Here we report the redox properties of the cyto-chrome P450BM3 wild-type holoenzyme, and its isolated FAD reductase and P450 heme domains, when immobilized in a didodecyldimethylammonium
Trang 1Redox properties of cytochrome P450BM3 measured by direct methods Barry D Fleming1, Yanni Tian1, Stephen G Bell1, Luet-Lok Wong1, Vlada Urlacher2and H Allen O Hill1
1 Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, Oxford, UK; 2 Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany
Cytochrome P450BM3 is a self-sufficient fatty acid
mono-oxygenase consisting of a diflavin (FAD/FMN) reductase
domain and a heme domain fused together in a single
polypeptide chain The multidomain structure makes it an
ideal model system for studying the mechanism of electron
transfer and for understanding P450 systems in general
Here we report the redox properties of the
cyto-chrome P450BM3 wild-type holoenzyme, and its isolated
FAD reductase and P450 heme domains, when immobilized
in a didodecyldimethylammonium bromide film cast on
an edge-plane graphite electrode The holoenzyme showed
cyclic voltammetric peaks originating from both the flavin
reductase domain and the FeIII/FeIIredox couple contained
in the heme domain, with formal potentials of)0.388 and
)0.250 V with respect to a saturated calomel electrode,
respectively When measured in buffer solutions containing
the holoenzyme or FAD-reductase domain, the reductase
response could be maintained for several hours as a result of protein reorganization and refreshing at the didodecyldi-methylammonium modified surface When measured in buffer solution alone, the cyclic voltammetric peaks from the reductase domain rapidly diminished in favour of the heme response Electron transfer from the electrode to the heme was measured directly and at a similarly fast rate (ks¢ ¼ 221 s)1) to natural biological rates The redox potential of the FeIII/FeII couple increased when carbon monoxide was bound to the reduced heme, but when in the presence of substrate(s) no shift in potential was observed The reduced heme rapidly catalysed the reduction of oxygen
to hydrogen peroxide
Keywords: cytochrome P450BM3; redox properties; electro-chemistry
The cytochrome P450 group of enzymes comprises a
variety of heme-containing monooxygenases that are
present in the majority of prokaryotic and eukaryotic
organisms [1] The primary reaction catalysed by these
enzymes is the hydroxylation of carbonÆhydrogen bonds
Substrate hydroxylation requires the activation of
dioxy-gen The two-electron reducing agent in biological systems
is almost exclusively NAD(P)H, and this reductive
activa-tion of oxygen by P450 occurs in two separate one-electron
additions The first electron reduces the substrate-bound
ferric heme, facilitating the rapid binding of dioxygen and
the formation of the ferrous–dioxygen intermediate The
second electron addition, followed by protonation, forms
the ferric hydroperoxy complex The O–O bond is cleaved,
with one O inserted into the substrate and the other
reduced to form water
Cytochrome P450BM3is a self-sufficient fatty acid
mono-oxygenase found in Bacillus megaterium [2,3] The native
function of wild-type P450BM3is to oxidize long-chain fatty
acids, but it has also been shown to oxidize many other substrates [4,5] The 119-kDa molecular mass holoenzyme has its diflavin (FAD/FMN) reductase domain and heme domain fused together in a single polypeptide chain, making the transfer of electrons highly efficient [6] The multi-domain structure has made it an ideal model system for studying the mechanism of electron transfer and for understanding P450 systems in general [7–10] Independent expression of the two domains has permitted their study in isolation [11]
Redox potentiometric studies have shown the electron flow for P450BM3 to follow the path NADPHfi FADfi FMN fi heme [12] The electron flow to the heme centre in P450BM3 was presumed to be regulated by a substrate-dependent increase (> 100 mV) in the redox potential of the heme, with the suitability of the substrate for catalytic transformation being reflected in the magnitude
of the increase in potential Thermodynamic arguments recently presented by Honeychurch et al [13], suggested that the binding of dioxygen to P450cam, another essential step in the P450 catalytic process, would be sufficient to enable electron cycling, regardless of whether camphor is present
Research on the electrochemistry of enzymes is driven partly by the desire to understand the details of electron transport in proteins and partly by the great potential uses
of enzymes in electrochemically based biosensors and bioreactors Electron transfer between an electrode and protein was initially accomplished in the pioneering work
of Eddowes & Hill [14] and Yeh & Kuwana [15] They showed that the problem of slow electron transfer between
Correspondence to B D Fleming, Department of Chemistry,
Inorganic Chemistry Laboratory, University of Oxford,
South Parks Road, Oxford OX1 3QR, UK.
Fax: + 44 1865 272690, Tel.: + 44 1865 275902,
E-mail: barry.fleming@chem.ox.ac.uk
Abbreviations: DDAB, didodecyldimethylammonium bromide; EPG,
edge-plane pyrolytic graphite; SCE, saturated calomel electrode.
Enzymes: cytochrome P450 (CYP102, P450 BM3 ) (EC 1.14.14.1).
(Received 14 July 2003, revised 19 August 2003,
accepted 21 August 2003)
Trang 2an electrode and a metalloprotein could be overcome by
use of an electron shuttle or mediator Since then, much
effort has been directed into developing suitably mediated
or modified electrode systems that facilitate biological
electrochemistry One such approach is the recently
documented technique of using synthetic surfactant-based
biomimetic membranes [16] These cast films have been
shown to be useful in attaining direct electrochemistry of
heme-containing proteins [17–22] In the present work
we employed this method to obtain the direct,
quasi-reversible electrochemistry of the wild-type
cyto-chrome P450BM3holoenzyme and its isolated
FAD-reduc-tase and heme domains
Materials and methods
Enzymes and chemicals
Wild-type cytochrome P450BM3 was overexpressed in the
Escherichia colistrain, DH5a (that contained the gene of the
wild-type cytochrome P450BM3) and the bacterial growth
and protein purification were carried out using published
procedures [23] The FAD-reductase domain was cloned,
expressed and purified according to published procedures
[24] The P450BM3heme domain was cloned and expressed,
using standard procedures, to encompass amino acid
residues 1–481 of P450BM3 The P450BM3 heme domain
was purified by DEAE Sepharose and Source-Q
anion-exchange chromatography, as described previously [23]
After purification, fractions with an A417/A280of > 1 were
collected and stored All samples were stored at)20 C in
40 mM phosphate buffer, pH 7.4, containing 50% v/v
glycerol Glycerol was removed immediately prior to
experiments by gel filtration on a Amersham-Pharmacia
PD-10 column equilibrated with 40 mMphosphate buffer,
pH 7.4
Chemicals and solvents were of reagent grade and used
without further purification unless stated otherwise
Dido-decyldimethylammonium bromide (DDAB) from Aldrich
was prepared and used as a 0.1-M stock solution in
chloroform
Apparatus and procedures
DC cyclic voltammetry experiments were performed at
room temperature in a standard two-compartment glass cell
with a working volume of 0.5 mL The working
compart-ment housed the platinum gauze counter electrode in
addition to the edge-plane pyrolytic graphite (EPG)
work-ing electrode A saturated calomel electrode (SCE) was used
as a reference in a sidearm that connected to the working
compartment via a Luggin capillary All potentials were
referred to the SCE An Autolab potentiostat (Eco Chemie,
Utrecht, the Netherlands) was used to record and control
the potential of the working electrode All measurements
were made in 40 mMpotassium phosphate buffer, pH 7.4
Voltammograms were taken in solutions that had been
deoxygenated by purified argon For buffered protein
solutions this was accomplished by blowing argon over
the solution for several hours An argon atmosphere was
maintained over the solution during the experiment, unless
stated otherwise
Preparation of P450/DDAB/EPG electrodes DDAB films were made by spreading 5 lL of the stock solution onto a freshly polished EPG electrode The chloroform was allowed to evaporate in air at room temperature for 1 h To incorporate the enzyme into the DDAB-modified electrode, the electrode was either placed into a solution of protein ( 10 lM) for 1 h at 4C,
or directly into the electrochemical cell containing buffered protein solution at room temperature
Results
A typical cyclic voltammogram, recorded for a P450BM3– DDAB-modified EPG electrode in oxygen-free phosphate buffer, pH 7.4, is shown in Fig 1 Three cyclic voltammo-grams are shown: the initial scan soon after electrode immersion, and then scans after 30 and 60 min This series
of scans highlights the evolution of the response over a period of 1 h Initially, the electrode response was domin-ated by a well-defined reversible couple centred at)0.388 V, with a broader, less intense couple observed at)0.250 V With time the response at the more negative potential diminished, whilst the intensity of the other increased, until after 1 h the current response of the couples, relative to each other, remained stable (over longer periods of time both couples decreased at approximately the same rate) A significant response at)0.250 V could still be observed for
up to 1 week for electrodes maintained in buffer solution at
4C A different result was observed for voltammograms measured using a DDAB-modified EPG electrode in a buffered solution of the holoenzyme (Fig 2) The intensity
of the couple at)0.388 V could be maintained for longer time-periods (up to 3 h in this case), with a slight increase in the signal centred on )0.250 V When the electrode was removed from the enzyme solution and placed into enzyme-free buffer solution, the electrode response observed was
Fig 1 Cyclic voltammetry of wild-type P450 BM3 holoenzyme immobi-lized at a didodecyldimethylammonium bromide (DDAB)-modified edge-plane pyrolytic graphite (EPG) electrode Cyclic voltammograms were recorded in deoxygenated phosphate-buffer solution (pH 7.4), at a scan rate of 0.1 VÆs)1, for wild-type P450 BM3 holoenzyme immobilized
at a DDAB-modified EPG electrode after being initially immersed (darkest line) and then 30 and 60 min (lightest line) later The arrows indicate the direction of current change for the respective peaks over the duration of the experiment.
Trang 3similar to that shown in Fig 1 If, after removing the
electrode from the enzyme solution it was stored in buffer
for any length of time, then the response was essentially
dominated by the signal at)0.250 V
To help identify the origins of the cyclic voltammetric
peaks observed with the holoenzyme, the individual
reduc-tase and heme domains were tested separately Figure 3
shows that when the FAD-reductase domain was
immobi-lized at a DDAB-coated EPG electrode, quasi-reversible
electrochemistry in buffer was possible The mid-point
potential of the reductase domain was)0.405 V As with
the holoenzyme, the reductase domain response diminished
quite rapidly over a period of 1 h when in buffer solution,
but could be maintained for a longer duration when in
protein solution
A typical cyclic voltammogram recorded for the
heme-domain DDAB-modified EPG electrode in buffer is shown
in Fig 4 The response consisted of one Faradaic couple
centred on )0.244 V, probably from the FeIII/FeII redox
couple This strong response could be maintained for many hours and for up to 1 week when the electrode was maintained in buffer solution at 4C The cyclic voltam-metric peaks were approximately symvoltam-metrical, having equal areas under both the reductive and oxidative cycle, and showed a linear current dependence with scan rate from 0.01 to 1 VÆs)1, as expected for thin films of electroactive species Using these peak areas, the concen-tration of the electroactive enzyme at the electrode surface was calculated to be of the order of nmolesÆcm)2 The peak separation was measured as a function of scan rate for the
FeIII/FeII redox couple in both the holoenzyme and the heme domain The trumpet plots, so-formed in this case, are shown in Fig 5 Significant peak separation was only observed when the scan rate exceeded 10 VÆs)1 From these data, a value for the apparent average electron-transfer rate constant (ks¢) could be calculated Generally,
ks¢ values are obtained from the peak (Epa) to peak (Epc) potential separation values in cyclic voltammograms based
on Laviron’s approach for diffusionless thin-layer voltam-metry [25] The ks¢ value for the FeIII/FeIIredox couple in
Fig 2 Cyclic voltammetry of wild-type P450 BM3 holoenzyme in
solu-tionusing a DDAB-modified EPG electrode Cyclic voltammograms
were recorded in a pH 7.4-buffered solution of wild-type P450 BM3
holoenzyme at a scan rate of 0.1 VÆs)1for a DDAB-modified EPG
electrode after being immersed for 1 (darkest line), 2 or 3 h (lightest
line), respectively The arrows indicate the direction of current change
for the respective peaks over the duration of the experiment.
Fig 3 Cyclic voltammetry of the P450 BM3 FAD-reductase domain
immobilized at a DDAB-modified EPG electrode The cyclic
vol-tammogram was recorded in deoxygenated phosphate-buffer solution,
pH 7.4, at a scan rate of 0.1 VÆs)1for the P450 BM3 reductase domain
immobilized at a DDAB-modified EPG electrode.
Fig 4 Cyclic voltammetry of the P450 BM3 heme domainimmobilized at
a DDAB-modified EPG electrode The cyclic voltammogram was recorded in deoxygenated phosphate-buffer solution, pH 7.4, at a scan rate of 0.1 VÆs)1 for the P450 BM3 heme domain immobilized at a DDAB-modified EPG electrode.
Fig 5 ‘Trumpet’ plots for the heme-domainrespon se The response from the P450 BM3 holoenzyme (j) and the heme domain (m) are shown The reductive and oxidative peak potentials are plotted against the scan rate This type of plot can be used to calculate the electron transfer rate constant.
Trang 4the holoenzyme and heme domain were determined to be
138 and 221 s)1, respectively The influence of pH on the
heme redox potential, measured by cyclic voltammetry, is
shown in Fig 6 These data are representative for the
heme response from both the holoenzyme and isolated
heme domain The mid-point potential became
increas-ingly more negative as the pH was increased from 3 to 10
Two linear regions of different slopes were observed, one
between pH 3 and pH 8, and the other between pH 8 and
pH 10 The slope of these regions was )33 and
)126 mVÆpH unit)1, respectively
After bubbling the buffer solution with CO for
15 min, the mid-point potential of the heme domain
was positively shifted by 50 mV (Fig 7) When purged
with argon, the original formal potential returned The
effect of substrate binding on the redox potential of
the heme domain was also investigated When any of the
substrates lauric acid, palmitic acid or octane were added
to the buffer solution, there was typically no change in
the cyclic v oltammetric peaks
The heme redox couple was very sensitive to the
presence of molecular oxygen Figure 8 shows the effect,
on the cyclic voltammogram for the heme domain
DDAB-modified EPG electrode, of adding 1, 3 or 5 mL of air into
the buffer solution A new couple, at a potential slightly
positive of the FeIII/FeII couple, was observed The
reduction of O2by bare or DDAB-coated EPG electrodes
occurred at more negative potentials ()0.5 to )0.7 V)
Thus, the presence of the heme significantly lowers the
overpotential required for O2reduction The magnitude of
the reduction peak was related to the amount of O2added
The oxidation peak height was less intense than the
reduction peak, which is characteristic of a mechanism
involving the rapid electrocatalytic reduction of O2 to
H2O2by the reduced heme-containing films as per Eqn (1)
and Eqn (2):
P450 FeIIþ O2! P450 FeII O2 ð1Þ
P450 FeII O2þ 2Hþþ 2ee ! P450 FeIIþ H2O2 ð2Þ
Discussion The isolated FAD reductase and heme domains of P450BM3 have been useful in identifying the voltammetric response observed with the P450BM3holoenzyme It was clear that the Faradaic couples centred on)0.388 and )0.250 V had their origin in the reductase and heme components, respectively The peak identification was aided by the fact that there was no significant shift in potential for the individual electroactive components compared with when they were fused together in the holoenzyme A close similarity in the redox potential has also been measured
by redox potentiometry for FAD and FMN in the isolated domains or the P450BM3 holoenzyme [12] These solution measurements were explained in terms of there being no significant change in the domain environments whether isolated or fused together The same could also be said here
of the reductase and heme domains when they are incorporated in the DDAB film at the electrode surface
Fig 6 Influence of pH on the heme redox potential Cyclic
vol-tammograms at 0.1 VÆs)1for wild-type P450 BM3 holoenzyme were
measured at different pH values The mid-point potentials observed for
each voltammogram were plotted against pH A similar trend was
observed for the isolated heme domain.
Fig 7 The effect of CO binding on the P450 BM3 heme redox potential Cyclic voltammograms were recorded in deoxygenated phosphate-buffer solution (at pH 7.4), before and after bubbling with CO for
15 min, at a scan rate of 0.1 VÆs)1for the P450 BM3 heme domain immobilized at a didodecyldimethylammonium bromide (DDAB)-modified EPG electrode Peaks shifted to the right in the presence of CO.
Fig 8 The effect of O 2 binding on the electrochemistry of the P450 BM3
heme domain Cyclic voltammograms were recorded in a phosphate-buffer solution, pH 7.4, after adding 0, 1, 3 or 5 mL of air, at a scan rate of 0.1 VÆs)1 for the P450 BM3 heme domain immobilized at a DDAB-modified EPG electrode The height of the reduction peak increased with the amount of air injected into the solution.
Trang 5The change in the peak intensities for the holoenzyme,
when measured in enzyme-free buffer solution, is of
particular interest and several explanations are considered
First, some reorganization at the electrode/solution
inter-face, presumably within the DDAB film, had taken place
The initially large response from the reductase domain
indicates that it is preferentially bound/oriented closest to
the electrode surface The further development of the
electrode response, lowering of the reductase peak vs
increase of the heme peak, indicates that this orientation
may be reversed, with the enzyme seemingly rotating to
allow the heme domain to take up a more favourable
electron transfer position near the electrode surface It is
also possible that the short-lived response in buffer is caused
by denaturation at the electrode surface This is suggested
by the results observed with the isolated reductase domain
Its response, both in the presence of protein and in buffer
only, was similar to that for the holoenzyme (except for the
heme component) When the reductase domain is present in
solution, it is plausible that the surface can be refreshed,
effectively replacing the denatured protein This could
account for the longer duration of the large Faradaic
currents observed When the protein is not present in
solution, denaturation takes place with no refreshment of
the electrode surface and hence the relatively rapid decrease
in current
With bare EPG electrodes, the direct electron transfer to
P450BM3is slow and often not observed The heme group is
deep within the protein structure and favourable orientation
at the electrode surface must occur to ensure electron
transfer When incorporated into the surfactant layer, the
direct, rapid and quasi-reversible electron transfer between
the P450 heme and electrode was observed The P450 heme
redox potential measured here (in both the holoenzyme and
the heme domain only) is of a similar value to previous
measurements for other P450 enzymes and heme-containing
proteins incorporated in DDAB films [16,17,21,22] The
broadness of the heme peak is considered to be caused by
dispersion of E values resulting from slight variations in
protein orientation at the electrode surface [17] The
differences in broadness between the reductive and oxidative
waves, evident in Figs 3 and 4, is typically the result of
nonideality observed with thin-film systems [26] The
potential of the heme FeIII/FeII redox couple ()0.252 V)
was much more positive than that measured in solution
()0.609 V) [12] This type of behaviour has been reported
for all cases where DDAB has been present as the
biomimetic membrane This is the result of interactions
between the protein and surfactant and/or
surfactant-related electrical double-layer effects on electrode potential
This effect was also evident, albeit to a lesser extent, with the
reductase response
Our results show that the process of immobilization
provides a very favourable environment for electron transfer
to the heme to occur The ks¢ values calculated for the P450
heme domain ( 200 s)1) were similar in magnitude to the
ks¢ value measured for the natural electron transfer process –
that between the FMN and heme for the P450BM3
holoenzyme in solution (223 s)1with myristate) [7] These
electron transfer results were dependent on the nature of the
substrate – with the less favoured substrate lauric acid
showing a lower k¢ value of 130 s)1 It has also been shown
that it takes more energy to transfer an electron to the P450 heme when no substrate is bound [12] Our results are all the more interesting given that no substrate was present Other
ks¢ values for heme-containing proteins, measured using electrode systems, are well below those reported here for the P450BM3heme domain For example, P450camin a DDAB film on a PG electrode was 25 s)1[17], whilst a variety of modified PG electrodes containing myoglobin showed ks¢ values ranging from 27 to 86 s)1[27]
The pH-dependent potential change observed in Fig 6 has been shown previously for other heme-containing proteins immobilized at electrode surfaces [18,19,22,27– 30] However, in contrast to these previously published data, the slopes measured in this study for the linear regions were both quite different to the)59 mVÆpH unit)1expected for a reversible one-electron transfer coupled to a single proton transfer A similar low-slope region has also been reported for myoglobin and hemoglobin in polyacrylamide films, but this was for pH values of < 5, a region where protein integrity might be questioned [27,28]
Previous redox potentiometry experiments on P450BM3 showed that the presence of a suitable substrate results in
an anodic (or positive) shift in redox potential in excess of
100 mV [12] A similar substrate-dependent anodic shift was reported for P450cam from electrochemical data [31] Our results indicate that no shift occurs in the formal potential of heme when in the presence of the substrates lauric acid, palmitic acid or octane Similar results, based
on cyclic voltammetric data, were reported recently for P450cam and P450cin in the presence of their natural substrates, camphor and cineole, respectively [22,32] The work with P450cin employed a similar procedure for enzyme immobilization as reported in this work [22] Interestingly, redox potentiometric data for P450cin also showed no indication of a substrate-dependent anodic shift There are reasonable thermodynamic arguments to suggest that substrate binding is not the only or main consideration in determining whether electron transfer to P450 will occur [13]
CO is known to rapidly bind specifically as a sixth ligand
to the reduced heme iron of P450BM3 [33] The fact that addition of CO to the buffer solution in our electrochemical experiments resulted in a peak shift of 50 mV confirmed that the observed response is from the heme domain Similar results were obtained for P450camimmobilized at a DDAB-modified PG electrode [17] and a glassy carbon electrode modified with sodium montmorillonite [32] For catalytic reactions involving P450 enzymes, the reduction of molecular oxygen to reactive oxygen species, such as H2O2, is typically an unwanted occurrence which dramatically reduces the efficiency of the desired catalytic process As shown in Fig 8, once generated, the ferrous heme rapidly binds dioxygen, but unfortunately catalytic reduction to H2O2 usually quickly follows The real challenge then, in any development of electrode-based bioreactors designed to utilize the monooxygenase capabi-lities of P450, is getting the second electron to be used in peroxoiron complex formation and not in H2O2 dissoci-ation Several groups have recognized the difficulties associated with overcoming this problem and have attemp-ted to utilize mediator-promoted and H2O2-driven pathways to achieve their desired oxidation reactions
Trang 6[30,34,35] Given the fast electron transfer rates and low
potentials necessary for the first electron reduction of the
P450BM3heme domain in this surfactant-electrode
ensem-ble, it follows that it should be the subject of ongoing study,
as in our laboratory However, it remains to be seen whether
an effective electrochemically driven bioreactor, fully
utili-zing Nature’s enzyme technology, can be achieved
Conclusions
We determined the redox properties of
cyto-chrome P450BM3 by direct electrochemistry The
holo-enzyme response at a DDAB-modified EPG electrode was
characterized by redox couples at)0.388 V and )0.250 V
These were identified as being direct electron transfer to the
individual flavin reductase domain and P450 heme domain,
respectively We have also shown that, although electron
transfer in the biological system is from FAD/FMN to the
heme, electron transfer can occur directly from the electrode
to the heme under electrochemical conditions The rate of
this electron transfer is very rapid, of the order of the rates
observed in the natural donor system The redox potential
of the heme did not appear to be affected by substrate
binding, but it was possible that substrate inclusion in the
surfactant layer may alter the surrounding charge
environ-ment experienced by the protein The reduction of
mole-cular oxygen was readily catalysed by the P450BM3
heme domain immobilized at the DDAB-modified
elec-trode
Acknowledgements
We would like to thank the European Union for the collaborative
grant We also thank ECEnzymes, BASF and Dr T Habicher.
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