Direct Electrochemistry of Cytochrome P450 Reductases in Surfactant and Polyion Films

Full Paper

Direct Electrochemistry of Cytochrome P450 Reductases inSurfactant and Polyion Films

Nasreen Sultana,a John B. Schenkman,b James F. Ruslinga, b*a Department of Chemistry, 55 N. Eagleville Rd., University of Connecticut, Storrs, CT 06269, USAb Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06032, USA*e-mail: [email protected]

Received: June 22, 2007Accepted: August 22, 2007

AbstractNADPH-cytochrome P450 reductase (CPR) serves as electron donor for cytochrome P450 catalyzed monooxygenasereactions utilizing flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) as electron transfercofactors. Here, stable films of human and rabbit CPRs with didodecyldimethylammonium bromide (DDAB),dimyristoylphosphatidyl choline (DMPC), and poly(diallyldimethylammonium) (PDDA) were made on pyrolyticgraphite (PG) electrodes for comparative structural and electrochemical studies. CD and UV-VIS absorbance spectrasuggested that near native CPR conformation is retained in PDDA films, and some conformational changes occur inDMPC or DDAB films. Cyclic voltammetry of these films gave quasireversible pairs of peaks at average formalpotential �0.246� 0.008 V vs. NHE. In human CPR-DDAB (H-CPR-DDAB), a second pair of peaks at þ0.317 V vs.NHE was found that depended strongly on identity of buffer and salt. Excepting H-CPR in DDAB, films showedsimilar voltammetry, formal potentials, and ks values. While CPR-PDDA films had near native CPR structures,electrochemical parameters did not differ significantly from CPR-DMPC films. The relative independence of filmvoltammetry from the influence of film materials for CPRs is in contrast with heme iron proteins that, while retainingnear native structures, have formal potentials that depend significantly on identity of the film material.

Keywords: NADPH cytochrome P450 reductase, Surfactant films, Voltammetry, UV-vis spectroscopy, Circulardichroism

DOI: 10.1002/elan.200704014

1. Introduction

Mammalian NADPH-cytochrome P450 reductases (CPR)are the natural electron donors for all microsomal cyto-chrome P450 (CYP or P450)-catalyzed monooxygenasereactions [1, 2]. They are flavoproteins containing flavinmononucleotide (FMN) and flavin adenine dinucleotide(FAD) as prosthetic groups and utilize NADPH as anelectron source [3, 4]. These flavoproteins are on the surfaceof the endoplasmic reticulum(ER) and nuclear membraneof liver cells and other tissues [5]. In eukaryotic cells, CPR istightly bound to the ER membrane with a 25 – 30-amino-acid-longN-terminal hydrophobic anchor.Only one formofNADPH-cytochrome P-450 reductase has been identifiedper species in mammals, other vertebrates and non-verte-brates [6]. Mammalian CPR consists of 676 amino acids [7].Human CPR exhibits 92 – 95% amino acid sequencehomology with other mammalian CPRs. This high degreeof physical and structural similarity in different organismssuggests evolutionary conservation of CPR [5, 8].CPRs have one molecule each of FAD and FMN, and

shuttle electrons from NADPH one at a time to the hemeiron of cyt P450s with FAD and FMN being the point ofentry and exit, respectively [1, 2]. Cytochrome P450, as theterminal electron acceptor, converts various lipophilic

substrates and drugs into oxidized metabolites by monoxy-genation.Using spectrophotometric titrations, themidpointpotentials of each flavin half-reaction in the native enzymehave been determined in rabbit [4] and rat [9] CPRs. Theenzyme cycles between 1e and 3e reduced levels (or 2e and4e), with the one-electron reduced semiquinone of the FMNbeing the highest oxidation state during catalytic turnover[10, 11]. Anaerobic redox titrations of human CPR [12]shows four redox couples at pH 7: FMNoxidized/semiquonone at�66� 8 mV, FMNsemiquinone/reduced at �269� 10 mV, FADoxi-

dized/semiquonone at �283� 5 mV, and FAD semiquinone/reduced at�382� 8 mV. These redox potentials are similar to those ofthe free flavins. The air-stable species represents a form ofNADPH-cytochrome P-450 reductase which contains twoflavins, one exists in the semiquinone state (a 1e – reducedform), and the other in the oxidized state [3, 9].There are several factors which influence the direct

transfer of electrons between CPR and acceptor proteins,such as the chemical structure, orientation and the redoxpotentials of the electroactive cofactors FMN and FADembedded in the enzyme. The structure [7, 13], electrontransfer process between the co-factors and cyt P450enzymes [14, 15] have been studied in solution by variousmethods.

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The objective of the present work was to immobilizemammalian CPRs on pyrolytic graphite electrodes toinvestigate comparative structure and voltammetry inseveral microenvironments using protein film voltammetry(PFV). Direct reversible voltammetry of many proteins hasbeen achieved in biomembrane-like films of dimyristoyl-phophatidylcholine (DMPC) and didodecyldimethylam-monium bromide (DDAB) on pyrolytic graphite electrodes[16 – 24]. These films are prepared by casting surfactant-biomolecule dispersions onto the electrodes. These stablefilms under most conditions preserve the conformationalintegrity of many proteins [16 – 18, 25]. A second type ofprotein film can be made by adsorbing alternate layers ofprotein and oppositely charged polyion until desired filmarchitecture is achieved, and is called the alternate electro-static layer-by-layer (LbL) technique [26, 27]. This methodgives films with better mechanical stability than lipid films,while also retaining enzyme activity and structure. Rever-sible electrochemistry and catalysis have also been achievedwith films of heme proteins and polyions constructed usingLbL [17, 18, 28 – 30].We previously reported a single pair of reversible CV

peak at �0.23 V vs. NHE at pH 7.4 (þ0.1 M KCl, 4 8C) forrabbit CPR in films constructed layer-by-layer with poly(-ethyleneimine), and showed that these peaks were nearlyidentical to those obtained from similar films made withmicrosomes genetically enriched in human monooxygenas-es [31]. Very recently, Shulka, et al. [32] reported voltam-metry of both rat and human CPR inDDAB films, althoughthis paper did not contain a detailed study of rabbit CPRvoltammetry.These protein-surfactant films gave two sets ofreversible CVpeaks at 25 8C for both reductases at pH~8 inDDAB films. The midpoints of these peaks were at �0.002and �0.278 V vs. NHE for human CPR and �0.020 and�0.254 V for rat CPR, and were attributed to FMN andFAD, respectively. Thus, it appeared that film materialexerts a significant influence on CPR voltammetry, and wewished to explore this effect in greater detail.In this paper, we report a comparison of human CPR and

rabbit CPR in LbL films with poly(diallyldimethylammo-nium) (PDDA) and in lipid films (DDAB and DMPC) onpyrolytic graphite electrodes and fused silica slides. Formalredox potentials and surface electron transfer rate constants(ks) are reported. The structural integrity of protein in thefilms was monitored by UV-visible spectroscopy andcircular dichroism.

2. Experimental

2.1. Materials

PDDA (poly (diallyldimethylammonium) (3 mg/mL) wasfrom Aldrich. Rabbit NADPH Cytochrome P450 Reduc-tase(CPR) 22 mM(1.8 mg/mL)was a gift fromDr. I. Jannsenat Univ. of CT. Health Center, and was prepared aspreviously described [33]. Pure Human CPR, recombinant51 mM(95%) (4.1 mg/mL)was obtained fromGentest,MA.

PSS (sodium polystyrene sulfonate) MW 70000, (3 mg/mLþ 0.5 M NaCl), FMN (99%) sodium salt MW 478.41 mg/mL, FAD (Disodium salt FW: 829.5), DMPC (Dimyr-istoylphophatidylcholine, 99%) 4 mM, DDAB (Didodecyl-dimethylammonium bromide, 98%) were from Sigma.

2.2. Film Preparation

Basal plane pyrolytic graphite (PG) disk electrodes (A¼0.16 cm2) were initially polished on 400 grit (Buehler) SiCpaper, then roughened using medium crystal bay (PH4 3 M001K) emery cloth, washed thoroughly with pure waterfollowed by sonication in deionized water for 30 s. Filmswere assembled using two methods, the alternate LbLmethod and the cast method.Quartz crystal microbalance was used to monitor the

enzyme film growth on gold QCM resonators [29] (USIJapan, resonator frequency9 MHzAT-cut, area¼ 0.16 cm2).Filmswere grown by theLbL electrostatic assemblymethodwith four bilayers of CPR and PDDA, denoted (CPR-PDDA)4. The resonator was first cleaned with piranhasolution for 30 s followed by thorough washing with water.The resonator was dried in a stream of nitrogen for 8 min.One side of the resonator gold surface was made negativelycharged by chemisorption of 5 mM of 3-mercaptopropionicacid (MPA) in ethanol. A 10 mLdrop of PDDA (3 mg/mLþ50 mMNaCl), was spread over the negatively charged goldsurface to form a monolayer. After 20 min of adsorptiontime, the resonator was washed thoroughly with water anddried under nitrogen stream before measuring frequency atroom temperature. Subsequent PSS (3 mg/mLþ 0.5 MNaCl) and CPR (1.0 mg/mL in 30 mM sodium phosphatebuffer pH 7.5) layers were assembled in the same way untilfilm architecture of (PDDA-PSS)2 PDDA(CPR-PDDA)4,as on PG electrodes, was achieved. The adsorption times forPSS and enzyme were 20 min and 1 h respectively. Forabsorption spectroscopy and circular dichroism, thickerH/R-CPR – PDDA films were formed using cast method onfused silica slides.Surfactant films were formed using cast method. 5 mL of

clear vesicle dispersions of 10 mMDDAB for voltammetry,or 2 mMDDABwith 50 mMNaBr for spectroscopy or 4 mMDMPC for both voltammetry and spectroscopy, weremixedwith 5 mL of R-CPR (1.8 mg/mL) or H-CPR (4 mg/mL) in30 mM sodium phosphate solution, pH 7.5, and was evenlyspread on the PG or fused silica slide surface, then coveredand dried overnight at 4 8C to form a film. NaBr in Protein-DDAB films cast onto fused silica stabilizes the structure ofheme proteins to near-native conformations [25, 34].

2.3. Apparatus and Measurements

Absorption spectroscopywas doneusing aHewlett-Packard8453UV-vis spectrophotometer with a diode array detector.A JASCO 710 spectropolarimeter was used to obtain CDspectra. All experiments were done at room temperature,

2500 N. Sultana et al.

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www.electroanalysis.wiley-vch.de

22� 2 8C. Cyclic voltammetry (CV) and square-wave vol-tammetry (SWV) on the H/R-CPR films were done in athree electrode cell at room temperature using CHI electro-chemical analyzers. The three electrode cell featured asaturated calomel reference electrode (SCE), a Pt wirecounter electrode, and a film-coated PG electrode, andohmic drop was compensated >95%. 100 mM sodiumphosphate buffer, pH 7.4 and 30 mM sodium phosphatebuffer, pH 7.5 were used for voltammetry experiments.Potentials in this paper were reported after conversion tothe NHE scale. Both CV and SWV scans were initially innegative directions from positive initial potentials, and weredone in 100 mM sodium phosphate buffer, pH 7.4 contain-ing 50 mM KCl. Solutions were purged with purifiednitrogen, and a nitrogen blanket maintained during scans.

3. Results and Discussion

3.1. Quartz Crystal Microbalance (QCM)

Layer by layer CPR-polyion film assembly was monitoredon a quartz crystal microbalance [27 – 29]. Layers weredeposited directly onto a 9 MHz gold-coated quartz reso-nator. A decrease in the frequency was observed for eachdried layer in [PDDA-PSS]2-PDDA-[R-CPR-PDDA]4 and[PDDA-PSS]2-PDDA-[H-CPR-PDDA]4 films (Fig. 1) indi-cating regular and reproducible film growth. These filmarchitectures were chosen because they gave good voltam-metry (see below).

Mass of each layer can be obtained from the expressionfor the 9 MHz quartz resonators [26]

DF¼�1.832� l08 M/A (1)

whereM (g) is the adsorbedmass,DF (Hz) is frequency shift,and A (0.16� 0.01 cm2) is the gold surface area on theresonator. The nominal thickness (d) of dry films wasestimated fromDF valueswith the empirical relation [26, 35]

d (nm)¼�0.016 (�0.002) DF (Hz) (2)

Table 1 shows the nominal average thicknesses and totalamounts of proteins in the films obtained from the QCMdata. R-CPR gave larger amount and bilayer thickness,possibly due to its higher purity.

3.2. UV-Vis Spectroscopy

R-CPR and H-CPR solutions were yellow, indicatingpresence of flavin in oxidized form [36]. Figures 2 to 4show UV-VIS absorption spectra of the CPRs in solutionsand films. The relative low molar absorptivity of thechromophores, e.g., compared to iron heme proteins, makesthe spectra of the CPRs in thin films rather noisy. Never-theless, films of [R-CPR-PDDA]4 (Fig. 2a) and [H-CPR-PDDA]4 (Fig. 2b), [R-CPR-DMPC] (Fig 3a) and [H-CPR-DMPC] (Fig. 3b) gave measurable peaks around 365 –385 nm, and 460 nm as did dissolved H-CPR (Fig. 4c) and

Fig. 1. QCM frequency shift for (*) MPA adsorption step, (&)PSS adsorption step, ( 3)PDDA adsorption and (~)R/H-CPR adsorptionstep for (a) [PDDA-PSS]2-PDDA-[R-CPR-PDDA]4 and (b) [PDDA-PSS]2-PDDA-[H-CPR-PDDA]4 films assembled layer by layer onAu/MPS surface. For some points, the error bars are smaller than the size of markers due to low standard deviation.

Table 1. QCM results for [CPR/PDDA]4 films on Au/MPS/[PDDA-PSS]2 surface on QCM gold electrodes. For calculations of totalconcentration, MW 78000 and 70000 were used for R-CPR and H-CPR respectively.

Enzyme M/A [a] (ng cm�2) Total thickness (nm) Bilayer thickness (nm) G [a] (pmol cm�2)

R-CPR 11.7� 1.3 45� 5 11.1� 2.5 150� 16H-CPR 9.1� 0.4 34� 1 8.6� 2.3 130� 5

[a] total amount of CPR.

2501Direct Electrochemistry of Cytochrome P450 Reductases



R-CPR (Fig. 4d). These peaks are characteristic of oxidizedforms of flavoproteins [37], and suggest the present of intactCPRs in these films. Blank polyion or lipid films gave noabsorbance in these spectral regions.In DDAB films, attempts to increase CPR concentrations

to obtain good spectra resulted in protein precipitation. The

resulting film spectra were of very poor quality, and R-CPR(Fig. 4a) showed no peaks at 365 nm and 460 nmwhich maybe due to structural alteration in the precipitate, or to thereduced form of flavoprotein [34]. H-CPR-DDAB filmsgave a peak at 365 nm and a very weak noisy peak at 460 nm(Fig 4b). It is difficult to make definitive conclusions about

Fig. 2. UV-vis Absorption spectra of (a) [R-CPR-PDDA] film and (b) [H-CPR-PDDA] film cast on fused silica slide.

Fig. 3. UV-vis Absorption spectra of (a) [R-CPR-DMPC] film and (b) [H-CPR-DMPC] film cast and dried on fused silica slide.

Fig. 4. UV-vis Absorption spectra obtained in solution and in films cast and dried on fused silica slide. A) (a)[R-CPR-DDAB] film (b)[H-CPR-DDAB] film and B) (c) H-CPR (4 mg/mL) solution (d) R-CPR (1.8 mg/mL) solution.




protein integrity in the CPR-DDAB films from thesespectral results. Spectral maxima of all films are comparedwith solution values in Table 2.

3.3. Circular Dichroism

UVcircular dichroism (CD) spectra reflectmajor secondarystructure features of protein polypeptide backbones [38].CD spectra ofR-CPR andH-CPR in solution (Fig. 5A), andin dry films showed two overlapped negative peaks at 209and 222 nm with a positive peak at 192 nm similar toreported solution spectra [39, 40]. These features are muchstronger for R-CPR, and reflect mixed secondary structuredominated by a-helix similar to that of other flavoproteins[41]. Protein-free films ofDMPCandPDDAshowed noCDpeaks between 190 and 260 nm, but DDAB film showedsome weak structure, possibly due to their layered structure[16]. UV CD spectra of rat liver CPR in solution waspreviously reported to showminima at 208 – 209 and 222 nmwith 34% a-helix, 16% beta sheet and 50% randomestimated by curve fitting [42]. However, such analysis ofsecondary structures of CPR in the films was not possiblebecause of inherent uncertainties in protein concentrationand path length [43]. Qualitatively, there are small but clearindividual differences between film and solutionCD spectra

in some cases. Specifically, Figure 5B, curve (f) [R-CPR-DDAB], Fig. 5C, curves (k) [H-CPR-DMPC] and (l) [R-CPR-DMPC] suggest some changes in the secondarystructure of CPRs in the lipid films relative to solution. Incontrast, CDs of CPRs in the PDDA films correspondedwell to solution spectra, suggesting similar secondarystructures in films and solutions.

3.4. Voltammetry

Cyclic voltammetry (CV) gave pairs of well-defined, chemi-cally reversible peaks for (PDDA-PSS)2-PDDA (R-CPR-PDDA)4; (PDDA-PSS)2-PDDA (H-CPR-PDDA)4; [R-CPR-DMPC]; [H-CPR-DMPC]; (Figs. 6 and 7). However,[R-CPR-DDAB] films gave a reversible pair of peaks withan overlapped reduction peak (Fig. 8) and [H-CPR-DDAB]films (Fig. 9) showed two reversible reduction and oxidation

Table 2. Comparison of UV-vis absorbance maxima.

Enzyme 1st peak (nm) 2nd peak (nm)

R-CPR [a] 384 467[R-CPR-PDDA] [b] 366 469[R-CPR-DMPC] [b] 382 465[R-CPR-DDAB] [b] – –H-CPR [a] 383 466[H-CPR-PDDA] [b] 366 466[H-CPR-DMPC] [b] 364 461[H-CPR-DDAB] [b] 366 460

[a] solution; [b] film.

Fig. 5. Circular dichroism spectra at pH 7.4 obtained at 0.2 nm resolution, 1.0 nm band width, 20 mdeg sensitivity, and accumulation of8 scans. (A) In 50 mM sodium phosphate buffer solution, (B) and (C) In films casted on fused silica slide. A: (a) buffer solution (b) H-CPR (0.1 mg/mL) solution and (c) R-CPR (0.5 mg/mL) solution B: (d) DDAB film (e) [H-CPR-DDAB] film (f) [R-CPR-DDAB] filmC: (g) PDDA film (h) DMPC film (i) [H-CPR-PDDA] film (j) [R-CPR-PDDA] film (k) [H-CPR-DMPC] film and (l) [R-CPR-DMPC] film.

Fig. 6. Background subtracted cyclic voltammograms at 0.1 V s�1

in 100 mM sodium phosphate buffer pH 7.4 containing 50 mMKCl of (a) [PDDA-PSS]2-PDDA-[R-CPR-PDDA] 4 film and (b)[PDDA-PSS]2-PDDA-[H-CPR-PDDA]4 film assembled by layer-by-layer method on PG electrodes after purging in nitrogen gasfor 20 min.




peak pairs. All films were stable and showed little change inpeak currents for over amonthwhen stored dry in the dark at48C. CVs of control films containing only PDDA, or DMPC,or DDABwithout protein gave no redox peaks. CVs of pureFMN and FAD in DDAB and DMPC films showedreversible pair of peaks at similar midpoint potentials tothose of the CPR films, i.e. close to �0.245 V vs. NHE.Reduction peak current increased linearly with scan rate

for all CPR films between 5 and 1000 mV s�1, and reductionand oxidation peak heights were equal. For each reversiblepeak pair, reduction-oxidation peak potential separations(DEp) were nearly constant at low scan rates and increasedwith the increase in the scan rate (Fig. 10a). These data areconsistent with a non-ideal quasireversible protein filmvoltammetry [18]. The constant non-kinetically related lowscan rate DEp was subtracted from the higher scan rate data[44] and the corrected peak separation data were analyzedby using the Butler –Volmer model for surface-confinedelectron transfer [45]. Resulting fits of theory to experiment

Fig. 7. Background subtracted cyclic voltammograms at 0.1 V s�1

in 100 mM sodium phosphate buffer pH 7.4 containing 50 mMKCl of (a) [H-CPR-DMPC] film and (b) [R-CPR-DMPC] filmassembled by cast method on PG electrodes after purging innitrogen gas for 20 min.

Fig. 8. Background subtracted cyclic voltammetry at 100 mV s�1

of PG electrode with [R-CPR (1.8 mg/mL)-DDAB (10 mM)] filmformed by cast method, in sodium phosphate buffer, 100 mM,pH 7.4þ 50 mM NaBr after purging in nitrogen gas for 20 min.

Fig. 9. Background subtracted cyclic voltammetry at 100 mV s�1

of PG electrode with [H-CPR (4 mg/mL)-DDAB (10 mM)] filmformed by cast method, in sodium phosphate buffer, 100 mM,pH 7.4þ 50 mM NaBr after purging in nitrogen gas for 20 min.

Fig. 10. Representative peak separation data from film voltammetry results in pH 7.4 bufferþ 50 mM KCl: (a) Influence of scan rateon reduction and oxidation peak potentials for [R-CPR-PDDA]4 films; (b) Influence of scan rate for [H-CPR-DMPC] films on correctedpeak separation (DEp) shown with theoretical line for ks¼ 76� 4 s�1, a¼ 0.5.




were good (Fig. 10b) and provided a value for the averageapparent electrochemical surface electron-transfer rateconstant ks.Our peak potentials of CPRs in DDAB films (Table 3)

differ from those of Shukla et al. [32] who reportedreversible peaks at �0.002 and �0.278 V vs. NHE for H-CRP and �0.020 and �0.254 V for R-CRP. In fact for R-CPR in pH 7.4 phosphateþ 50 mM NaBr we found areversible pair of peaks with an overlapped reductionpeak, and forH-CPRreversible peaks atþ0.32 and�0.24 Vfor H-CPR. The difference in these experiments was thatShukla used a rather exotic buffer, 50 mM bis-tris propane-and 2-amino-2-methylpropane hydrochlorideþ 10 mMNaCl at pH ~8. We briefly surveyed CVs of both CPRs inpH 7.9 phosphate and Tris buffers with 10 mM NaCl. Wenever observed the two sets of peaks reported byShukla, nordid we observe the two overlapped peaks that we found atpH 7.4 with NaBr in the electrolyte. In TRIS bufferpH 7.9þ 10 mM NaCl, we found overlapped peaks similarto those in Figure 8. These results suggest that CVs of CPRsin DDAB films are very sensitive to the components of theelectrolyte solution.CD and UV-vis spectra similar for the CPRs in PDDA

films (Figs. 2 and 5C) to solution spectra (Figs. 4b and 5A)suggest that the proteins in these films have structuressimilar to those of the dissolved proteins. In the DMPCfilms, the UV-vis spectra (Fig. 3) are consistent with intactproteins, but the CD spectra (Fig. 5C) suggest that there areconformational differences relative to solution. Precipita-tion of CPRs at higher concentrations in DDABdispersionssuggests strong interactions, and UV-vis spectra of theseprecipitated materials (Fig. 4A) were featureless and unin-formative.Whether the cofactors remain associatedwith theprotein structures in these films is uncertain. The CDspectrum of the human CPR-DDAB film (Fig. 5B) wassimilar to those in solution, but that of rabbit CPR-DDABsuggested conformational changes from the solution secon-dary structure.Taken together, spectral data suggest that near native

CPR conformation is retained in the PDDA films, but not inthe DMPC or DDAB films. The extent of conformationaldifference in the DMPC films is not extensive, based on thesimilarities of VIS spectra with solution and relatively small

differences in CD spectra. However, strong interactionsmay occur between DDAB and the CPRs, especially rabbitCPR, as evidenced by the lack of clear VIS spectra andchange in the rabbit CPR CD spectrum from solution.With these caveats about the nature of protein structure in

the films, we note that CV gave an average midpointpotential of �0.246� 0.008 mV for R-CPR and H-CPR inall the films (Table 3). In addition, formal potentials and ks

were identical in PDDA films for R-CPR and H-CPR.Formal potentials and ks were very similar in DMPC filmsforR-CPRandH-CPR. InDDABfilms,where the strongestprotein-lipid interactions are suspected, formal potentials ofR-CPR and H-CPR were very similar, but H-CPR had thelarger ks (Table 3). These results suggest that the smallstructural differences reflected in the spectra do not have alarge influence on the electrochemical characteristics of theproteins in the films. This is in distinct contract to iron hemeproteins in which film materials exert a relatively largeinfluence on formal potentials despite retaining near nativesecondary structures in the films [16, 18, 25]. However, thelow ks for R-CPR in DDAB could be related to significantconformational changes.The average midpoint potential of �0.246 V of R-CPR

and H-CPR in the films suggest association with the redoxcouples [FMNsq/FMNred] at �0.269 V, [FADox/FADsq] at�0.283 V obtained from redox potentiometric titrations atpH 7 [12]. The reversible pair of peaks at 0.317 V inH-CPR-DDAB films is, however, a mystery. The fact that changingsalt and buffer identity of the electrolyte solution can makethis peak disappear, or shift several hundred mV suggeststhe possibility of an artifact. This is under further inves-tigation.We do not observe any high potential redox coupleby CV in DMPC or PDDA films.

4. Conclusions

In summary, we compared direct quasireversible electro-chemistry between PG electrodes and two CPRs in twotypes of surfactant and one polyion film constructed layer bylayer. Excepting H-CPR inDDAB, films showed peak pairsat �0.246 V vs. NHE with similar voltammetry, formalpotentials, andks values, despite the fact that some structuralchanges relative to solution were found in DDAB andDMPC films. The LbL CPR-PDDA films had spectraindicating near native structure, but their electrochemicalparameters in these films did not differ significantly fromDDAB and DMPC films. The independence of filmvoltammetry from the influence of film materials is incontrast with film voltammetry of iron heme proteins whichdepends significantly on film materials [16, 18, 25].

5. Acknowledgements

This work was supported by US PHS grant No. ES03154from the National Institute of Environmental HealthSciences (NIEHS), NIH, USA. The authors thank Dr.

Table 3. Average formal potentials (E8’) and heterogeneouselectron transfer rate constant (ks) of CPRs in films from CV inanaerobic phosphate buffer pH 7.4þ 50 mM KCl.

Film E8’ (V) vs. NHE GT

(pmol cm�2)ks

(s�1)

[R-CPR-PDDA]4 �0.246� 0.004 86� 2 56� 4[R-CPR-DMPC] �0.256� 0.001 60� 9 74� 3[R-CPR-DDAB] �0.239� 0.004 105� 1 24� 6[H-CPR-PDDA]4 �0.247� 0.002 26� 3 56� 5[H-CPR-DMPC] �0.253� 0.001 51� 6 76� 4[H-CPR-DDAB] �0.237� 0.010 [a] 197� 10 81� 13 [a]

0.317þ 0.004 [b] 40� 7 [b]

[a] peak I; [b] peak II.




Ingela Jansson of University of Connecticut Health Centerfor the gift of R-CPR.

6. References

[1] Cytochrome P450 (Eds: J. B. Schenkman, H. Greim), Spring-er, Berlin 1993.

[2] Cytochrome P450: Structure, Mechanism and Biochemistry(Ed: P. R. Ortiz de Montellano), Kluwer Academic/Plenum,New York 2005.

[3] T. Iyanagi, H. S. Mason, Biochemistry 1973, 12, 2297.[4] T. Iyanagi, N. Makino, H. S. Mason, Biochemistry 1974, 13,

1701.[5] T. D. Porter, C. B. Kasper, Biochemistry 1986, 25, 1682.[6] D. L. Simmons, P. A. Lalley, C. B. Kasper, J. Biol. Chem.

1985, 260, 515.[7] M. Haniu, M. E. McManus, D. J. Birkett, T. D. Lee, J. E.

Shively, Biochemistry 1989, 28, 8639.[8] M. Wang, D. L. Roberts, R. Paschke, T. M. Shea, B. S. S.

Masters, J.-J. P. Kim, Proc. Natl. Acad. Sci. USA 1997, 94,8411.

[9] J. L. Vermilion, M. J. Coon, J. Biol. Chem. 1978, 253, 2694.[10] B. S. S. Masters, M. H. Bilimoria, H. Kamin, J. Biol. Chem.

1965, 240, 4081.[11] W. L.Backes, C. E. Reker-Backes, J. Biol. Chem. 1988, 263,

247.[12] A. W. Munro, M. A. Noble, L. Robledo, S. N. Daff, S. K.

Chapman, Biochemistry 2001, 40, 1956.[13] P. A. Hubbard, A. L. Shen, R. Paschke, C. B. Kasper, J.-J. P.

Kim, J. Biol. Chem. 2001, 276, 29163.[14] A. Gutierrez, A. Grunau, M. Paine, A. W. Munro, C. R.

Wolf, G. C. K. Roberts, N. S. Scrutton, Biochem. Soc. Trans.2003, 31, 497.

[15] F. P. Guengerich, in CytochromeP450: Structure, Mechanismand Biochemistry (Ed: P. R. Ortiz de Montellano), PlenumPress, New York 2005, pp. 377 – 463.

[16] J. F. Rusling, Acc. Chem. Res. 1998, 31, 363.[17] J. F. Rusling, in Protein Architecture: Interfacing Molecular

Assemblies and Immobilization Biotechnology (Eds: Y. Lvov,H. Mçhwald), Marcel Dekker, New York 2000, pp. 337 – 354.

[18] J. F. Rusling, Z. Zhang, in Biomolecular Films:Design,Functions, and Applications (Ed: J. F. Rusling), MarcelDekker, New York 2003, pp. 44 – 52.

[19] Z. Zhang, A.-E. F. Nassar, Z. Lu, J. B. Schenkman, J. F.Rusling, J. Chem. Soc., Faraday Trans. 1997, 93, 1769.

[20] M. Tominaga, J. Yanagimoto, A.-E. F. Nassar, J. F. Rusling,N. Nakashima, Chem. Lett. 1996, 523.

[21] A.-E. F. Nassar, J. F. Rusling, M. Tominaga, J. Yanagimoto,N. Nakashima, J. Electroanal. Chem. 1996, 416, 183.

[22] B. Munge, S. K. Das, R. Ilagan, Z. Pendon, J. Yang, H. A.Frank, J. F. Rusling, J. Am. Chem. Soc. 2003, 125, 12457.

[23] Q. Huang, Z. Lu, J. F. Rusling, Langmuir 1996, 12, 5472.[24] Z. Lu, Q. Huang, J. F. Rusling, J. Electroanal Chem. 1997,

423, 59.[25] P. M. Guto, J. F. Rusling, Electrochem. Commun. 2006, 8, 455.[26] Y. Lvov, in Protein Architecture (Eds: Y. Lvov, H. Mçhwald),

Marcel Dekker, New York 2000, pp. 125 – 167.[27] G. Decher, Science 1997, 277, 1232.[28] B. Munge, C. Estavillo, J. B. Schenkman, J. F. Rusling,

ChemBioChem 2003, 4, 82.[29] Y. M. Lvov, Z. Lu, J. B. Schenkman, X. Zu, J. F. Rusling, J.

Am. Chem. Soc. 1998, 120, 4073.[30] X. Zu, Z. Lu, Z. Zhang, J. B. Schenkman, J. F. Rusling,

Langmuir 1999, 15, 7372.[31] N. Sultana, J. B. Schenkman, J. F. Rusling, J. Am.Chem. Soc.

2005, 127, 13460.[32] A. Shukla, E. M. J. Gillam, P. V. Bernhardt, Electrochem.

Commun. 2006, 8, 1845.[33] I. Jansson, I. Stoilov, M. Sarfarazi, J. B. Schenkman, Phar-

macogenetics 2001, 11, 793.[34] S. Wiwatchaiwong, H. Matsumura, N. Nakamura, M. Yohda,

H. Ohno, Electroanalysis 2007, 19, 561.[35] Y. Lvov, K. Ariga, I. Ichinose, T. Kunitake, J. Am. Chem. Soc.

1995, 117, 6117.[36] P. Macheroux, in Methods in Molecular Biology: Flavopro-

tein Protocols (Eds.: S. K.Chapman, G. A. Reid), N. J.Humana Press, Totowa 1999, pp. 1 – 2.

[37] G. C. M. Smith, D. G. Tew, C. R. Wolf, Proc. Natl. Acad. Sci.USA 1994, 91, 8710.

[38] G. Holzwarth, P. Doty, J. Am. Chem. Soc. 1965, 87, 218.[39] S. M. Kelly, T. J. Jess, N. C. Price, Biochim. Biophy. Acta

2005, 1751, 119.[40] J. A. DQAnna Jr., G. Tollin, Biochemistry 1972, 11, 1073.[41] D. E. Edmondson, G. Tollin, Biochemistry 1971, 10, 113.[42] J. A. Knapp, J. D. Dignam, H. W. Strobel, J. Biol. Chem.

1977, 252, 437.[43] P. McPhie, Anal. Biochem. 2001, 293, 109.[44] J. Hirst, F. A. Armstrong, Anal. Chem. 1998, 70, 5062.[45] E. Laviron, J. Electroanal. Chem. 1979, 101, 19.




Direct Electrochemistry of Cytochrome P450 Reductases in Surfactant and Polyion Films

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Transcript of Direct Electrochemistry of Cytochrome P450 Reductases in Surfactant and Polyion Films