in different positions.
Unsaturated substituents and more bulky bicyclic compounds such as p-ethylbiphenyl and 2-
ethylnaphthalene are also active substrates. 9, 12, 17
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Asp223
S
O
CH3
O
S
Asp223
CH3
H
MoVI
H
H
S
H
O
ethylbenzene
H
S
S
O
N
O
S
CH3
MoV
S
N
S
HO
His192
H
N
H
Asp223
O
N
S
MoVI
S
O
S
S
His192
O
H
N
Asp223
N
His192
S
O
O
2H+ 2e-
S
,
MoIV
Asp223
S
CH3HO
S
H
S
MoIV
O
H
N
O
S
H
2O
S
N
H
2O
His192
S
H
N
CH3
N
OH
-1-phenylethanol
H
His192
S
Scheme 1. Proposed catalytic mechanism18, 19 of ethylbenzene hydroxylation by EBDH.
The catalytic mechanism of Mo containing enzymes are generally associated with oxido ligand
transfer reactions in which an O-atom is exchanged directly between the Mo active site and the
substrate coupled with 2-electron transfer with the metal.4, 20-23 In contrast, the proposed EBDH catalytic
mechanism is more complicated.10, 18 Based on quantum chemical modeling and embedding this model
4
ACS Paragon Plus Environment

Page 5 of 24
The Journal of Physical Chemistry
1
2
into the EBDH structure (QM/MM model), the mechanism shown in Scheme 1 was proposed.10, 17, 19, 24, 25
The Mo active site can be reached through the 25 Å long tunnel shaped pocket lined by mostly non-
polar residues.12 On the basis of crystallographic evidence Asp223 is coordinated to the Mo ion and
His192 is a potential H-bond donor to the predicted oxido ligand in oxidized EBDH.10, 18 The proposed
mechanism involves H-abstraction generating an intermediate comprising an ethylbenzyl radical and a
hydroxido-coordinated MoV ion followed by a second single electron transfer from the radical to MoV to
generate a second intermediate involving MoIV and a ethylbenzyl secondary carbocation. The OH- ligand
rebounds by combining with the adjacent carbocation to form the product 1-(S)-phenylethanol. The
enantioselectivity of this transformation is typically very high across a number of related substrates.9, 10
The redox potentials of EBDH have been determined26 and they follow the same trend reported
for the highly homologous DMS dehydrogenase (R. sulfidophilum).27 The highest potential cofactor is the
heme (Eo = +255 mV vs NHE)26 to which electrons are ultimately drawn after substrate oxidation,
although several of the intermediate redox transitions via the Fe-S clusters are endergonic. The heme is
clearly the site of electron egress from EBDH and it is this site that interacts with electron acceptors both
in vivo and in synthetic systems. The ferrocenium ion has been found to be an effective synthetic
electron partner for EBDH in solution assays.6, 9
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Coupling of the EBDH catalytic reaction to an electrochemically regenerated electron acceptor
has recently been reported to allow bulk scale hydroxylation.11 In the present study, we report the
mediated electrocatalytic voltammetry of EBDH with two different substrates, namely ethylbenzene
and p-ethylphenol, in the presence of the synthetic redox mediator hydroxymethylferrocene
(ferrocenemethanol, FMox/red, +420 mV vs NHE, Scheme 2) employing a Au working electrode modified
with the heterocyclic thiol 5-(4’-pyridinyl)-1,3,4-oxadiazole-2-thiol (Hpyt). Digital simulation was
employed to explore the kinetics of the coupled homogeneous chemical reactions at a variety of sweep
5
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 6 of 24
1
2
3
4
5
6
rates as well as different substrates concentrations and it provides new insights to the EBDH catalytic
mechanism.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
ACS Paragon Plus Environment

Page 7 of 24
The Journal of Physical Chemistry
1
2
3
4
5
Experimental Section
6
7
8
9
Materials. EBDH was purified from cells of strain EbN1 (Aromatoleum aromaticum) grown on
ethylbenzene as previously described.5 Ferrocene methanol (FM) was synthesized by based on the
previously reported procedure28 in which commercially available ferrocene carbaldehyde is reduced by
NaBH4 in methanol, washed with water extracted into chloroform and evaporated to dryness.
Ethylbenzene, p-ethylphenol and toluene were purchased from Aldrich and were used as received. All
other reagents used were of analytical grade purity and used without any further purification. The stock
solutions of ethylbenzene, p-ethylphenol and toluene were prepared in tert-butyl alcohol. All other
solutions were prepared in purified water (Millipore, resistivity 18.2 MΩ.cm). Phosphate buffer solution
was prepared using 50 mM Na2HPO4 and NaH2PO4. A mixture of buffers (10 mM MES, 10 mM Bis-Tris, 10
mM Tris and 10 mM CHES) was used for pH dependent experiments across the range 5.5 < pH < 9.5 and
the desired pH was obtained by addition of dilute acetic acid or NaOH.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Electrochemical Measurements and Electrode Cleaning. Cyclic voltammetry (CV) was
carried out with a BAS 100B/W electrochemical workstation using a three-electrode system consisting of
a Au working electrode, a platinum wire counter electrode, and a Ag/AgCl reference electrode.
Potentials are cited versus normal hydrogen electrode (NHE). The gold working electrode was first
cleaned according to a published protocol29 then modified with 5-(4’-pyridinyl)-1,3,4-oxadiazole-2-thiol
as described.30 A platinum wire counter electrode and Ag/AgCl reference electrode were used (+196 mV
vs NHE). Unless otherwise stated, electrochemical solutions were purged with argon for at least 30 min
prior to the series of experiments and all experiments were performed under a blanket of argon gas.
The electro-active surface area of the Au electrode (A = 0.055 cm2) was determined by CV of 1
mM ferrocene methanol (FM) in 0.1 M KCl solution at different sweep rates using the Randles-Sevcik
equation (eq. 1).31 The diffusion coefficient (Do) of FM is 6.7 × 10-6 cm2 s-1,32 ip is the measured current
7
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 8 of 24
1
2
maximum, the number of electrons n=1, concentration of analyte Co = 10-6 mol cm-3, and ν is the sweep
3
4
5
6
rate (V s-1).
7
8
9
ip = (2.69 × 105)n3/2ADo1/2Coν1/2
(1)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The variation of the observed limiting catalytic current (ilim) as a function of substrate
concentration (Csub) followed Michaelis-Menten kinetics and the data were fit to the eq. (2).33
푖
max[Sub]
푖lim
=
(2)
퐾M,app + [Sub]
where imax is the saturation limiting current and KM,app is the apparent Michaelis constant.
The pH dependence of the catalytic current was modeled by eq. (3) which is applicable for an
enzyme that is deactivated by either deprotonation (pKa1) or protonation (pKa2).34
푖opt
(
)
푖lim pH =
(3)
−pH)
1 + 10(pH−p퐾 ) + 10(p퐾
a1
a2
Enzyme Electrode Preparation. A 5 µL aliquot of 21 µM EBDH in Tris-HCl buffer solution (pH
8) was dropped on a freshly prepared, inverted Au/Hpyt electrode and this was allowed approximately
30 min to dry to a thin film at 4 oC in the refrigerator. To prevent protein loss the electrode surface was
carefully covered with a perm-selective dialysis membrane presoaked in water. The dialysis membrane
was pressed onto the electrode Teflon cap and fastened to the electrode with a rubber O-ring to
prevent leakage of the internal membrane solution. The resulting modified electrode was stored at 4 oC
in 50 mM phosphate buffer solution (pH 7.0) when not in use.
Electrochemical Simulation. The DigiSim (version 3.03b) program was employed35 to simulate
the experimental cyclic voltammograms of FM obtained at different sweep rates and different substrate
concentrations. The experimental parameters restrained in each case were the working electrode
surface area (0.055 cm2) and the double-layer capacitance (2 µF). Semi-infinite diffusion was assumed
and all pre-equilibration reactions were disabled. The diffusion coefficients for EBDH (all forms) and
substrates (ethylbenzene and p-ethylphenol) were 9×10-8 and 7×10-7 cm2 s-1 respectively. The apparent
8
ACS Paragon Plus Environment

Page 9 of 24
The Journal of Physical Chemistry
1
2
diffusion coefficient used in the simulation for FM (5×10-7 cm2 s-1) is one order of magnitude lower
compared to the reported experimental value used in equation 1 (6.7×10-6 cm2 s-1).32 This value
effectively models the attenuated mass transport of FM from the bulk, across the membrane to the
diffusion layer as described previously.36 The heterogeneous rate constant was 0.05 cm s-1 determined
from simulating the sweep rate dependence of the anodic peak to cathodic peak separation (in the
absence of EBDH). The apparent redox potential of FM+/0 was determined from a control experiment
without substrate present. All of the above mentioned parameters were then held constant during
simulations of different sweep rate and substrate concentration dependent catalytic voltammograms
and only the homogenous rate constants (k1-k4; k-1-k-4) in Scheme 2 were allowed to vary.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1-phenylethanol
N
N
N
FMox
k-4
S
S
O
N
N
N
k3
e-
k-3
k4
O
N
EBDHred
N
S
:
EBDHred
O
N
1-phenylethanol
N
N
FMred
FMox
S
S
k-2
k2
EBDHint
O
N
N
N
N
k'-4
O
:
EBDHox
e-
Ethylbenzene
EBDHox
N
N
S
O
N
N
N
k1
N
k'4
k-1
S
O
Ethylbenzene
FMred
electrode
Au/pyt
membrane
Dialysis
Scheme 2. Mediated Electrochemically Driven Catalysis of EBDH with ethylbenzene.
9
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 10 of 24
1
2
3
4
5
Results and Discussion
6
7
8
9
Mechanism for Mediated Electrocatalysis of EBDH. A simplified double substrate (ping-
pong) mechanism was used to model the reaction (Scheme 2). EBDH is trapped beneath the dialysis
membrane in proximity to the electrode surface and reacts with both substrate and oxidized form of the
mediator (ferrocenium, FMox). The substrate (ethylbenzene or p-ethylphenol) and mediator (FMred) in
the bulk solution are under diffusion control but must cross the membrane to access the enzyme or
electrode. FM is a single electron transfer mediator so two consecutive outer sphere redox reactions are
required to fully restore EBDH to its active (oxidized) form. We have assumed that the EBDH-substrate
reaction follows Michaelis-Menten kinetics and it is associated with the substrate binding (k1/k-1),
turnover (k2/k-2), product release (k3/k-3) and reactivation by FMox (two consecutive, one-electron, outer
sphere redox reactions k4/k-4 and k’4/k’-4). The rate constants as k1/k-1, k2/k-2 and k3/k-3 are mediator
independent whereas k4/k-4 and k’4/k’-4 are substrate independent. The EBDH-FM reaction proceeds by
an outer-sphere electron transfer mechanism and no pre-equilibrium between enzyme and mediator is
considered. This is a simpler case than normally associated with a true ping-pong mechanism where
non-covalent interactions would drive the formation of an enzyme-electron partner complex. FMox,
FMred, EBDHox and EBDHred are the oxidized and reduced forms of mediator and enzyme, respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Catalytic Voltammetry. An example of the mediated catalytic voltammetry of EBDH with p-
ethylphenol is illustrated in Figure 2. It was reported that p-ethylphenol is a better substrate for EBDH
than ethylbenzene due to the hydroxyl substituent in the para position, which may H-bond with
Asp485.37 Although EBDH has seven electro-active cofactors (Mo, heme b and five iron-sulfur clusters,
Figure 1)12 no ‘non-turnover’ redox responses were observed from these cofactors at the Au/Hpyt
electrode (data not shown). Most of these cofactors are deeply buried inside the protein structure
(Figure 1) and not accessible to the electrode surface.12 Alternatively the small molecular weight redox
10
ACS Paragon Plus Environment

Page 11 of 24
The Journal of Physical Chemistry
1
2
3
4
5
6
mediator FM is capable of shuttling electrons between EBDH and the electrode where its heterogeneous
electron kinetics are facile.
7
8
9
0.2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
b
0.1
a
0
–0.1
200
300
400
500
E/ mV vs. NHE
Figure 2. CVs obtained for 50 μM FM at Au/Hpyt/EBDH electrode in (a) absence and (b) presence of 200
µM p-ethylphenol in 50 mM phosphate buffer (pH 7) at a sweep rate of 5 mV s−1.
At the Au/Hpyt/EBDH electrode, in the absence of substrate, a reversible FMox/red redox wave at
+420 mV is observed with a peak to peak separation of ca. 50 mV (Fig. 2a) in phosphate buffer (50 mM,
pH 7) which is characteristic of a single electron transfer redox process.32 This current is governed by the
diffusion of both oxidized and reduced forms of FM between the electrode and bulk solution via the
membrane. Upon introduction of p-ethylphenol (400 µM) to the electrochemical cell a complicated
amplified anodic wave emerges (Fig. 2b). This is indicative of a catalytic homogeneous reaction coupled
to heterogeneous electron transfer (EC′ mechanism).31 Here p-ethylphenol is oxidized to p-(1-
hydroxyethyl)phenol37 yielding a reduced form of the enzyme EBDHred which is oxidized again by
electrogenerated FMox at the electrode surface thus leading to an amplified anodic current through
11
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 12 of 24
1
2
3
4
5
6
7
8
9
mediator recycling. The catalytic waveform is dependent upon the diffusion of FMred to the Au/Hpyt
electrode, reaction between p-ethylphenol and EBDHox, and the reaction between FMox and EBDHred. In
a control experiment, we found that the response of 50 µM FM at the Au/Hpyt electrode, in the absence
of EBDH, was unaltered by addition of 400 µM p-ethylphenol (Supporting Figure S1). This illustrates that
p-ethylphenol is electro-inactive in the experimental potential window of +250 to +600 mV vs NHE and
also it does not undergo any chemical reaction with FM in either its oxidised or reduced form.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.2 B
0.2 A
e
e
0.1
a
0.1
a
0
0
–0.1
200
–0.1
200
300
400
500
300
400
500
E/ mV vs. NHE
E/ mV vs. NHE
Figure 3. CVs obtained for varying concentrations of (A) ethylbenzene and (B) p-ethylphenol in the
presence of 50 µM FM at Au/Hpyt/EBDH electrode (a) 0, (b) 25, (c) 50, (d) 100 and (e) 200 µM substrate
in 50 mM phosphate buffer (pH 7) at a sweep rate of 5 mV s-1.
Enzyme-Substrate Reaction. The EBDH - substrate reaction was examined by varying the
concentrations of its natural substrate ethylbenzene (Figure 3A) and also of p-ethylphenol (Figure 3B) in
the presence of 50 µM FM at the Au/Hpyt/EBDH electrode. At a concentration of 25 µM ethylbenzene, a
catalytic pre-wave emerges at ca. +300 mV in the presence of 50 µM FM at Au/Hpyt/EBDH electrode
(Figure 3A, curve b). The pre-wave grows steadily in magnitude relative to the higher potential
12
ACS Paragon Plus Environment

Page 13 of 24
The Journal of Physical Chemistry
1
2
3
4
5
6
7
8
9
(uncoupled) FMox/red wave as the substrate concentration rises (50 to 400 µM). The pre-wave is
associated with the coupled catalytic EBDH-ethylbenzene reaction but depletion of ethylbenzene in the
vicinity of the electrode (under the membrane) results in the CV reverting to that of the uncoupled
reversible FMox/red response at ca. +450 mV when the supply of substrate is exhausted. Similar
phenomena were reported by our group recently with the mediated voltammetry of other
molybdoenzymes on different electrode surfaces.20, 38 It is apparent that the shape of the transient wave
becomes more sigmoidal at higher ethylbenzene concentrations (400 µM) and the cathodic wave
diminishes (Figure 3A, curve e). However, even at the highest substrate concentrations there is still a
slight shoulder due to reduction of some persistent FMox in the diffusion layer. The kinetics of this
reaction are considered in greater detail later. We also investigated the catalytic activity of
Au/Hpyt/EBDH electrode towards p-ethylphenol (Figure 3B) with identical conditions as indicated in
Figure 3A. At low p-ethylphenol concentrations a prewave emerges at ca. +300 mV which grows in
magnitude with substrate concentration.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The apparent Michaelis constant (KM,app) was calculated from the dependence of the high
potential catalytic current from substrate concentration at pH 7 using equation 2. The anodic currents at
+350 vs NHE were chosen for ethylbenzene and p-ethylphenol, respectively, which increase linearly up
to ca. 100 μM substrate and then approach an asymptote at around 400 μM for both ethylbenzene and
p-ethylphenol (Supporting Figure S2). The calculated KM,app values were found to be 81 and 102 µM for
ethylbenzene and p-ethylphenol, respectively. The KM,app value obtained here for p-ethylphenol is about
2.5-fold higher than that found from spectrophotometry (40 µM) using ferrocenium tetrafluoroborate
as an electron acceptor.9, 17 However, the KM,app value obtained for ethylbenzene (81 µM) is more than
100-fold higher than the published value from a solution assay using stopped-flow spectrophotometry
13
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 14 of 24
1
2
(0.45 µM).17 The variation is most likely attributable to mass transport limitations of the substrate and
mediator crossing the membrane as observed previously.36
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
-0.02
4
5
6
8
9
10
11
7pH
Figure 4. pH dependence of the maximum oxidation current in the presence of 400 μM ethylbenzene at
the Au/Hpyt/EBDH electrode at a sweep rate of 5 mV s−1. The solid curve is obtained from a fit to the
experimental points using Eq. (3).
pH Profile. The electrocatalytic activity of Au/Hpyt/EBDH electrode towards the oxidation of
ethylbenzene was investigated within the range of pH 5.5 to pH 9.5 in 40 mM mixed buffer solution.
Figure 4 illustrates the activity/pH profile. The catalytic current exhibits a pH optimum of 8 which is
similar to that obtained from the solution assay using ferrocenium tetrafluoroborate as electron
acceptor (pH optimum 7.5).18 The bell shaped catalytic profile was modeled with equation 3 for a system
that is deactivated by either protonation (pKa1 = 5.9) or deprotonation (pKa2 = 9.8). Furthermore, the pH
dependence was reversible and independent of the direction of titration. Catalytic activity was fully
restored when the pH was returned to its optimal value.
14
ACS Paragon Plus Environment

Page 15 of 24
The Journal of Physical Chemistry
1
2
3
a
4
0.2
5
6
7
8
9
0.1
0
b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
300
400
500
600
E/ mV vs. NHE
Figure 5. CVs obtained for 400 µM ethylbenzene and 50 µM FM in the (a) absence and (b) presence of 5
mM toluene at Au/Hpyt/EBDH electrode in 50 mM phosphate buffer (pH 7) at a sweep rate of 5 mV s-1.
Catalytic Inhibition. It has been reported that several compounds including toluene, indole,
styrene and anisole act as effective competitive inhibitors of EBDH as they bind strongly to active site
but do not undergo hydroxylation thus blocking access by other substrates.9 As shown in Figure 5a the
initial catalytic activity of the Au/Hpyt/EBDH electrode in the presence of FM and ethylbenzene (400
µM) is greatly suppressed upon addition of 5 mM toluene (Figure 5b) leaving only the transient FMox/red
response now uncoupled from the inhibited EBDH/ethylbenzene reaction.
Simulation of the Catalytic Voltammetry
The ultimate goal of electrochemical simulation is to deduce the kinetic constants defined in
Scheme 2 that reproduce all voltammetry performed at different sweep rates and concentrations of
each substrate. The coupled homogeneous chemical reactions are mostly bimolecular so the
concentrations of each reactant will have an effect on the observed reaction rates and CV. The relevant
values derived from the simulations are given in Table 1.
15
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 16 of 24
1
2
3
4
5
6
0.2 µΑ
0.2 µΑ
A
B
–1
–1
2 mV s
2 mV s
7
8
–1
–1
5 mV s
5 mV s
9
–1
10 mV s
–1
10 mV s
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
–1
20 mV s
–1
20 mV s
–1
–1
50 mV s
50 mV s
200
300
500
E / mV 4v0s0. NHE
200
300
500
E / mV 4v0s0. NHE
Figure 6. Experimental (solid lines) and simulated (dotted lines) CVs for (A) 10 µM FM and 200 µM
ethylbenzene and (B) 50 µM FM and 50 µM ethylbenzene at the Au/Hpyt/EBDH electrode in 50 mM
phosphate buffer solution (pH 7) at different sweep rates.
Table 1. Kinetic Parameters Derived from Electrochemical Simulation (published values in
brackets).
Ethylbenzene p-ethylphenol
k1 (M-1 s-1) 1.0 × 105
5.0 × 105
200
k-1 (s-1)
k2 (s-1)
k-2 (s-1)
k3 (s-1)
100
2 (1)
2 (4.8)
4.2 × 10-3
1
4.2 × 10-3
0.77
k-3 (M-1 s-1) 1.5 × 10-3
k4 (M-1 s-1)
2.1× 10-3
7.0 × 106
k-4 (M-1 s-1)
1.4 × 102
7.0 × 106
1.4 × 102
k’4 (M-1 s-1)
k’-4 (M-1 s-1)
Sweep Rate and Mediator Concentration Dependence. The voltammetric sweep rate is a
powerful variable as it defines the timescale of each experiment. At high sweep rates all homogeneous
16
ACS Paragon Plus Environment

Page 17 of 24
The Journal of Physical Chemistry
1
2
3
4
5
6
7
8
9
reactions are bypassed and only the reversible heterogeneous electron transfer reaction (the FMox/red
couple) will be actuated. As the sweep rate is decreased, electro-generated FMox in the reaction layer
close to the electrode surface has time to react with EBDHred (the product of substrate turnover) and the
CV takes on a more complex waveform. Under these conditions FMred is regenerated by EBDHred (as well
as the intermediate half reduced form EBDHint) then electrochemically reoxidized which leads to an
amplification of the anodic current and concomitant loss of the cathodic wave altogether because FMox
formed at the electrode surface is continually consumed by the enzyme. Under ideal conditions an
electrochemical steady state results and a sigmoidal waveform is observed. In effect the electrochemical
reaction becomes biased completely toward oxidation. Here an ideal steady state is never seen due to a
combination of substrate and mediator mass transport limitations.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The sweep rate dependent CVs of the Au/Hpyt/EBDH/FM system in the presence of 200 μM
ethylbenzene and two different concentrations of FM (10 μM in Figure 6A and 50 μM in Figure 6B) are
illustrated. At the higher bulk FM concentration (Figure 6B) a cathodic peak around 360 mV emerges
with increasing sweep rate due to accumulation of FMox in the reaction layer. A combination of the
faster sweep rate (greater flux of FM to the electrode) and the shorter timescale of the sweep
(uncoupling the FMox:EBDHred reaction) results in this buildup of FMox at the electrode at high potential
and the observation of a significant return cathodic peak current.
In Figure 6A the effect of the lower FM concentration (10 μM) is clear. Firstly, no cathodic wave
is seen even at the highest sweep rate because FMox is always the limiting reagent and is immediately
consumed by EBDHred and EBDHint at this lower FM concentration (10 μM). Secondly only a single anodic
peak is observed which corresponds to the prewave seen at higher FM concentrations. No reversible
FMox/red response is observed at +420 mV as the concentration of FMred at the electrode surface is very
low and the EBDH:FM reaction is tightly coupled. In this case the rising prewave is due to the catalytic
ethylbenzene oxidation which then collapses due to depletion of ethylbenzene from the reaction layer
17
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 18 of 24
1
2
3
4
5
6
(enzyme oxidation outrunning mass transport). At this point catalysis is halted and the prewave is
replaced by the transient, reversible FMox/red couple.
7
8
9
All other sweep rate dependent simulated CVs recorded at various ethylbenzene and p-
ethylphenol concentrations are given in the Supporting information (Figures S3 to S14). Also of note is
that the anodic wave appears to shift to higher potential as the sweep rate increases. This can only be
understood by realising that the waveform is a convolution of two different processes; FMred diffusing
from the bulk straight to the electrode plus FMred ‘rebounding’ from EBDHox back to the electrode for
reoxidation after homogeneous electron transfer.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The homogeneous outer sphere redox reactions between FMox and the one and two-electron
reduced forms of EBDH (EBDHred k4 and EBDHint k4’ ) were the same. In this case the (ferrous) heme
cofactor (see Figure 1) is the likely group that reacts with FMox with intramolecular electron transfer
within EBDH being non-rate limiting. The heme bears the highest redox potential within EBDH26, 27 and is
the thermodynamic sink for electrons generated by substrate oxidation. The FMox-EBDH reaction should
be independent of substrate (see Scheme 2).
Substrate concentration dependence. Figure 7 depicts the effect of increasing concentrations of
ethylbenzene (Figure 7A) and p-ethylphenol (Figure 7B) in presence of 50 µM FM at Au/Hpyt/EBDH
electrode. As shown in Figure 7A, at low ethylbenzene concentrations the reversible high potential
(uncoupled) FMox/red wave is dominant along with a small prewave which collapses at higher potential
due to the depletion of substrate as discussed above. It is apparent that the reversible uncoupled
FMox/red wave is gradually subsumed by the prewave with increasing substrate concentrations as the
FMox-EBDHred reaction grows in significance thus scavenging all FMox from the reaction layer before it
can be reduced on the cathodic sweep. At the substrate concentrations shown in Figure 7 the most
obvious feature is that the prewave appears to move to higher potential with increasing concentration
18
ACS Paragon Plus Environment

Page 19 of 24
The Journal of Physical Chemistry
1
2
3
4
5
6
but this is actually a result of the catalytic reaction being sustained further along the potential axis by
the greater supply of substrate from the bulk.
7
8
9
0.1 µΑ
B
A
0.1 µΑ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25 µΜ
50 µΜ
25 µΜ
50 µΜ
100 µΜ
200 µΜ
100 µΜ
200 µΜ
200
300
500
E / mV 4v0s0. NHE
200
300
500
E / mV 4v0s0. NHE
Figure 7. Experimental (solid lines) and simulated (dotted lines) CVs for varying concentrations of (A)
ethylbenzene and (B) p-ethylphenol in the presence of 50 µM FM at the Au/Hpyt/EBDH electrode in 50
mM phosphate buffer solution at a sweep rate of 5 mV s-1. The deviations in absolute current between
simulation and experiment at high potentials (> +400 mV) are due to a small background contribution
from water oxidation.
Analysis of Kinetics Parameters. The kinetics parameters were obtained from
electrochemical simulation in Table 1 and compared with some of the values reported by conventional
solution assays with ferrocenium tetrafluoroborate as the electron acceptor.9, 17 The k4/k-4 and k’4/k’−4
values (EBDH reaction with FM) must be independent of substrate, while the k1/k-1, k2/k-2 and k3/k-3
values (EBDH reaction with substrates/products) may differ for each substrate. The simultaneous
determination of multiple variables is problematic in any simulation due to some parameters having no
effect on the simulated CV in some cases. Most steps were practically irreversible so the reverse rate
constants (k−2, k−3, k−4 and k’−4) are only estimates i.e. some of the values are merely upper bounds.
19
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 20 of 24
1
2
3
4
5
6
7
8
9
There was very little variation in the substrate dependent parameters as might be expected
from the similarity of the CVs (see Figure 7). The substrate binding rate (k1) of ethylbenzene was
somewhat lower than p-ethylphenol. There are no comparable values in the literature for this system.
Given the uncertainty introduced by mass transport limitations of the membrane,36 through which both
substrates must pass, we cannot attribute the apparent differential ‘on-rates’ for each substrate to
structural interactions with EBDH. The observed deviation of the KM,app values from those determined by
solution assays can be rationalized if the values of k1 obtained from the simulations are indeed lower
than their true values (by an order of magnitude). Nevertheless the values determined here for k1 are
lower bounds.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A five-fold faster turnover number (k2) of p-ethylphenol over ethylbenzene has been reported
previously from solution assays.9 Molecular modelling has suggested that the hydroxyl group of the
substrate may H-bond with Asp485 37
which may accelerate the hydroxylation reaction and lead to
,
resonance stabilisation of the carbocation intermediate. The outer sphere electron transfer rate
constants k4 and k’4 are well-defined because they the simulations are very sensitive to these
parameters at both lower and higher concentration of substrates and mediator. The reactions are
particularly fast compared with recent systems that we have investigated and demonstrate that FM is an
ideal synthetic electron partner for EBDH.
Conclusions
The mediated electrocatalytic voltammetry of EBDH in reactions with its native substrate
ethylbenzene and also with p-ethylphenol was demonstrated using FM as an artificial electron acceptor
for the first time. Both transient and sigmoidal voltammograms were obtained depending upon the
substrate concentrations and sweep rates. Catalytic inhibition was demonstrated with the competitive
inhibitor toluene. A bell shaped catalytic pH profile was obtained with an optimal value of pH 8 for EBDH
in reaction with ethylbenzene. Moreover, digital simulation enabled to determine a single set of
20
ACS Paragon Plus Environment

Page 21 of 24
The Journal of Physical Chemistry
1
2
3
4
5
6
apparent rate and equilibrium constants that modelled the electrochemical system over all
experimental conditions.
7
8
9
Acknowledgement
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We gratefully acknowledge financial support from the Australian Research Council
(DP120101465 & DP150104672), the German research foundation and the Synmikro Centre Marburg.
Supporting Information Available. Control experiments are shown for the electrochemical activity of
p-ethylphenol on a Au/Hpyt electrode. Michaelis-Menten plots are shown for increasing concentrations
of the substrates ethylbenzene and p-ethylphenol. Comparisons of experimental and simulated
voltammetry are shown at various sweep rates for both ethylbenzene and p-ethylphenol.
21
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 22 of 24
1
2
3
4
5
References
6
7
8
9
1.
2.
3.
Hille, R. Molybdenum enzymes containing the pyranopterin cofactor: An overview. Met. Ions
Biol. Syst. 2002, 39, 187-226.
Bernhardt, P. V. Exploiting the versatility and selectivity of Mo enzymes with electrochemistry.
Chem. Commun. 2011, 47, 1663-1673.
Hille, R.; Hall, J.; Basu, P. The mononuclear molybdenum enzymes. Chem. Rev. 2014, 114, 3963-
4038.
Hille, R. The mononuclear molybdenum enzymes. Chem. Rev. 1996, 96, 2757-2816.
Johnson, H. A.; Pelletier, D. A.; Spormann, A. M. Isolation and characterization of anaerobic
ethylbenzene dehydrogenase, a novel Mo-Fe-S enzyme. J. Bacteriol. 2001, 183, 4536-4542.
Kniemeyer, O.; Heider, J. Ethylbenzene dehydrogenase, a novel hydrocarbon-oxidizing
molybdenum/iron-sulfur/heme enzyme. J. Biol. Chem. 2001, 276, 21381-21386.
Ball, H. A.; Johnson, H. A.; Reinhard, M.; Spormann, A. M. Initial reactions in anaerobic
ethylbenzene oxidation by a denitrifying bacterium, strain EB1. J. Bacteriol. 1996, 178, 5755-
5761.
Rabus, R.; Widdel, F. Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons
by new denitrifying bacteria. Arch. Microbiol. 1995, 163, 96-103.
Knack, D.; Hagel, C.; Szaleniec, M.; Dudzik, A.; Salwinski, A.; Helder, J. Substrate and inhibitor
spectra of ethylbenzene dehydrogenase: perspectives on application potential and catalytic
mechanism. Appl. Environ. Microbiol. 2012, 78, 6475-6482.
Szaleniec, M.; Dudzik, A.; Kozik, B.; Borowski, T.; Heider, J.; Witko, M. Mechanistic basis for the
enantioselectivity of the anaerobic hydroxylation of alkylaromatic compounds by ethylbenzene
dehydrogenase. J. Inorg. Biochem. 2014, 139, 9-20.
Tataruch, M.; Heider, J.; Bryjak, J.; Nowak, P.; Knack, D.; Czerniak, A.; Liesiene, J.; Szaleniec, M.
Suitability of the hydrocarbon-hydroxylating molybdenum-enzyme ethylbenzene dehydrogenase
for industrial chiral alcohol production. J. Biotechnol. 2014, 192, 400-409.
Kloer, D. P.; Hagel, C.; Heider, J.; Schulz, G. E. Crystal structure of ethylbenzene dehydrogenase
from Aromatoleum aromaticum. Structure 2006, 14, 1377-1388.
Krafft, T.; Bowen, A.; Theis, F.; Macy, J. M. Cloning and sequencing of the genes encoding the
periplasmic-cytochrome B-containing selenate reductase of Thauera selenatis. DNA Sequence
2000, 10, 365-377.
McDevitt, C. A.; Hugenholtz, P.; Hanson, G. R.; McEwan, A. G. Molecular analysis of dimethyl
sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide
reductase family of microbial molybdopterin-containing enzymes. Mol. Microbiol. 2002, 44,
1575-1587.
Lledo, B.; Martinez-Espinosa, R. M.; Marhuenda-Egea, F. C.; Bonete, M. J. Respiratory nitrate
reductase from haloarchaeon Haloferax mediterranei: biochemical and genetic analysis.
Biochim. Biophys. Acta, Gen. Subj. 2004, 1674, 50-59.
Rothery, R. A.; Workun, G. J.; Weiner, J. H. The prokaryotic complex iron–sulfur molybdoenzyme
family. Biochim. Biophys. Acta 2008, 1778, 1897-1929.
Szaleniec, M.; Hagel, C.; Menke, M.; Nowak, P.; Witko, M.; Heider, J. Kinetics and mechanism of
oxygen-independent hydrocarbon hydroxylation by ethylbenzene dehydrogenase. Biochemistry
2007, 46, 7637-7646.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
22
ACS Paragon Plus Environment

Page 23 of 24
The Journal of Physical Chemistry
1
2
3
4
5
6
7
8
9
18.
19.
20.
21.
22.
Szaleniec, M.; Borowski, T.; Schuehle, K.; Witko, M.; Heider, J. Ab inito modeling of ethylbenzene
dehydrogenase reaction mechanism. J. Am. Chem. Soc. 2010, 132, 6014-6024.
Szaleniec, M.; Salwinski, A.; Borowski, T.; Heider, J.; Witko, M. Quantum chemical modeling
studies of ethylbenzene dehydrogenase activity. Int. J. Quantum Chem. 2012, 112, 1990-1999.
Chen, K.-I.; McEwan, A. G.; Bernhardt, P. V. Mediated electrochemistry of dimethyl sulfoxide
reductase from Rhodobacter capsulatus. J. Biol. Inorg. Chem. 2009, 14, 409-419.
Kalimuthu, P.; Leimkühler, S.; Bernhardt, P. V. Xanthine dehydrogenase electrocatalysis:
autocatalysis and novel activity. J. Phys. Chem. B 2011, 115, 2655-2662.
Kalimuthu, P.; Tkac, J.; Kappler, U.; Davis, J. J.; Bernhardt, P. V. Highly sensitive and stable
electrochemical sulfite biosensor incorporating a bacterial sulfite dehydrogenase. Anal. Chem.
2010, 82, 7374-7379.
Pietsch, M. A.; Hall, M. B. Theoretical studies on models for the oxo-transfer reaction of
dioxomolybdenum enzymes. Inorg. Chem. 1996, 35, 1273-8.
Szaleniec, M.; Witko, M.; Heider, J. Quantum chemical modelling of the C-H cleavage mechanism
in oxidation of ethylbenzene and its derivates by ethylbenzene dehydrogenase. J. Mol. Catal. A:
Chem. 2008, 286, 128-136.
Szaleniec, M.; Witko, M.; Tadeusiewicz, R.; Goclon, J. Application of artificial neural networks
and DFT-based parameters for prediction of reaction kinetics of ethylbenzene dehydrogenase. J.
Comput.-Aided Mol. Des. 2006, 20, 145-157.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
23.
24.
25.
26.
27.
Hagel, C. PhD Thesis, Technical University of Darmstadt, 2006.
Creevey, N. L.; McEwan, A. G.; Hanson, G. R.; Bernhardt, P. V. Thermodynamic characterization
of the redox centers within dimethylsulfide dehydrogenase. Biochemistry 2008, 47, 3770-3776.
Broadhead, G. D.; Osgerby, J. M.; Pauson, P. L. Ferrocene derivatives. V. Ferrocenealdehyde. J.
Chem. Soc. 1958, 650-6.
Tkac, J.; Davis, J. J. An optimized electrode pre-treatment for SAM formation on polycrystalline
gold. J. Electroanal. Chem. 2008, 621, 117-120.
28.
29.
30.
Paulo, T. d. F.; da Silva, M. A. S.; Pinheiro, S. d. O.; Meyer, E.; Pinheiro, L. S.; Freire, J. A.; Tanaka,
A. A.; de Lima Neto, P.; Moreira, I. d. S.; Diogenes, I. C. N. 5-(4-pyridinyl)-1,3,4-oxadiazole-2-thiol
on gold: SAM formation and electroactivity. J. Braz. Chem. Soc. 2008, 19, 711-719.
Bard, A. J.; Faulkner, L. R. Electrochemical methods: fundamentals and applications. 2001; p null.
Anicet, N.; Bourdillon, C.; Moiroux, J.; Saveant, J.-M. Electron transfer in organized assemblies of
biomolecules. Step-by-step avidin/biotin construction and dynamic characteristics of a spatially
ordered multilayer enzyme electrode. J. Phys. Chem. B 1998, 102, 9844-9849.
Situmorang, M.; Hibbert, D. B.; Gooding, J. J.; Barnett, D. A sulfite biosensor fabricated using
electrodeposited polytyramine: application to wine analysis. Analyst 1999, 124, 1775-1779.
Brody, M. S.; Hille, R. The kinetic behavior of chicken liver sulfite oxidase. Biochemistry 1999, 38,
6668-6677.
Rudolf, M.; Feldber, S. W. DigiSim version 3.03b. Bioanalytical System, West Lafayette 2004.
Kalimuthu, P.; Kappler, U.; Bernhardt, P. V. Catalytic voltammetry of the molybdoenzyme sulfite
dehydrogenase from Sinorhizobium meliloti. J. Phys. Chem. B 2014, 118, 7091-7099.
Dudzik, A.; Kozik, B.; Tataruch, M.; Wojcik, A.; Knack, D.; Borowski, T.; Heider, J.; Witko, M.;
Szaleniec, M. The reaction mechanism of chiral hydroxylation of p-OH and p-NH2 substituted
compounds by ethylbenzene dehydrogenase. Can. J. Chem. 2013, 91, 775-786.
Kalimuthu, P.; Heath, M. D.; Santini, J. M.; Kappler, U.; Bernhardt, P. V. Electrochemically driven
catalysis of Rhizobium sp. NT-26 arsenite oxidase with its native electron acceptor cytochrome
c552. Biochim. Biophys. Acta, Bioenerg. 2014, 1837, 112-120.
31.
32.
33.
34.
35.
36.
37.
38.
23
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 24 of 24
1
2
3
4
5
6
7
Table of Contents Graphic
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1-(
S
)-phenylethanol
ethylbenzene
CH3
CH3
OH
Asp223
H
H
H
Asp223
H
2O
S
O
O
S
MoIV
S
O
O
S
S
MoVI
S
S
O
H
2O
S
-2H+
2 FMox
2 FMred
electrode
2e-
24
ACS Paragon Plus Environment