Journal of Electroanalytical Chemistry xxx (2011) xxx–xxx

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Journal of Electroanalytical Chemistry

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Electrochemical investigations of fungal cytochrome P450

Preety Vatsyayan a, Mitun Chakraborty a, Sandip Bordoloi b, Pranab Goswami a,⇑
a
Department of Biotechnology, Indian Institute of Technology Guwahati, Assam 781 039, India
b
Center for Energy, Indian Institute of Technology Guwahati, Assam 781 039, India

a r t i c l e i n f o a b s t r a c t

Article history: The electrochemical investigations of fungal (Aspergillus terreus MTCC 6324) cytochrome P450 monoox-
Received 20 April 2011 ygenase (CYP) (EC 1.14.14.1) immobilized on multi-walled carbon nanotubes–NafionÒ–polyethyleneim-
Received in revised form 14 August 2011 ine (MWCNT-NF/PEI) modified glassy carbon electrode (GCE) were carried out to understand the
Accepted 30 August 2011
electrochemistry of the CYP and its application potential in electrochemical-based devices. In deoxygen-
Available online xxxx
ated condition, the formal potential for Fe(III)/Fe(II) redox couple of CYP was about 0.53 V (vs. Ag/AgCl
reference electrode, pH 8). A positive shift in the redox potential of the bioelectrode to 0.475 V was
Keywords:
observed in the presence of oxygen and substrate. The electrocatalytic activity towards n-hexadecane
Cytochrome P450
Direct electrochemistry
and inhibition with piperonyl butoxide (PBO) confirmed the involvement of Fe(III/II) system of CYP in
Bioelectrocatalysis the redox process. The current response increased linearly from 1 lM to 100 lM of n-hexadecane with
n-Hexadecane detection limit 0.1 lM. The IC50 for PBO was 2.7 lM. The surface coverage of CYP immobilized on
Aspergillus terreus MWCNT-NF/PEI modified GCE was approximately 3.45  1010 mol cm2. The electron transfer rate con-
Multi-walled carbon nanotubes stant (ks) was 1.0 ± 0.2 s1, indicating facilitation of the electron transfer between CYP and GCE.
Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction of a wide range of physiologically relevant drugs as reviewed by

Fleming et al. [9]. The major driving force for electrochemical stud-
CYP forms a large family of heme enzymes that catalyzes a wide ies of CYP catalyzed reactions is their scope for applications in
range of redox reactions such as, epoxidation, hydroxylation and developing amperometric biosensors, bioreactors, and bio-fuel
heteroatom oxidation [1–5]. The redox active center consisting of cells [6,7,10,11]. The success for developing such electrochemical
iron-protoporphyrin IX (heme) of CYP is involved in these cataly- devices depends largely on the efficiency of the enzyme, method
ses. Initially, upon binding of the substrate to CYP, the Fe(III) in of fabrication of the bioelectrode, which influence the electron
the native heme acquires an electron from a redox co-factor, transport kinetics between the redox center of the enzyme and
usually NAD(P)H, through an electron transport chain in the cells the electronic unit and also on the stability of the construct
and is subsequently reduced to Fe(II)–heme state. This reduced [12,13]. Investigations on the enzymes from the sources that have
heme is re-oxidized to a state containing Fe(III) O–O radical after not been adequately studied may unearth efficient enzymes appli-
binding with molecular oxygen and eventually rearranged to a cable to these potential technological ventures. The sources of CYP
high-energy oxidizing species Fe(IV) = O+–heme after gaining so far used for electrochemical studies are mostly limited to bacte-
another electron from the redox co-factor. The substrate already ria and higher organisms [6–8,10]. The electrochemical studies on
bound to the CYP is oxidized by this oxyferryl species with the con- CYP from fungal sources are yet to be pursued passably. Fungi are
comitant regeneration of the native Fe(III)–heme thus completing widespread in nature and utilize a wide range of substrates. Cyto-
the catalytic cycle. It has been possible to establish the CYP cata- chrome P450 enzymes are the foremost enzymes involved in these
lyzed redox reactions electrochemically that eliminate the need metabolisms. Thus the electrochemical studies on fungal
of reductant co-factors in the reactions [6–8]. Some of the promis- cytochrome P450 may throw light on their potentialities for bio-
cuous examples include reduction of molecular oxygen, epoxida- sensor or bio-fuel cell applications. In this paper we are reporting
tion of styrene, hydroxylation of fatty acids, steroids, and sensing for the first time the electrochemical investigations of fungal CYP
from Aspergillus terreus. The CYP from A. terreus is highly active to-
wards n-hexadecane and many other organic compounds like,
Abbreviations: Aspergillus terreus, A. terreus; atomic force microscopy, AFM; methylated organic solvents, steroids, aromatic hydrocarbons,
catalase, CAT; cyclic voltammetry, CV; cytochrome P450, CYP; differential pulse etc. [14]. This broad substrate specific property of CYP supports
voltammetry, DPV; glassy carbon electrode, GCE; multi-walled carbon nanotubes,
MWCNT; NafionÒ, NF; piperonyl butoxide, PBO; polyethyleneimine, PEI.
its catalytic flexibility towards various chemical substrates and
⇑ Corresponding author. Tel.: +91 361 2582202; fax: +91 361 2582249. thus, offers potential scope for its application in areas like, bio-
E-mail address: pgoswami@iitg.ernet.in (P. Goswami). electrocatalytic transformations and bio-fuel cells. The microsomal

1572-6657/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jelechem.2011.08.020

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2 P. Vatsyayan et al. / Journal of Electroanalytical Chemistry xxx (2011) xxx–xxx

Fig. 1. AFM images of different layers of modifications of GCE for immobilization of CYP. (a) Bare GCE, (b) GCE/MWCNT-NF, (c) GCE/MWCNT-NF/CYP, and (d) GCE/MWCNT-
NF/CYP/PEI. Scales in all axes are in nm.

enzyme preparation employed in this investigation consists of the 2.2. Fabrication of MWCNT-NF/CYP/PEI modified GCE and apparatus
single CYP (MW110 kDa), highly induced in the cells by the
n-hexadecane substrates and the typical characteristics confirmed Fabrication of CYP bioelectrode was carried out by a similar pro-
by CO-difference spectra [14]. The bioelectrode used for these cedure described earlier by us [15]. Briefly, GCE (5 mm diameter)
studies was constructed by immobilizing the microsomal CYP in was polished with 3 lm alumina slurry, and then ultrasonically
an electro-active surface over GCE prepared with nanocomposite cleaned successively in distilled water and ethanol to remove
of MWCNT-NF. The enzyme adsorbed on the nanocomposite was adsorbed alumina particles and finally, air dried. A total of 10 mg
stabilized by encapsulation with PEI following similar fabrication MWCNT was added to 1 ml of 5% NF and sonicated for about
procedure used to construct large catalase based bioelectrode [15]. 3.5 h to obtain a stable and uniform suspension. Ten ll of this sus-
pension was layered on GCE and allowed to dry under clean air at
room temperature. Once dried, 15 ll of CYP solution (0.5 mg ml1;
2. Experimental
specific activity 2.2 ± 0.07 U mg1 of microsomal preparation in
50 mM tris buffer, pH 8) was layered on the MWCNT-NF nano-
2.1. Reagents
composite and kept overnight at 4 °C. Finally, 5 ll of PEI (10% in tris
buffer pH 8) was layered and dried at room temperature. The
Microsomal CYP (EC 1.14.14.1) was isolated from n-hexadecane
buffer pH of 8 was used throughout this study due to the optimum
grown cells of A. terreus MTCC 6324 as described elsewhere [14].
stability of CYP at this pH. Separate GCE with each layer as control
MWCNT (10–15 nm outer diameter, 2–6 nm inner diameter and
were prepared simultaneously and dried as described earlier. Elec-
0.1–10 lm length), PEI (50%) and n-hexadecane were obtained
trochemical experiments were performed with potentiostat (Auto-
from Sigma, NF from Du Pont (USA), and PBO was obtained from
lab PGSTAT 302 N, Eco Chemie B.V., The Netherlands) driven with
Fluka. Fifty mM tris buffer, pH 8 was used throughout the experi-
GPES software. A conventional three-electrode cell was used with
ment. A stock (300 lM) of hydrocarbon substrate (n-hexadecane)
an Ag/AgCl/saturated KCl reference electrode, a platinum wire as
was prepared in tris buffer with triton X-100 (5%) and sonicated
counter electrode, and a glassy carbon disk (modified and unmod-
for 0.5 h. During the substrate sensitivity assays, the maximum
ified) as working electrode. All experiments were carried out at
concentration of triton X-100 did not exceed 0.25% of the final
25 °C.
concentration. CAT (EC 1.11.1.6) was isolated and purified from
the same strain of A. terreus; similar to the procedure described
elsewhere [16]. The stock solution of CAT (1 mg ml1) was pre- 2.3. Surface morphology studies of the fabricated bioelectrode by AFM
pared in 50 mM sodium phosphate buffer (pH 7.5). All solutions
were prepared with deionized water (Milli Q). Solutions were de- GCE fabricated with each layer were mounted on the specimen
aerated by bubbling high-purity (99.99%) argon gas through them holders with electro-conductive tape and were placed in standard
prior to the experiments. sample plates. AFM was performed using an ambient air scanning

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 P. Vatsyayan et al. / Journal of Electroanalytical Chemistry xxx (2011) xxx–xxx 3

redox couple of CYP [17] was observed when GCE/MWCNT-NF/

CYP/PEI electrode was used in deoxygenated condition with con-
tinuous argon gas purging (Fig. 2). The ratio of anodic to cathodic
peak current (Ipa and Ipc) was approximately 1.0 (Fig. 2 background
subtracted CV), indicating that CYP undergoes a near reversible re-
dox process at the GCE modified with MWCNT-NF/CYP/PEI film.
Thus, MWCNT-NF film provides a three-dimensional nano-elec-
trode ensemble for sufficient adsorption of CYP and a suitable envi-
ronment for electron transfer with underlying GCE. The poly-
cationic layer of PEI indirectly supported electron transfer by pro-
moting better contact between the enzyme and the electro-active
nano-composite matrix through electrostatic interaction with the
underlying negatively charged CYP-adsorbed MWCNT-NF film.
Fig. 2. The cyclic voltammogram of microsomal CYP immobilized on MWCNT-NF/ The formal potential (E°) of CYP was estimated as a midpoint of
PEI modified GCE in deoxygenated condition. Arrow head points to the background
CV reduction and oxidation peak potentials (Epc and Epa) at approx-
subtracted redox peaks. Background subtraction was carried out by GPES software
tool. The voltammograms were recorded in 50 mM tris buffer (pH 8) and scan rate imately 565 mV and 500 mV, respectively. The separation of Epc
of 100 mV s1. and Epa (DEp = 65 mV) indicates a single electron transfer reaction.
The surface coverage (C) of 3.45  1010 mol cm2 was calculated
probe microscope (Agilent Technologies 5500). Images were re- for CYP at the modified GCE using the equation C = Q/nFA, where Q
corded with typical contact mode using PicoScan 5 software. is the charge obtained by integrating the peak current area, n is the
number of electrons involved, F is Faraday’s constant and A is elec-
trode area. To obtain the kinetic parameters of CYP redox at
3. Results and discussion
MWCNT-NF/PEI modified GCE, the effect of scan rate was investi-
gated. Fig. 3 shows the CV at different scan rates. The peak current
3.1. Surface characterization of the fabricated bioelectrode
(Ip) vs. scan rate plot, shown in the inset (a) of Fig. 3, exhibits a lin-
ear relationship (R2 = 0.9935), as expected for a surface-confined
AFM was used to characterize the surface morphology of the
redox process. The peak to peak separation is approximately
fabricated GCE (Fig. 1). Fig. 1a shows the bare and smooth GCE
60 mV at scan rates less than 50 mV s1, suggesting facile charge
surface. Layering of MWCNT-NF nanomatrix on bare GCE
transfer kinetics over this range of sweep rates. On the other hand,
(Fig. 1b) provides sufficient cavities for the uniform adsorption of
it was found that at scan rates greater than 100 mV s1, DEp in-
CYP (Fig. 1c). Further, the addition of PEI on the nanomatrix-ad-
creased with the increase of scan rate. The values of peak to peak
sorbed CYP layer packed the enzyme effectively within the cavities
potential separations were proportional to the logarithm of the
of the nano-matrix as evident from the Fig. 1d, thus providing addi-
scan rate for scan rates greater than 100 mV s1 (inset b of
tional physical stability to the immobilized CYP on the electrode. It
Fig. 3). Based on the Laviron’s theory, the transfer coefficient (a)
is suggested that the electrostatic interaction developed between
and heterogeneous electron transfer rate constant (ks) were esti-
the positively charged PEI and the underlying negatively charged
mated by measuring the variation of peak potential with scan rate
nanomatrix packed the CYP layer closer to the electrode surface
[18]. The a and ks of CYP were calculated as 0.6 ± 0.1 and
and thereby provided the stability to the fabricated bioelectrode.
1.0 ± 0.2 s1, respectively showing a significant electron transfer
kinetics between the redox center of immobilized CYP and the
3.2. Cyclic voltammetry of MWCNT-NF/CYP/PEI modified GCE
electronic unit. Thus, by following the current protocol, we are
reporting here for the first time, an efficient electrical conductivity
The electrochemical behavior of microsomal CYP in MWCNT-
between the redox center of microsomal CYP from fungal source
NF/PEI film was studied by CV. A well defined CV peak at approx-
and electrode.
imately 0.53 V [(Epa + Epc)/2], characteristic of heme Fe(III)/Fe(II)

Fig. 3. Cyclic voltammograms of MWCNT-NF/CYP/PEI modified GCE at different scan rates in 50 mM tris buffer (pH 8). The scan rates were 10–800 mV s1. Inset: (a) Plot of
peak currents vs. scan rates and (b) variation of peak potentials vs. the logarithm of the scan rates.

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4 P. Vatsyayan et al. / Journal of Electroanalytical Chemistry xxx (2011) xxx–xxx

CYP with the substrate and molecular oxygen that promote facile
electron transfer process. In substrate-free CYP, the transition from
a low spin form (six co-ordinate complex) to a high spin form (five
co-ordinate complex) upon reduction may result in larger inner
sphere reorganization energy, than in substrate-bound CYP which
is in high spin form in both oxidation states. Therefore, relative to
substrate bound CYP, the substrate-free form has a less favorable
spin state which is stated as one of the reason for the potential shift
on substrate binding [19]. Further, oxygen is the natural co-sub-
strate of CYP and binds to the ferrous iron (FeII) at a very fast rate
[20,21], thus making the overall process more thermodynamically
favorable. The change in E° following substrate binding thus gave
birth to the idea of a substrate-dependent thermodynamic switch
Fig. 4. The cyclic voltammograms of GCE/MWCNT-NF/CYP/PEI in absence (a) and being present in P450 enzyme systems, which may be speculated
presence (b) of oxygen and n-hexadecane. (a0 ) and (b0 ) depict the background as the nature’s way of preventing the futile cycling of electrons
subtracted peaks of (a) and (b), respectively. Voltammograms were recorded in [22]. Early voltammetric works with P450cam at a graphite elec-
50 mM tris buffer (pH 8) and scan rate of 100 mV s1. trode [17] and in biomembrane-like films [23] have shown that
the presence of a substrate/inhibitor results in a significant shift
3.3. Electro-catalytic behavior of CYP immobilized on MWCNT-NF/PEI in the value of E° to more positive values (in a range of 45 mV–
modified GCE 100 mV). However, voltammetric studies in the presence of
substrate (or inhibitors) with CYP from other sources have not pro-
In well oxygenated solution and in presence of n-hexadecane, vided as conclusive or consistent results. Thus, the response profile
(substrate of CYP [14]) a positive shift of 55 mV was observed of the CYP bioelectrode shown in Fig. 4 is similar to the one from
in the redox peak for MWCNT-NF/CYP/PEI bioelectrode the bacterial sources and is first of its kind for CYP from fungal
(E°’ = 0.475, DEp = 62 mV). The increase in Ipc is also evident from source.
the background subtracted CV accompanied by a decrease of Ipa Although, the increase in Ipc was observed with increasing
(Fig. 4). Further with increasing concentration of n-hexadecane, substrate concentration by CYP bioelectrode but the outcome
the voltammetric behavior of the CYP-bioelectrode changes obvi- was more significant when CAT (5 lg ml1) was co-adsorbed with
ously, with an increase in the Ipc and the subsequent disappearance CYP in a concentration which can effectively neutralize the H2O2
of the Ipa (Fig. 5a) which, indicates the electrocatalytic reduction of generated on the electrode (by the electrochemical reduction of
CYP immobilized on GCE/MWCNT-NF/PEI and the increases in the oxygen) and can thus effectively inhibit the peroxide shunt path-
rate of dioxygen binding to the heme. The positive shift in the way, without having an interference in the redox of CYP. The Ipc
redox potential of CYP is likely to be caused by the binding of thus generated (Fig. 5b) can be used to co-relate the complete

Fig. 5. Substrate dependent response of CYP immobilized on MWCNT-NF/PEI modified GCE to different n-hexadecane concentration. (a) Cyclic voltammograms of MWCNT-
NF/CYP/PEI modified GCE with successive addition of substrate (ranging from 10–100 lM n-hexadecane), (b) DPV curves of GCE/MWCNT-NF/CYP-CAT/PEI with different
substrate concentration at 0.51 V, (c) response curve of CYP immobilized on MWCNT-NF/PEI GCE and (d) linear calibration curve for determination sensitivity of
immobilized CYP.

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 P. Vatsyayan et al. / Journal of Electroanalytical Chemistry xxx (2011) xxx–xxx 5

positive shift in the redox potential of CYP in the presence of oxy-

gen and substrate is also one of the interesting finding, which sup-
ports the theory of thermodynamic switch present in CYP catalytic
systems. The results have established the potential of the CYP from
A. terreus and their fabrication procedure for their application in
bioelectronic devices, such as bio-fuel cells.

Acknowledgment

This work was carried out with the financial support from the
Department of Biotechnology (DBT), Government of India.

References

[1] A.I. Archakov, G.I. Bachmanova, Cytochrome P450 and Active Oxygen, Taylor
and Francis, London, 1990.
[2] T. Omura, Biochem. Biophys. Res. Commun. 266 (1999) 690.
Fig. 6. Inhibitor studies with PBO. DPV curves at different PBO concentrations (at [3] D.F.V. Lewis, Guide to Cytochromes P450: Structure and Function, Taylor and
fixed substrate concentration of 100 lM and 0.51 V). Inset: Linear curve for Francis, London and NewYork, 2001.
determination of IC50. [4] P.R. Ortiz de Montellano, J.J. De Voss, in: P.R. Ortiz de Montellano (Ed.),
Cytochrome P450: Structure, Mechanism and Biochemistry, third ed., Kluwer
Academic/Plenum Publishers, New York, 2005, p. 183.
CYP activity. Saturation and linear response curves were obtained [5] J.B. van Beilen, E.G. Funhoff, Curr. Opin. Biotechnol. 16 (2005) 308.
[6] V.V. Shumyantseva, T.V. Bulko, A.I. Archakov, J. Inorg. Biochem. 99 (2005) 1051.
within the n-hexadecane concentration from 0.1 lM to 350 lM [7] N. Bistolas, U. Wollenberger, C. Jung, F.W. Scheller, Biosens. Bioelectron. 20
(Fig. 5c) and 10 lM to 100 lM (R2 = 0.975) (Fig. 5d), respectively (2005) 2408.
by using DPV at 0.51 V. The detection limit was nearly 0.1 lM. [8] A.K. Udit, H.B. Gray, Biochem. Biophys. Res. Commun. 338 (2005) 470.
[9] B.D. Fleming, D.L. Johnson, A.M. Bond, L.L. Martin, Expert Opin. Drug Metab.
Further, to confirm the correlation between increasing Ipc and Toxicol. 2 (2006) 581.
CYP activity, quantitative analysis of the bioelectrocatalytic prod- [10] V.V. Shumyantseva, T.V. Bulko, S.A. Usanov, R.D. Schmid, C. Nicolini, A.I.
uct of substrate n-hexadecane using sensitive technique is Archakov, J. Inorg. Biochem. 87 (2001) 185.
[11] D. Djuricic, B.D. Fleming, H.A.O. Hill, Y. Tian, in: A.J.L. Pombeiro, C. Amatore
warranted. Nonetheless, CYP inhibition studies using PBO (specific
(Eds.), Trends in Molecular Electrochemistry, Marcel Dekker, USA and Fontis
inhibitor of CYP [24]) was carried out where different concentra- Media SA, Switzerland, 2004, pp. 189.
tions of PBO were layered on the fabricated bioelectrode. The [12] J.F. Rusling, Acc. Chem. Res. 31 (1998) 363.
[13] I. Willner, E. Katz, Angew. Chem. Int. Ed. 39 (2000) 1180.
bioelectrode was then allowed to dry followed by washing with
[14] P. Vatsyayan, A.K. Kumar, P. Goswami, P. Goswami, Bioresour. Technol. 99
distilled water. DPV with different inhibitor concentrations in pres- (2008) 68.
ence of a fixed concentration of substrate at 0.51 V showed a lin- [15] P. Vatsyayan, S. Bordoloi, P. Goswami, Biophy. Chem. 153 (2010) 36.
ear decrease in Ipc with increasing PBO concentration (Fig. 6). IC50 [16] J.A. Calera, J. Sanchez-Weatherby, R. Lopez-Medrano, F. Leal, FEBS Lett. 475
(2000) 117.
for CYP activity was observed at 2.7 ± 0.3 lM concentration of PBO. [17] J. Kazlauskaite, A.C.G. Westlake, L.L. Wong, H.A.O. Hill, Chem. Commun. 18
(1996) 2189.
[18] E. Laviron, J. Electroanal. Chem. 101 (1979) 19.
4. Conclusions [19] M.J. Honeychurch, H.A.O. Hill, L.L. Wong, FEBS Lett. 451 (1999) 351.
[20] E.I. Iwuoha, S. Joseph, Z. Zhang, M.R. Smyth, U. Fuhr, P.R. Ortiz de Montellano, J.
In this study, direct electrochemistry and electrocatalytic prop- Pharm. Biomed. Anal. 17 (1998) 1101.
[21] S. Joseph, J.F. Rusling, Y.M. Lvov, T. Friedberg, U. Fuhr, Biochem. Pharmacol. 65
erty of a CYP from fungal source (A. terreus) were established using
(2003) 1817.
MWCNT-NF/PEI modified GCE. The hydrocarbon substrate depen- [22] S.N. Daff, S.K. Chapman, K.L. Turner, R.A. Holt, S. Govindaraj, T.L. Poulos, A.W.
dent increase in the current of the fabricated bioelectrode was also Munro, Biochemistry 36 (1997) 13816.
demonstrated. The involvement of Fe(III)/Fe(II) system of CYP in this [23] Z. Zhang, A.E.F. Nassar, Z. Lu, J.B. Schenkman, J.F. Rusling, J. Chem. Soc. Faraday
Trans. 93 (1997) 1769.
redox process has also been confirmed by using specific CYP inhib- [24] E. Hodgson, P.E. Levi, in: D.G. Jones (Ed.), Piperonyl Butoxide: The Insecticide
itor in the bioelectrocatalytic reaction. Besides, a significant Synergist, Academic Press, London, 1998, p. 41.

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