Control of electrochemical and ferryloxy formation kinetics of cyt P450s in polyion films by heme iron spin state and secondary structure

Article abstract of DOI:10.1021/ja9065317

Voltammetry of cytochrome P450 (cyt P450) enzymes in ultrathin films with polyions was related for the first time to electronic and secondary structure. Heterogeneous electron transfer (hET) rate constants for reduction of the cyt P450s depended on heme iron spin state, with low spin cyt P450cam giving a value 40-fold larger than high spin human cyt P450 1A2, with mixed spin human P450 cyt 2E1 at an intermediate value. Asymmetric reduction-oxidation peak separations with increasing scan rates were explained by simulations featuring faster oxidation than reduction. Results are consistent with a square scheme in which oxidized and reduced forms of cyt P450s each participate in rapid conformational equilibria. Rate constants for oxidation of ferric cyt P450s in films by t-butyl hydroperoxide to active ferryloxy cyt P450s from rotating disk voltammetry suggested a weaker dependence on spin state, but in the reverse order of the observed hET reduction rates. Oxidation and reduction rates of cyt P450s in the films are also likely to depend on protein secondary structure around the heme iron.

Full text of DOI:10.1021/ja9065317

Published on Web 10/20/2009
Control of Electrochemical and Ferryloxy Formation Kinetics
of Cyt P450s in Polyion Films by Heme Iron Spin State and
Secondary Structure
Sadagopan Krishnan,† Amila Abeykoon,† John B Schenkman,‡ and
James F Rusling*,†,‡,§
Department of Chemistry, UniVersity of Connecticut, Storrs, Connecticut 06269, Department of
Cell Biology, UniVersity of Connecticut Health Center, Farmington, Connecticut 06032, and
School of Chemistry, National UniVersity of Ireland at Galway, Ireland
Received August 3, 2009; E-mail: james.rusling@uconn.edu
Abstract: Voltammetry of cytochrome P450 (cyt P450) enzymes in ultrathin films with polyions was related
for the first time to electronic and secondary structure. Heterogeneous electron transfer (hET) rate constants
for reduction of the cyt P450s depended on heme iron spin state, with low spin cyt P450cam giving a value
40-fold larger than high spin human cyt P450 1A2, with mixed spin human P450 cyt 2E1 at an intermediate
value. Asymmetric reduction-oxidation peak separations with increasing scan rates were explained by
simulations featuring faster oxidation than reduction. Results are consistent with a square scheme in which
oxidized and reduced forms of cyt P450s each participate in rapid conformational equilibria. Rate constants
for oxidation of ferric cyt P450s in films by t-butyl hydroperoxide to active ferryloxy cyt P450s from rotating
disk voltammetry suggested a weaker dependence on spin state, but in the reverse order of the observed
hET reduction rates. Oxidation and reduction rates of cyt P450s in the films are also likely to depend on
protein secondary structure around the heme iron.
Introduction
as CYP1A2, CYP2E1, CYP3A4, CYP2D6, CYP2C9, and
CYP2C19 are responsible for about 95% of human oxidative
Cytochrome P450s (cyt P450s) are metabolic enzymes
containing a heme iron cofactor (Figure 1). They are the major
enzymes for oxidative metabolism of lipophilic xenobiotic
chemicals including drugs and environmental pollutants, and
play a key role in metabolism-mediated toxicity.1-8 Cyt P450s
have a cysteinyl residue coordinated to the heme iron and a
distal water molecule bound above that can freely exchange
with ligands and substrates. Lipophilic substrates bind in a
hydrophobic pocket of the protein in the distal region, displacing
the water.1,3 About 57 human cyt P450s had been identified by
2007,9 and their highest concentrations are in the liver, which
is the major site of xenobiotic metabolism.4 Cyt P450s denoted
drug metabolism.1,10,11
Cyt P450s catalyze a wide variety of selective oxidations such
as heteroatom oxygenation, carbon hydroxylation, epoxidation,
dealkylation, and others.1-3 In the cyt P450 catalytic cycle,3,9
after substrate binding in the heme pocket, the cyt P450
NADPH-reductase reduces the substrate-bound ferric cyt P450
to ferrous cyt P450. This follows dioxygen binding to form a
ferrous cyt P450-dioxygen complex. Then, a second electron
transfer from NADPH-reductase and protonation yields a
FeIII-hydroperoxo complex. In some cases, Cytochrome b5 has
been shown to act as the second electron donor instead of the
cyt P450 NADPH-reductase.3,9 Bacterial P450cam used in this
study requires the iron-sulfur protein putidaredoxin as a redox
partner to deliver electrons to cyt P450s.3 Protonation and
heterolytic cleavage of the FeIII-hydroperoxo complex yields
+•(P450sFeIVdO). This ferryloxy species is presumed to be
the active oxidant that transfers oxygen to bound substrates.1,3
The presumed +•(P450sFeIVdO) form can also be generated
directly by reaction with hydrogen peroxide or organic peroxides
in the reverse of a process called the peroxide shunt.1,3 The use
of the peroxide shunt pathway to hydroxylate the substrates
bypasses complex electron delivery involving NADPH and cyt
P450 reductases.3,9
† Department of Chemistry, University of Connecticut.
‡ Department of Cell Biology, University of Connecticut Health Center.
§ School of Chemistry, National University of Ireland at Galway.
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A R T I C L E S
Krishnan et al.
Figure 1. Crystal structures of (A) cyt P450 1A2 (PDB:2HI4),51 (B) cyt P450 2E1 (PDB:3E4E),52 and (C) cyt P450cam (PDB:2CPP)53 showing the heme
iron in red. Structures were obtained from the protein data bank.54
Hill et al. first reported direct reversible voltammetry of
purified, dissolved bacterial cyt P450cam at low temperatures.12
Cyt P450s have generated interest as highly regio- and stereo-
selective synthetic catalysts for organic synthesis.13 Thus,
electron transfer from doped tin oxide to putidaredoxin provided
electrons to cyt P450cam to drive biocatalytic conversion of
camphor to 5-exohydroxycamphor in solution.14 Electrochemi-
cally mediated R-hydroxylation of lauric acid by cyt P450 4A1
fusion protein with cyt P450 reductase or by purified P450 4A1
plus NADPH-reductase dissolved in solution was achieved.15
Electrocatalytic dehalogenation of haloalkanes by cyt P450cam
in solution using ferredoxin as an electron mediator has been
demonstrated.16 Synthetic applications have been reviewed
recently.17,18
structure and facilitates reversible electron exchange with the
electrode. Advantages include avoidance of electron mediators
and of diffusion of large protein molecules, minimizing or
eliminating electrode fouling by denatured protein, and economy
of enzyme use.
We reported the first direct reversible protein film voltam-
metry of cyt P450cam using thin surfactant films on pyrolytic
graphite (PG) electrodes,23 as well as the first direct reversible
voltammetry of this enzyme in protein-polyion films con-
structed layer-by-layer (LbL).24 We subsequently pursued direct
cyclic voltammetry and qualitative mechanistic studies of
electrochemical biocatalysis using bacterial and human cyt
P450s in these films.25
A number of subsequent studies addressed other cyt P450s
by direct film voltammetry. Farmer et al. reported electrocata-
lytic reduction of nitrite, nitric oxide, and nitrous oxide using
thermophilic cyt P450 119 in didodecyldimethylammonium
bromide (DDAB)-poly(styrene sulfonate) (DDAPSS) films.26
They also demonstrated electrocatalytic conversion of CCl4 to
CH4 using this thermophilic P450 CYP119 in DDAPSS films
at high temperatures.27 Other studies of cyt P450s on electrodes
have been aimed at applications including drug metabolism
sensors,28-30 drug sensors,31,32 monitors of cyt P450-drug
interactions,33 and clinical uses.34
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Cyt P450s in Polyion Films
A R T I C L E S
Scheller et al.35 used Laviron’s method,36 to estimate
heterogeneous electron transfer (hET) rate constants for cyt
P450cam adsorbed to clay modified glassy carbon in the range
5-152 s-1 at scan rates 0.4-12 V s-1. This large variation of
hET rate with scan rate suggests poor agreement of the Laviron
model to the data. Electron transfer kinetics of cyt P450 2B4 at
glassy carbon electrodes modified with nonionic detergent and
colloidal clay37 yielded a rate constant of 80 s-1. Gilardi et al.38
reported hET rate constants for cyt P450 2E1 of 5 ( 0.5 s-1
immobilized on glassy carbon, and 2.0 ( 0.5 s-1 on glassy
carbon/poly(diallyldimethyl ammonium chloride) (PDDA) or
thiol-modified gold with PDDA as an underlayer. A ks of 10 (
0.5 s-1 was obtained for cyt P450 2E1 covalently linked via
cysteine to maleimide on cysteamine modified gold.38
Scheme 1. Representation of LbL Films of Cyt P450s and Polyions
on PG Electrodesa
a Polyions are purple strands, and proteins are green/red ribbon structures.
conformations.20,21,44,46 Kinetics of reduction and oxidation
processes were measured by voltammetry for human cyt P450s
1A2 and 2E1, bacterial cyt P450cam, and the heme proteins
myoglobin (Mb) and catalase. Results are explained by differ-
ences in FeIIIP450 spin states, secondary structures, and rapid
conformational equilibria coupled to electron transfer.
Direct electrochemistry of human cyt P450s 2C9, 2C18, and
2C19 in DDAB films on an edge plane pyrolytic graphite was
reported without hET rates.39 Martin et al. studied the influence
of pH on the hET rate constant of human cyt P450 2C9 in
DDAB films on edge-plane pyrolytic graphite.40 Using Laviron’s
method, they found ks of 139 s-1 at pH 6.0, 150 s-1 at pH 7.4,
and 165 s-1 at pH 8.2. Gray et al.41 found a hET rate constant
of 10 s-1 at 10 V s-1 for cyt P450 BM3 in sodium dodecyl
sulfate films on basal plane pyrolytic graphite, and compared
voltammetry of wild type cyt P450 BM3 with its mutant.42
Direct voltammetry of human cyt P450 2B6 on zirconium
dioxide nanoparticles on glassy carbon was also reported.43
Experimental Section
Bacterial Pseudomonas putida cyt P450 101 (cyt P450cam, MW
47 500),47 human cyt P450s 1A2 (MW 58 200),48 and 2E1 (MW
56 900)49 were expressed from DH5R Escherichia coli containing
the relevant cDNA, then isolated and purified according to the
referenced literature. Horse heart myoglobin (Mb, MW 16 900),
poly(ethyleneimine) (PEI), poly(diallyldimethyl ammonium chlo-
ride) (PDDA), poly(sodium 4-styrene sulfonate) (PSS), catalase
(Bovine liver, MW 247 500), and t-butyl hydroperoxide (t-BuOOH)
were from Sigma.
A CHI 660A electrochemical analyzer was used for voltammetry
at 25 °C. CHI software was used to simulate thin film voltammo-
grams (see Supporting Information for full details). Thin film
voltammetry was done in 3-electrode cell as described previously.50
Electrolyte was 50 mM pH 7.0 potassium phosphate buffer or
appropriate buffers of other pH25b containing 0.1 M NaCl. Buffers
Considering these numerous prior reports, a major challenge
remains to understand how the electronic structure of the heme
iron and the secondary structure around it controls the dynamics
of electron exchange of cyt P450s with electrodes and of
forming the active ferryloxy form +•(P450sFeIVdO). Elucidat-
ing these relationships is the goal of the work described herein.
Such information can provide insights into biological electron
transfer processes of cyt P450 enzymes, and how they vary with
enzyme structure, and are also important for electrochemical
catalytic synthesis and sensing applications of cyt P450s.
were purged with purified nitrogen before voltammetry. Rotating
50
disk voltammetry was done at 1000 rpm and scan rate 0.1 V s-1
.
Polyion/enzyme films were assembled layer-by-layer (LbL) on
basal plane pyrolytic graphite (PG) electrodes as previously
described (see Supporting Information for details).25 Assembly of
LbL polyion-enzyme film was monitored at each step using a quartz
crystal microbalance (QCM).25 Spectroscopy of P450/polyion LbL
films was done with films fabricated on aminoalkylsilated fused
silica slides as previously described.55
In the present paper, we compare for the first time three
different cyt P450s with regard to kinetics of direct electron
transfer and formation of +•(P450-Fe(IV)dO) in ultrathin
enzyme/polyion films constructed layer-by-layer (LbL) (Scheme
1). We chose the LbL method44,45 for enzyme immobilization
because of its versatility, facilitation of direct electron transfer
for enzymes,20,21 and ability to stabilize proteins in near native
Results
LbL Assembly of Enzyme/Polyion Films. Table 1 describes
LbL enzyme film architectures that were constructed for this
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A R T I C L E S
Krishnan et al.
Figure 2. UV-vis spectra of films on aminosilane-functionalized fused silica slides: (A) CO difference spectrum for PEI(/PSS/cyt P450 1A2)6 film after
reducing to the ferrous form and purging the pH 7 buffer with CO, (B) ferric form of enzyme in PEI(/PSS/cyt P450 1A2)6, and (C) ferric form of enzyme
in PSS(/PEI/cyt P450cam)6 film.
Table 1. Average Characteristics of Enzyme Layer-by-Layer Assemblies from QCM Studies
film architecture
thickness (nm)
amount of enzymea (nmol cm-2
)
enzyme conc in film (µmol cm-3
)
PEI(/PSS/1A2)4
PEI(/PSS/2E1)4
PSS(/PEI/cam)4
PEI(/PSS/Mb)2
PEI(/PSS/Mb)4
PEI(/PSS/Mb)6
PSS(/PEI/catalase)4
PSS(/PEI/hemin)4
24 ( 1
24 ( 3
9 ( 1
0.11 ( 0.01
0.12 ( 0.02
0.05 ( 0.003
0.12 ( 0.01
0.21 ( 0.04
0.32 ( 0.03
0.015 ( 0.001
6.6 ( 0.82
46 ( 4
51 ( 8
55 ( 4
9 ( 2
130 ( 11
130 ( 24
140 ( 13
12 ( 1
16 ( 3
24 ( 2
13 ( 1
19 ( 2
3500 ( 430
a Average ( SD for films on 3 resonators.
work. QCM mass measurements on gold resonators (Supporting
Information, Figure S1) having alkylthiol carboxylate groups
to mimic the surface of pyrolytic graphite confirmed regular
and reproducible assembly of polyion/enzyme films as described
elsewhere.56 The nominal thickness of dry films and amount of
enzyme in the film (Table 1) were estimated by QCM. While
the films are constructed layer-by-layer, the final film structure
features considerable mixing of neighboring layers (Scheme 1),
which actually facilitates charge transport through the film.20,21,44
Cyt P450s 1A2 and 2E1 films had a similar nominal thickness
of ∼24 nm, whereas cyt P450cam films were about 9 nm thick.
Though cyt P450 1A2, cyt P450 2E1, and cyt P450cam are of
similar size, the bacterial cyt P450cam formed relatively thinner
films than the human cyt P450s (Table 1). Dividing the amount
of enzyme in each film in nmol cm-2 by film thickness reveals
that the cyt P450 films have very similar enzyme concentrations
(Table 1). The observed nominal film thickness of polyion and
catalase films was 13 nm. With neutral buffer solutions from
which to grow these enzyme films, the human cyt P450s (pI >
8) have a net positive charge, whereas bacterial cyt P450cam
(pI 4.6) and catalase (pI 5.8) have a net negative charge. These
observations are consistent with layer thickness for these
enzymes controlled by relative surface charges of the film
components.57
polyions (∼1.5 nm) deposited first on aminoalkylsilated fused
silica slides.46,55 As on the electrodes, this protects the protein
from adsorbing on the bare surface and ensures that the spectra
are characteristic of protein in the bulk of the film. Carbon
monoxide binds to ferrous cyt P450s to form a cyt P450
FeII-CO complex that has a characteristic difference absorbance
band near 450 nm that gave these enzymes their name.58
Denatured cyt P450s show the FeII-CO band near 420 nm. To
verify that P450 enzymes in the LbL films retained structural
integrity, CO-difference spectra were measured for human cyt
P450s in LbL films grown on aminoalkylsilated glass slides.
Figure 2A shows the CO difference spectrum for PEI(PSS/P450
1A2)6 film with the absorbance band near 450 nm consistent
with that reported for native cyt P450 enzyme in solution, and
for P450cam in LbL films.55
Spectroscopic studies of ferric cyt P450 films were done to
confirm heme iron spin states. Figure 2B shows the UV-vis
spectrum of the PEI(/PSS/cyt P450 1A2)6 film, and Figure 2C
is that of the PSS(/PEI/cyt P450cam)6 film. The absorbance
maximum at 394 nm for the PEI(/PSS/cyt P450 1A2)6 film
(Figure 2B) corresponds to the high spin heme iron in cyt P450
1A2,51,59 and that of 420 nm for the PSS(/PEI/cyt P450cam)6
film (Figure 2C) confirms the low spin electronic state of heme
iron in cyt P450cam as reported previously.23,53,60,61
For myoglobin, 4-bilayer PSS/Mb films gave a thinner
assembly of ∼16 nm attributed to the smaller protein size (Table
1). In order to study the influence of LbL film thickness on the
voltammetry, to be discussed below, we also assembled 2 and
6 bilayer films of PSS/Mb layers having the thicknesses 9 and
24 nm, respectively. Mb concentrations in these three films were
similar (Table 1).
Voltammetry of Cyt P450 Films. While the LbL films are
made one layer at a time, significant interlayer mixing in
polyion-proteinfilmsmadebyLbLhasbeenwelldocumented.24,45
This mixing (cf. Scheme 1) facilitates charge transport through
the film most likely by keeping the average distance between
heme centers of neighboring protein molecules within a range
Spectroscopic Characterization. Spectra were taken of films
similar to those used for voltammetry, with the first 2 layers of
(58) Omura, T.; Sato, R. J. Biol. Chem. 1964, 239, 2379–2385.
(59) Sandhu, P.; Guo, Z.; Baba, T.; Martin, M. V.; Tukey, R. H.;
Guengerich, F. P. Arch. Biochem. Biophys. 1994, 309, 168–177.
(60) Poulos, T. L.; Finzel, B. C.; Howard, A. J Biochemistry 1986, 25,
5314–5322.
(56) Zhou, L.; Yang, J.; Estavillo, C.; Stuart, J. D.; Schenkman, J. B.;
Rusling, J. F. J. Am. Chem. Soc. 2003, 125, 1431–1436.
(57) Schenkman, J. B.; Jansson, I.; Lvov, Y.; Rusling, J. F.; Boussaad, S.;
Tao, N. J. Arch. Biochem. Biophys. 2001, 385, 78–87.
(61) (a) Peterson, J. A. Arch. Biochem. Biophys. 1971, 144, 678–693. (b)
Harris, D.; Loew, G. J. Am. Chem. Soc. 1993, 115, 8775–8779.
9
16218 J. AM. CHEM. SOC. VOL. 131, NO. 44, 2009

Cyt P450s in Polyion Films
A R T I C L E S
Figure 3. Background subtracted thin film voltammograms of cyt P450
enzymes in films on pyrolytic graphite electrodes in anaerobic 50 mM
potassium phosphate buffer + 0.1 M NaCl, pH 7.0: (A) PEI(/PSS/cyt P450
2E1)4 and (B) PSS(/PEI/cyt P450cam)4 .
in which electron hopping between them is relatively efficient.25
Efficient electron transport through LbL films of redox proteins
and enzymes is reflected by nearly reversible CVs.20,21
Figure 3A,B represents the background subtracted cyclic
voltammograms (CV) of PEI(/PSS/P450 2E1)4 and PSS(/PEI/
P450cam)4 films on pyrolytic graphite electrodes with increasing
scan rates in anaerobic pH 7.0 buffer. Well-defined quasire-
versible cyt P450 heme FeIII/FeII redox peaks can be observed
from these CVs.
Peak currents were proportional to scan rate and oxidation-
reduction peak current ratios were close to 1. Table 2 gives
formal potential (Eo′) values, amount of electroactive enzymes
in the films obtained from the integration of the reduction peak
and using Faraday’s law,21 and the percentage of electroactive
enzyme.25
Figure 4. Influence of cyclic voltammetry scan rate for enzyme films on
experimental (b) peak separation (∆Ep) corrected for scan rate independent
nonkineticcontribution.ThetheoreticallineswerecomputedforButler-Volmer
theory for the rate constant (ks) values shown and R ) 0.5.
Notably, the formal potential of the cyt P450cam film was
similar when either PEI or PDDA was the polycation in the
film, suggesting that the type of polycation has little effect on
the voltammetry (Table 2). Furthermore, since the outer layer
of each film is protein, the outer layer charge will be
characteristic of the protein, while internal charge is completely
compensated by protein-polyion interactions.44 The formal
potential was most negative for the catalase film while that of
the hemin film fell in the midrange of all the proteins examined
(Supporting Information Figure S3). E0′ values for the enzymes
are similar to those reported previously in LbL films.25b,c,38,62
The amounts of electroactive cyt P450s in the films were
obtained from the ratio of electroactive surface concentration
to total concentration from QCM and are in the range 15-40%
(Table 2). For hemin alone, the electroactive fraction was very
small (2%).
Table 2 also shows the shift in formal potential of cyt P450s
and myoglobin films upon CO binding to form the cyt
P450-FeII-CO complex. Complex formation following electron
transfer results in a positive shift of midpoint potential that is
characteristic of heme iron enzymes.20-24,35,40 For hemin, this
shift was slightly higher than for protein films (Table 2).
Catalytic oxygen reduction from the CVs of cyt P450 films
was observed from the increase in reduction peak current in
the presence of oxygen over that of anaerobic buffer (Figure
S5). Also, positive formal potential shifts in the CVs of protein
films were observed with lowering pH in the pH range 4-9
(Figure S6) suggesting proton coupled electron transfer. The
change in formal potential per pH unit was in the range 43-50
mV pH-1 for these films which is slightly less than the
theoretical 59 mV pH-1 for a reversible one electron process
coupled to proton transfer.
Figure 5. Apparent heterogeneous electron transfer rate constant (ks’s) for
films of cyt P450s, myoglobin (Mb), and catalase (Cat) from CVs in pH 7
buffer + 0.1 M NaCl. For Mb, the ks values for different films thicknesses
utilizing 2, 4, or 6 bilayers are shown.
Heterogeneous Electron Transfer (hET) Rate Constants. For
the estimation of hET rate constants, we used CVs of enzyme
films at scan rates 0.005-1.4 V s-1 where good quality data
were obtained. Following the suggestion of Hirst and Arm-
strong,63 we first subtracted the constant nonkinetic component
from the oxidation-reduction peak separation (∆Ep) at lower
scan rates from that at higher scan rates and fit the corrected
peak separations to Butler-Volmer theoretical peak separation
versus scan rate theory for surface confined voltammetry.36
Average standard hET rate constants (ks’s) were estimated using
the corrected ∆Ep and the working curve for R ) 0.5 in Figure
4, ref 36. Good fits of theory to experiment (Figure 4) indicated
reliable estimates of ks.
Figure 5 compares the ks values obtained at pH 7 for the
enzyme films. Among the cyt P450s, cyt P450cam gave the
largest hET rate constant, while cyt P450 2E1 was intermediate,
and cyt P450 1A2 was the smallest. The electron transfer rate
for the cyt P450 2E1 film is significantly larger than that reported
for this enzyme on bare glassy carbon, a glassy carbon/
polycation surface, or a gold electrode with a thiol monolayer
and polycation surface.38 This suggests that faster electron
transfer of cyt P450s can be achieved within the LbL architec-
ture. Mb and catalase films gave hET rate values similar to cyt
(62) Shen, L.; Hu, N. Biomacromolecules 2005, 6, 1475–1483.
(63) Hirst, J.; Armstrong, F. A. Anal. Chem. 1998, 70, 5062–5071.
9
J. AM. CHEM. SOC. VOL. 131, NO. 44, 2009 16219

A R T I C L E S
Krishnan et al.
Figure 6. Trumpet plots showing the influence of scan rate on CV peak potentials in anaerobic pH 7.0 buffer. Experimental oxidation (red diamonds) and
reduction (blue circles) peak potentials are shown for (A) PEI(/PSS/P450 1A2)4, (B) PEI(/PSS/P450 2E1)4, (C) PSS(/PEI/P450cam)4, (D) PSS(/PEI/catalase)4,
and (E) PEI(/PSS/myoglobin)4 films. The lines were obtained by best fit simulations using an Ereduction/Eoxidation mechanism using the reduction and oxidation
rate constants shown in each plot.
Table 2. Electrochemical Parameters and Formal Potential Shift of Enzyme Films upon CO Binding in 50 mM Phosphate Buffer Plus 0.1 M
NaCl at pH 7.0
film assembly
E0′ vs SCE (V)
Γa (nmol cm-2
)
% electroactive enzyme
+E0′ shift in CO (mV)
PEI(/PSS/P450 1A2)4
PEI(/PSS/P450 2E1)4
PSS(/PEI/P450cam)4
PSS(/PDDA/P450cam)4
PEI(/PSS/Mb)4
-0.330 ( 0.010
-0.347 ( 0.009
-0.363 ( 0.002
-0.366 ( 0.004
-0.337 ( 0.005
-0.467 ( 0.005
-0.370 ( 0.004
0.016 ( 0.005
0.030 ( 0.004
0.021 ( 0.004
0.019 ( 0.003
0.085 ( 0.007
0.003 ( 0.001
0.13 ( 0.02
15 ( 4
25 ( 3
42 ( 8
38 ( 6
40 ( 3
20 ( 7
2.0 ( 0.3
36 ( 4
50 ( 4
46 ( 3
48 ( 5
49 ( 4
PSS(/PEI/catalase)4
PSS(/PEI/hemin)4
58 ( 3
a Γ ) surface concentration of enzyme in the film from CV; av ( SD for n ) 4-6 electrodes.
P450 2E1. Also, Mb films of thickness between 9 and 24 nm
gave hET rate constants that were essentially the same,
indicating that there is little effect of film thickness in this range
on the measured ks values. This result confirms the validity of
comparing hET rate constants for the cyt P450 films of different
thicknesses.
different electrochemical rate constant for its oxidation compared
to the reduction rate constant. Thus, taking the ks estimated from
the Laviron model as the starting reduction rate constant (now
denoted as ks,red) and evaluating multiples of this value for the
oxidation rate (ks,ox), we arrived at the best fit illustrated by the
theoretical lines that gave good fits to the experimental trumpet
plots for all proteins (Figure 6, see Figure S7 for simulated CVs).
As discussed later, these simulations are consistent with a square
scheme in which oxidized and reduced forms of the proteins
are involved in conformational equilibria that are fast with
respect to the time scale of the voltammetry.
The linear relation of peak current to scan rate, similarity of
oxidation and reduction peak heights, and good fits of ∆Ep
versus scan rate (Figure 4) are consistent with surface confined
species, and suggest a nearly reversible thin film voltammetry
model.20,21 However, the so-called trumpet plots of individual
oxidation and reduction peak potentials versus scan rate should
be symmetric for a simple quasireversible surface electron
transfer process,19b,c but they were distinctly unsymmetrical for
the cyt P450 films (Figure 6). Reduction peaks shifted more
rapidly than oxidation peaks with increasing scan rate, which
is also inconsistent with a simple electron transfer process.
Thus, digital simulations of CVs were done to fit trumpet
plots. The two simplest mechanisms likely to explain the data
were considered: ECreduction/Eoxidation (where E ) electron transfer,
C ) chemical step) and Ereduction/Eoxidation. While both mecha-
nisms were capable of explaining the general trends observed
in peak potentials, the Ereduction/Eoxidation simulation gave the best
fits by far. These simulations embody the concept that the
reduced species could be rapidly converted into a different form,
e.g., by a conformation change, and so it could take on a
Kinetics of Ferryloxy Cyt P450 Formation. Rotating disk
voltammetry was used to monitor the oxidation of cyt P450s to
their active ferryloxy forms. A well studied example of this
reaction is the oxidation of ferric Mb by t-BuOOH which
64-67
·
produces the ferryloxy species MbFeIVdO, a strong oxidant.
Cyt P450s can be activated a similar way.1-3 On the basis of
knowledge of these processes, a simplified Michaelis-Menten
reaction pathway for RDV similar to that proposed for Mb in
films can be represented by Scheme 2.50
(64) Allentoff, A. J.; Bolton, J. L.; Wilks, A.; Thompson, J. A.; Ortiz de
Montellano, P. R. J. Am. Chem. Soc. 1992, 114, 9744–9749.
(65) Reeder, B. J.; Svistunenko, D. A.; Cooper, C. E.; Wilson, M. T.
Antioxidants Redox Signaling 2004, 6, 954–966.
(66) Chen, Y.-R.; Mason, R. P. Biochem. J. 2002, 365, 461–469.
(67) Van der Zee, J. Biochem. J. 1997, 322, 633–639.
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16220 J. AM. CHEM. SOC. VOL. 131, NO. 44, 2009

Cyt P450s in Polyion Films
A R T I C L E S
Scheme 2. Oxidation Pathway of Cyt P450s by Organic Peroxides
a fraction of electroactive hemin that was very low (Table 2).
These data show that electrochemical properties of hemin LbL
films are very different from those of protein films. Taken
together with the spectroscopy and many studies showing that
cytP450enzymesretainenzymeactivityinsimilarfilms,20,30,45,55,56
results strongly support the view that the cyt P450s retain near
native structures in the films. This is also consistent with the
many other enzymes demonstrated to maintain native structures
and enzyme activities in LbL films.20,21,30,44-46
via RDV
Equation 1 in Scheme 2 describes binding of substrate (t-
BuOOH) to ferric cyt P450 enzyme forming a cyt P450-substrate
complex characterized by Michaelis dissociation constant KM,
followed by formation of the ferryloxy radical cation and
conversion of t-BuOOH into t-BuOH characterized by kcat, eq
2, Scheme 2. The formed ferryloxy species is subsequently
reduced by the electrode regenerating ferric cyt P450 (eq 3,
Scheme 2).
Figure 7 shows the representative steady state RDVs showing
increasing limiting currents with increasing t-BuOOH concen-
tration in pH 7.0 buffer due to this pathway. t-BuOOH is reduced
by cyt P450s in the polyion films according to Scheme 2 to
give the catalytic reduction limiting current that increases with
increasing t-BuOOH concentration.
Electrochemical Dynamics. Well defined quasireversible CVs
of cyt P450 films (Figure 3) enabled a comparative study of
the effect of cyt P450 structure on the kinetics of direct electron
transfer for the first time. Demonstration of good model fitting
is an essential test confirming the estimation of reliable hET
rate constants (ks).20,21 After a correction was made for
nonkinetic peak separations that do not depend on scan rate,63
plots of peak separation versus scan rate agreed well with
Butler-Volmer theory for surface confined electron transfer
(Figure 4), and peak currents were proportional to scan rate.
The values of ks obtained from this first level of analysis
correlate with heme spin state. Cyt P450cam having predomi-
nantly low spin heme iron53,60 gave the largest electron transfer
rate (95 ( 11 s-1, Figure 5), high spin cyt P450 1A2 (>90%)59
gave the smallest electron transfer rate (2.3 ( 0.4 s-1), and cyt
P450 2E1 with mixed spin states (∼80% high spin)49,52 gave
an intermediate value (18 ( 4 s-1).
The catalytic current Icat in rotating disk voltammetry is related
to kinetic parameters (Scheme 2) by an electrochemical version
of the Michaelis-Menten equation (eq 4):19a
nFAΓ(kcat/KM)Cs
Icat
)
(4)
(1/KM)Cs + 1
On the basis of biochemical studies, Guengerich et al.68-70
reported that low spin forms of cyt P450s 1A2 and 2E1 undergo
significantly faster reduction than the corresponding high spin
forms, although these correlations were difficult to uncouple
from other structural factors. Cyt P450 2E1 is reduced by
NADPH-cyt P450 reductase70 about 2.5 times faster than cyt
P450 1A2 consistent with our finding that mixed spin cyt P450
2E1 is reduced about 7 times faster than high spin cyt P450
1A2 using the electrode as electron donor. Thus, qualitatively,
the dependence of hET rates in the films on cyt P450 heme
spin states seems consistent with literature precedents.
where n is the number of electrons in the electrochemical
reaction (n ) 2 in our case, Scheme 2), F is Faraday’s constant
(96 485 C mol-1), A is electrode area (0.2 cm2), Γ is the
electroactive surface concentration of enzyme in the film (in
nmol cm-2), Cs is substrate concentration in solution (in mM),
kcat is the catalytic rate constant (s-1) assumed to be rate limiting,
and KM is the Michaelis-Menten dissociation constant. Icat is
the catalytic current obtained by extrapolating limiting current
(IL) to infinite rotation rate at each Cs. Here, to conserve enzyme,
we used limiting currents at one large rotation rate to ap-
proximate Icat to obtain relative Michaelis parameters50 for
comparisons between enzymes.
In addition, voltammetry studies by Feng and Schultz showed
that free low spin heme iron complexes (not bound to proteins)
gave faster electron transfer rates than high spin complexes by
about 0.3-1.3 orders of magnitude.71 They explained that the
reduction of high spin heme complexes involves greater
activation energy than the reduction of low spin heme com-
plexes. They also showed that the electron self-exchange rate
is several-fold larger for low spin hemes compared to high
spin.71 Unlike the natural reductase-P450 electron transfer
involving a prior protein-protein interaction on a membrane,
in thin film voltammetry an electron is transferred from the solid
electrode to heme irons of the cyt P450 near the surface, and
self-exchange within the film propagates charge transfer.20,21
Thus, correlation of hET rate constants to spin state the same
as in model heme iron complexes is reasonable.
Figure 8 shows the turnover rates from RDV versus t-BuOOH
concentration for all the films. Fitting these experimental
turnover plots with the electrochemical Michaelis-Menten
equation (eq 4), using nonlinear regression, provided apparent
KM and kcat values. The ratio kcat/KM has units of a second order
rate constant and is a relative measure of catalytic efficiency
(Table 3). These data suggested the following order for rate of
ferryloxy protein formation: P450 1A2 > P450 2E1 > myoglobin
> P450cam . catalase.
Discussion
Enzyme Characterization in Films. Near native enzyme
structures in the films were confirmed by UV-vis spectroscopy
reflecting known high and low spin ferric heme iron bands of
cyt P450s and by characteristic 450 nm bands of reduced cyt
P450-CO complexes (Figure 2). CO binding to reduced cyt
P450s in the films was confirmed by positive midpoint potential
shifts in CVs (Table 2).
We also need to consider the structural factors (Figure 1)
that may influence the electron transfer rates. Considering
While the spectroscopic data argue conclusively against it,
in rare cases loss of heme from the protein in LbL films might
be envisioned. Relevant to this issue, hemin films (Figure S3,
Table 1) had peak potentials that changed very differently with
increasing scan rate from the protein films (Figure S4), gave
larger CO potential shifts than the proteins (Table 2), and had
(68) Yamazaki, H.; Ueng, Y.-F.; Shimada, T.; Guengerich, F. P. Biochem-
istry 1995, 34, 8380–8389.
(69) Yamazaki, H.; Johnson, W. W.; Ueng, Y.-F.; Shimada, T.; Guengerich,
F. P. J. Biol. Chem. 1996, 271, 27438–27444.
(70) Guengerich, F. P.; Johnson, W. W. Biochemistry 1997, 36, 14741–
14750.
(71) Feng, D.; Schultz, F. A. Inorg. Chem. 1988, 27, 2144–2149.
9
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A R T I C L E S
Krishnan et al.
Figure 7. Representative steady state RDVs at 1000 rpm for protein films on PG electrodes for t-BuOOH reduction in anaerobic 50 mM potassium phosphate
buffer + 0.1 M NaCl, pH 7.0 at 25 °C. RDVs for increasing t-BuOOH concentration (in µM) for (A) PEI(/PSS/P450 2E1)4, (B) PSS(/PEI/P450cam)4, and
(C) PSS(/PEI/catalase)4 films.
This asymmetry was best fit by using digital simulations of
the redox process (Figure 6) using an Ereduction/Eoxidation mecha-
nism. As mentioned earlier, ECreduction/Eoxidation simulations were
able to explain trends, but gave much worse fits to the data
than Ereduction/Eoxidation simulations. Using the Occam’s razor
principle, we relied on Ereduction/Eoxidation simulations for a second
level kinetic analysis, realizing that results need to be interpreted
in a chemically logical manner. Data for cyt P450 2E1 films
with the highest asymmetry (Figure 6B) were fit reasonably well
by simulations using 18 s-1 for ks,red and a g100-fold larger
ks,ox(g1800 s-1). This simulation remained the same upon further
increases of ks,ox to 18 × 106 s-1. On the other hand, when both
the reduction and oxidation values were the same (18 s-1) or
Figure 8. Michaelis-Menten fits (eq 4) of relative turnover rate (IL/Γ) vs
when ks,ox of only 10-fold (180 s-1) larger than the reduction
t-BuOOH concentration for cyt P450, myoglobin, and catalase films. Points
rate (18 s-1), nearly symmetrical trumpet plots were found that
are experimental data, and solid lines represent the best fit of eq 4 by
nonlinear regression. RDV parameters are as in Figure 7.
gave poor fits to experimental data.
A simulation of cyt P450cam with ks,ox g 950 s-1 and ks,red
Table 3. Apparent Relative kcat, KM, and kcat/KM Values Obtained
from Michaelis-Menten Fitting of RDV Data for Reduction of
of 95 s-1 gave the best fit to experimental data (Figure 6C).
Similarly, the less asymmetric cyt P450 1A2 (Figure 6A),
catalase (Figure 6D), and Mb (Figure 6E) trumpet plots were
t-BuOOH
film assembly
kcat (s-1
)
KM (mM)
104 kcat/ KM (M-1 s-1
)
best fit using a ks,ox 10 times larger than ks,red
.
PEI(/PSS/1A2)4
PEI(/PSS/2E1)4
PSS(/PEI/cam)4
PEI(/PSS/Mb)4
PSS(/PEI/catalase)4
83 ( 7
74 ( 5
49 ( 4
15 ( 2
7 ( 1
1.05 ( 0.11
1.43 ( 0.1
2.0 ( 0.24
0.36 ( 0.03
15.4 ( 3.7
7.9 ( 0.8
Simulations using the ECreduction/Eoxidation mechanism were
tested for cyt P450 2E1 films featuring the same ks,red and ks,ox
as used above, and with a reversible chemical (C) step using
various values of forward (kf) and reverse (kb) chemical rate
constants. Among all trials, the asymmetry was best reproduced
using kf 3.6 s-1 and kb 0.018 s-1 (Figure S8) for the chemical
equilibrium, but shifts in Ep,red with scan rate fit poorly compared
to the Ereduction/Eoxidation best fit (Figure 6B).
Asymmetry in trumpet plots on the order of that for cyt
P450cam and cyt P450 1A2 was found by Hirst and Armstrong
for the S. acidocaldarius (Sa Fd) ferredoxin and A. Vinelandii
ferredoxin-I mutant (D15N) adsorbed on edge plane pyrolytic
graphite electrodes.63 They attributed this to the conformational
or orientational changes coupled to electron transfer that gate
electron transfer at scan rates well above those used in our work.
A square scheme incorporating conformational equilibria coupled
to the electron transfer was suggested,63 but this mechanism
was not tested with simulations.
The Ereduction/Eoxidation simulation model used here requires an
oxidized form of the enzyme reduced at a moderate rate, and a
reduced form that is oxidized at a faster rate. This is an
elaboration of the Butler-Volmer surface voltammetry model36
that assumes that a redox system can be characterized by a single
ks that is the rate constant for ET at its formal potential.36 The
Ereduction/Eoxidation model can be interpreted in terms of two
separate redox couples, one for reduction and another for
oxidation. In this way, different electrochemical rate constants
for the reduction and oxidation can be rationalized. These two
redox couples can arise if oxidized and reduced forms of the
5.2 ( 0.4
2.5 ( 0.2
4.2 ( 0.5
0.045 ( 0.006
differences between the cyt P450s,72 human cyt P450s 1A2 and
2E1 share ∼30% sequence identity, and cyt P450 1A2 has no
sequence identity with cyt P450cam. The structure of cyt
P450cam features flexible helices,73,74 while human cyt P450s
have more compact secondary structures (Figure 1). This
conformational flexibility of cyt P450cam structure may lead
to subconformations with the result of entropically favored faster
direct electron transfer rates between the electrode and cyt
P450cam by facilitating a shorter average ET path. We are
currently pursuing quantum mechanical/molecular mechanics
(QM/MM) modeling of cyt P450s to better understand these
structural influences on ET rates.
Asymmetry in the CV trumpet plots (Figure 6) features much
smaller shifts in oxidation peak potentials compared to reduction
peak potentials. This is most prominent for the cyt P450 2E1
film (Figure 6B) compared to cyt P450 1A2 and cyt P450cam
(Figure 6A,C). Catalase and myoglobin films exhibited the
smallest asymmetry in trumpet plots (Figure 6D,E).
(72) Sequence identity between P450s was determined using the BLAST
program from the following website:http://blast.ncbi.nlm.nih.gov/
Blast.cgi.
(73) Dunn, A. R.; Dmochowski, I. J.; Bilwes, A. M.; Gray, H. B.; Crane,
B. R. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12420–12425.
(74) Prasad, S.; Mazumdar, S.; Mitra, S. FEBS Lett. 2000, 477, 157–160.
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16222 J. AM. CHEM. SOC. VOL. 131, NO. 44, 2009

Cyt P450s in Polyion Films
A R T I C L E S
documented in the literature. Lisdat et al.82 reported electron
hopping in direct electron exchange of cytochrome c with
electrodes in polyelectrolyte multilayer films. We realize that
the hET rate constants reported in this paper should be
considered characteristic of the charge transport properties of
the entire film, and not only the proteins. However, since ks
was independent of thickness of the films, internal charge is
compensated, and outer layers are always protein and will have
charge characteristic of the given protein, we can justify the
use of kinetic constants for the relative comparisons for cyt P450
films we have discussed above. Clearly, the rate constant values
should not be taken as absolute.
Scheme 3. Square Scheme Explaining Cyt P450 Thin Film
Voltammetry
enzymes are involved in separate conformational equilibrium.75
Conformational equilibria and their influence on thin film
voltammetry have been well documented for cytochrome c,76,77
myoglobin,78 and several ferredoxins.63 This concept implies a
square scheme mechanism63,76 (Scheme 3). The horizontal
reactions in Scheme 3 are electrochemical steps, and the vertical
transitions are likely to be conformational equilibria. The fact
that the Ereduction/Eoxidation simulations fit our data for the proteins
implies that the conformational equilibria are fast relative to
the CV time scale. This is also consistent with findings above
for ferredoxins in which the proposed conformational-based
asymmetry in trumpet plots63 was only manifested at much
higher scan rates than was possible for the cyt P450 films.
The square scheme (Scheme 3) implies reduction of the cyt
P450 enzyme with heme iron in FeIII state [P(Ox)1] to FeII heme
[P(Red)1] by an electron supplied by the electrode. Conforma-
tional change produces P(Red)2 which is oxidized to P(Ox)2
with ks,ox . ks,red. This explains the smaller shifts in experimental
Ep,ox with increasing scan rate compared to the shifts in Ep,red
(Figure 6B). Since peak potentials are also pH dependent (Figure
S6), the participation of protons in the conformational changes
is also possible.
Certainly, the above electron transfer model remains an
approximation as it does not consider electron self-exchange
or “hopping”79 through the films of 9-24 nm. We roughly
estimated the distance between adjacent redox centers of cyt
P450s in the films. From the experimentally obtained amount
of protein molecules per layer from QCM, protein diameter,80
and film thickness, we estimated an upper limit of protein heme
group separation from its adjacent neighbor at e4 nm assuming
a uniform protein distribution and absence of specific interac-
tions. However, we know that strong electrostatic and other
secondary interactions exist in LbL films as well as interlayer
mixing of neighboring protein layers.25,44 This may further
decrease the effective distance between adjacent protein redox
centers in LbL films as would intermolecular electron transfer
via water channels,81 all of which favor efficient electron self-
exchange between protein redox centers. The role of electron
hopping in direct electron transfer in protein LbL films has been
Oxidation to Ferryloxy Cyt P450s. Increasing RDV limiting
currents with increasing t-BuOOH concentration are due to the
enzyme-catalyzed reduction of t-BuOOH (Scheme 2, Figure
7A,B). Catalase films gave very small catalytic currents for the
peroxide and serve as a negative control (Figure 7C).
The kcat for oxidation of human cyt P450s is about 5-fold
greater than that of myoglobin (Table 3), and ∼1.5 times larger
than for cyt P450cam. The two human cyt P450s had compa-
rable binding affinity for t-BuOOH as shown by KM, but cyt
P450cam had (Table 3) less binding affinity. Myoglobin showed
strong binding of t-BuOOH (lowest KM, 0.36 mM), and catalase
showed the weakest binding (highest KM). The binding trends
are most likely controlled by the nature of the protein secondary
structure around the heme group, as discussed below.
The ratio kcat/KM, which can be considered relative catalytic
efficiency,19 was larger for the human cyt P450s than for cyt
P450cam and myoglobin. Again, this can be related to differ-
ences in the amino acids close to the active heme group, which
are different in cyt P450cam than in human cyt P450s. Due to
the stronger binding of peroxide to Mb, catalytic efficiency of
Mb was slightly larger than that of cyt P450cam.
Overall, the trend of catalytic efficiency was cyt P450 1A2
> P450 2E1 > myoglobin > P450cam . catalase (Table 3).
This order of +•(P450sFeIVdO) formation rate for cyt P450s
is the reverse of that obtained for hET rate, and reflects an ∼3.5-
fold faster oxidation of the high spin heme iron cyt P450
compared to low spin cyt P450cam. Thus, the dependence on
spin state is weaker than for the hET rate constant.
Significantly, cyt C on surfaces having high spin heme iron
gave higher activity for peroxide reduction than low spin cyt
C.83-85 On the basis of quantum mechanical simulations, Boyes
et al.86 reported stabilization of low spin cyt P450 that could
lead to lower catalytic efficiency compared to high spin P450s.
Thus, kcat/KM in the order for cyt P450 1A2 films seems
consistent with the influence of spin state on peroxidase activity
of other heme enzymes.
The influence of amino acid residues in the distal and
proximal regions of the heme in enhancing the formation rate
of +•(P450sFeIVdO) must also be considered. Cyt P450s with
their active site residues around the heme distal region that
participate in peroxide reduction are shown in Figure 9.51-53
Peroxidases and Mb have proximal histidine coordinated to the
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A R T I C L E S
Krishnan et al.
Figure 9. Heme structures with distal and proximal region amino acid residues for (A) cyt P450 1A2 (PDB:2HI4),51(B) cyt P450 2E1 (PDB:3E4E),52and
(C) cyt P450cam (PDB:2CPP).53
heme, whereas in cyt P450s the proximal iron-binding ligand
is cysteine thiolate (Figure 9).3b
However, spin state does not control all aspects of these
reactions, and oxidation and reduction rates of cyt P450s in the
films are also likely to depend on protein secondary structure
around the heme group.
Recently, Roth and Cramer87 proposed alternate mechanisms
involving reversible O-O heterolysis/homolysis for peroxide
activation of heme peroxidases. A recent study on cyt P450cam
using QM/MM showed that proton coupled electron transfer
played a role in formation of ferryloxy radical cation (Scheme
2).88 As we can presently only speculate on the influence of
enzyme structure, we plan future QM/MM studies to address
the involvement of specific residues in the distal/proximal heme
regions of cyt P450 films in formation of +•(P450sFe(IV)dO).
Acknowledgment. This work was supported financially by U.S.
PHS Grant ES03154 from the National Institute of Environmental
Health Sciences (NIEHS), NIH. We thank Dr. Eli Hvastkovs for
providing purified cyt P450s and Dr. Dominic Hull for assistance
in using CHI simulation software. We thank Professor Jose Gascon
and L. Menikarachchi of the University of Connecticut for their
help in representing protein structures. J.F.R. thanks Science
Foundation Ireland for a Walton Research Fellowship in 2009.
Conclusions
We have shown for the first time that rates of electrochemical
reduction of cyt P450s in thin LbL polyion films depend
significantly on spin state, with low spin cyt P450cam giving
the largest rate. Observed asymmetry in CV trumpet plots for
the enzyme films was explained by a square scheme supported
by simulations featuring faster oxidation than reduction. To a
lesser extent and in the opposite order, the rate of oxidation of
cyt P450s by an organic peroxide also correlated with spin state.
Supporting Information Available: Detailed procedures used
for simulations of voltammetry and making layer-by-layer films.
Eight additional figures giving data for QCM, CVs of cyt P450
films without background subtraction, CVs and trumpet plot
for hemin, CVs of cyt P450 films upon catalytic oxygen
reduction and at various pH values, and simulation results with
an alternative mechanism. This material is available free of
charge via the Internet at http://pubs.acs.org.
(87) Roth, J. P.; Cramer, C. J. J. Am. Chem. Soc. 2008, 130, 7802–7803.
(88) Chen, H.; Hirao, H.; Derat, E.; Schlichting, I.; Shaik, S. J. Phys. Chem.
B 2008, 112, 9490–9500.
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