Proc. Natl. Acad. Sci. USAVol. 92, pp. 7705-7709, August 1995Biochemistry

Electrocatalytically driven w-hydroxylation of fatty acids usingcytochrome P450 4A1

(electromotive force/electroactive mediator/hydrogen peroxide/bioreactor/biosensor)

KEVIN M. FAULKNER, MANJUNATH S. SHET, CHARLES W. FISHER, AND RONALD W. ESTABROOKDepartment of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9038

Contributed by Ronald W Estabrook, May 2, 1995

ABSTRACT The cyclic enzymatic function of a cyto-chrome P450, as it catalyzes the oxygen-dependent metabo-lism of many organic chemicals, requires the delivery of twoelectrons to the hemeprotein. In general these electrons aretransferred from NADPH to the P450 via an FMN- andFAD-containing flavoprotein (NADPH-P450 reductase). Thepresent paper shows that NADPH can be replaced by anelectrochemically generated reductant [cobalt(H) sepulchratetrichloridel for the electrocatalytically driven co-hydroxyla-tion of lauric acid. Results are presented illustrating the useof purified recombinant proteins containing P450 4A1, suchas the fusion protein (rFP450[mRat4Al/mRatOR]L1) or asystem reconstituted with purified P450 4A1 plus purifiedNADPH-P450 reductase. Rates offormation of 12-hydroxydo-decanoic acid by the electrochemical method are comparableto those obtained using NADPH as electron donor. Theseresults suggest the practicality of developing electrocatalyti-cally dependent bioreactors containing different P450s ascatalysts for the large-scale synthesis of stereo- and regio-selective hydroxylation products of many chemicals.

The gene superfamily of hemeproteins called cytochromeP450 (P450) catalyzes the NADPH- and oxygen-dependentoxygenation of a great variety of chemical structures (1). Theunique oxygen chemistry associated with regio- and stereo-specific reactions catalyzed by P450s, as well as the greatdiversity of P450s and their associated specificity for thechemical substrates they oxidize, provides an opportunity todevelop catalysts for specialty chemical syntheses as well asbiosensors for detection and disposal of hazardous chemicals,such as bioremediation of environmental contaminants andpollutants. Recently, we have described (2-4) the expression inEscherichia coli of a number of enzymatically active recombi-nant fusion proteins engineered to contain the heme domainof a mammalian P450 linked to the flavin domains of ratNADPH-P450 reductase. Relatively large amounts of thesefusion proteins can be isolated and purified for study. Theseself-contained enzymatic units have properties comparable tothe native reconstituted P450s.The present study describes the ability to catalyze the

enzymatic w-hydroxylation of lauric acid when an electrocata-lytic system consisting of a platinum electrode and a redoxmediator is employed with purified recombinant proteinscontaining P450 4A1. Results are presented illustrating the useof the purified fusion protein [rFP450(mRat4Al/mRat-OR)L1], where rat P450 4A1 is linked to rat NADPH-P450reductase, here termed rFP4504A1, or a reconstitution systemcontaining purified P450 4A1, engineered to contain a histi-dine hexapeptide domain at the carboxyl terminus of theprotein, [rP4504A1(His)6], together with purified NADPH-P450 reductase. Under these conditions the P450-containingproteins catalyze the .-hydroxylation of lauric acid at rates

similar to those obtained when NADPH is used as the sourceof reducing equivalents.The ability to apply electrochemistry to drive the enzymatic

function of P450s for specific oxygenation reactions opens anumber of opportunities for the large-scale usage of P450s inbioreactor systems as well as the development of amperometricbiosensors.

MATERIALS AND METHODSChemicals. All reagents were used without further purifi-

cation. All solutions were prepared with deionized water.Glycerol, cobalt(III)sepulchrate trichloride [Co(sep)3+], ru-thenium(III) acetylacetonate [Ru(acac)3], potassium ferricya-nide [Fe(CN)6,, Aldrithol-4, meso-2,3-dimercaptosuccinicacid, 5'-deoxy-5'-[methylthio]adenosine, and xylenol orangewere purchased from Aldrich. CHAPS {3-[(3-cholamidopro-pyl)dimethylammonio]-1-propanesulfonate}, horse heart cy-tochrome c (type VI), bovine liver catalase, methyl viologen,methylene blue, flavin mononucleotide, and adenosine 5'-O-[2-thio]diphosphate were purchased from Sigma. Phenosafra-nine was purchased from British Drug Houses (Poole, En-gland). Platinum gauze (52 mesh) and potassium phosphatewere purchased from Fisher Scientific. Radioactive [14C]lauricacid (52.8 mCi/mmol; 1 Ci = 37 GBq) was purchased fromAmersham.

Preparation of Recombinant Enzyme. The recombinantfusion enzyme, rFP450[mRat4A1/mRatOR]L1 (here abbre-viated rFP4504A1), containing the heme domain of rat P4504A1 and the flavin domains of rat NADPH-P450 reductasewas expressed and purified according to methods describedpreviously (2-4). A carboxyl-terminal histidine-tagged P450,rP4504A1(His)6, was expressed and purified according tomethods described by Jenkins and Waterman (5). Concentra-tions of the P450 enzymes were determined spectrophoto-metrically (reduced-CO bound minus oxidized enzyme) (6).The P450 was reduced under anaerobic conditions with so-dium dithionite in the presence of a trace of added methylviologen. Recombinant rat cytochrome b5 (rHMw(His)4b5),here termed b5, was purified from E. coli membrane fractionsas described (7). The cDNA for rat liver NADPH-P450reductase (pOR263) was obtained from Charles Kasper of theUniversity of Wisconsin and recombinant rat NADPH-P450reductase (rHMwmRatOR) was purified from E. coli mem-brane fractions as described by Shen et al. (8).

Electrochemical Methods. Cyclic voltammetry and electrol-ysis were carried out using a model CV-27 Voltammograph(Bioanalytical Systems, West Lafayette, IN). Direct electro-chemistry was performed using a Au button working electrode(1.5-mm diameter) and a reaction volume of 200-400 ,ul ofanalyte (buffer plus redox active compound). A platinum wireserved as the auxiliary electrode, and a premade Ag/AgClelectrode (Bioanalytical Systems) served as the referenceelectrode.

Abbreviation: NHE, normal hydrogen electrode.

7705

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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Cyclic voltammetry was performed from 2 mV/sec to 500mV/sec, at 22°C, under anaerobic conditions. The redoxpotentials cited, E1l2, are the average of the cathodic and anodicpeak potentials for a given analyte vs. normal hydrogenelectrode (NHE), using the Eil2 value obtained for theFe(CN)3-/Fe(CN)'- couple as an internal standard. Thebuffer containing the supporting electrolyte used in the elec-trochemistry of Co(sep)3+, Ru(acac)3, Fe(CN)3-, and cyto-chrome c was 0.02 M potassium phosphate (pH 6.8) containing0.20 M NaCl. Cyclic voltammetry studies using the puri-fied recombinant fusion protein rFP4504A1, purifiedrP4504A1(his)6, or purified NADPH-P450 reductase werecarried out using 0.025 M potassium phosphate buffer (pH 7.5)containing 10% glycerol, 0.20 M KC1, and 0.05% CHAPS. Topromote direct protein electrochemistry, the Au surface of theelectrode was modified with various thiol reagents as describedby Allen et al. (9). Solutions of the modifiers were always madefresh in 0.025 M potassium phosphate buffer (pH 7.5).

Controlled potential electrolysis experiments were carriedout using a reaction mixture containing an electrochemicalmediator and a solution of the P450 enzyme dissolved in abuffer containing 0.05 M Tris-HCl buffer (pH 7.5), 0.01 MMgCl2, 0.10 M KCI, and the indicated concentration of[14C]lauric acid. Reactions were performed at 22°C whilestirring under air. For these experiments the working electrodewas a 1 x 2.5 cm piece of platinum gauze attached to aplatinum wire. The auxiliary electrode was a 25-cm piece ofcoiled platinum wire separated from the bulk solution by aglass-fritted tube filled with 3 M KCI. The Ag/AgCl referenceelectrode was fitted with a Vycor tip as received (BioanalyticalSystems). The tip of the auxiliary electrode compartment fritand the reference electrode tip were situated in the center ofa semicircle formed by the platinum gauze to minimize thedistance between all points on the working electrode and theauxiliary electrode. At closed circuit, the working electrodewas poised at -650 mV vs. Ag/AgCl (-450 mV vs. NHE).Other conditions for controlled potential electrolysis of thereaction mixture containing the mediator and P450 are asindicated in the text. Where indicated, purified b5, NADPH-P450 reductase, and catalase were added to the reactionmixture.

Lauric Acid Metabolism. Aliquots of 0.4 or 0.5 ml wereremoved during electrolysis from the reaction mixture at thetimes specified and added with mixing to 0.1 ml of 6 M HCI.This mixture was then extracted with 5 ml of ethyl acetate. Theseparated ethyl acetate layer was evaporated by a stream ofnitrogen gas and the residue was dissolved in 160 ,ul of 99.9%hexane/0.1% acetic acid, ofwhich 80 ,lwas injected for HPLCanalysis. Normal-phase HPLC was performed using a 3.9 x300 mm, 10-mm ,uPorasil column (Waters chromatographydivision of Millipore) and employing a linear gradient of 0-5%isopropyl alcohol in hexane containing 0.1% acetic acid for50 min. ,Radioactivity of the fractions was detected with aHewlett-Packard Flow One Radioactive detector.Other Methods. The rate of H202 formation was deter-

mined using the xylenol orange method as described (10, 11).

RESULTSDirect Electron Transfer. Cyclic voltammetry was per-

formed on Fe(CN)3-, Ru(acac)3, and Co(sep)3+ to calibratethe three-electrode system and to determine electrochemicalparameters. Using a Au electrode the Eil2 values determinedfor Fe(CN)3-, Ru(acac)3, and Co(sep)3+ were +415 mV, -290mV, and -350 mV (vs. NHE), respectively. These E/2 valuesare consistent with those reported in the literature (12-14) andindicate reversible redox reactions.

Cyclic voltammetry of horse heart cytochrome c was per-formed using an Aldrithiol-4 (4-mercaptopyridine)-modifiedAu electrode (9). A reversible redox couple was obtained, with

an E/2 of +250 mV (vs. NHE), identical to the literature value(9). Similarly, cyclic voltammetry was attempted using a solu-tion of rP4504Al(his)6 (200 ,uM) to determine the feasibilityof direct electron transfer from the electrode to the hemedomain of a P450. No cyclic voltammetry was observed for thepurified rP4504A1(his)6, the purified flavoprotein, NADPH-P450 reductase, or the purified fusion protein, rFP4504A1.Other Au electrode surface modifiers, such as meso-2,3-dimercaptosuccinic acid, 5'-deoxy-5'-[methylthio]adenosine,and adenosine 5'-O-[2-thio]diphosphate were tested, but noneof these promoted rapid electron transfer to a P450 or fla-voprotein as detected by cyclic voltammetry from 2 mV/sec to100 mV/sec.Mediator-Promoted Electrolysis. The electrolysis method

was tested in the presence of an electrochemically activemediator, Co(sep)3+. For this experiment a solution contain-ing purified rFP4504A1 and lauric acid was stirred in air, anda submerged 1 x 2.5 cm segment of platinum gauze served asthe working electrode. When the circuit was closed to initiatethe reaction, using a potential of -450 mV (vs. NHE), thecurrent jumped to about 2.2 mA and then progressivelydecreased, attaining a steady-state value of about 0.8 mA after5 min. Sampling of the reaction mixture followed by analysisusing HPLC showed that lauric acid was hydroxylated to form12-hydroxydodecanoic acid (c-hydroxylauric acid). The timecourse for this reaction, plotted together with the change incurrent, is shown in Fig. 1. The short delay in the start of thehydroxylation reaction can be attributed to the time requiredto reduce the mediator from the cobaltic (3+) to the cobaltous(2+) state. Spectrophotometric measurements confirm thistime-dependent reduction of the mediator. Further, when themediator, Co(sep)3+, was incubated at room temperature inthe reaction system for 10 min with the circuit closed, and thenan aliquot of rFP4504A1 was added to initiate the reaction, notime lag in the appearance of 12-hydroxydodecanoic acid wasobserved. Comparison of the rate of lauric acid hydroxylationobtained under similar conditions using NADPH as electrondonor showed that the rate was very similar and the same

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FIG. 1. Measurement of w-hydroxylation of lauric acid by thefusion protein, rFP4504A1, using an electrocatalytic method for thegeneration of electrons. A platinum gauze working electrode waspoised at -450 mV (vs. NHE) as described in the text and the changein current was measured (A). One-half micromolar rFP4504A1 wasincubated with 17 ,uM [14C]lauric acid and 1 mM Co(sep)3+ asdescribed in the text. Aliquots of the reaction mixture were removedat the times indicated and the conversion of lauric acid to 12-hydroxydodecanoic acid was determined by HPLC analysis (0).

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Proc. Natl. Acad. Sci. USA 92 (1995) 7707

product was formed-i.e., 1.1 nmol of 12-hydroxydodecanoicacid formed per min/nmol of rFP4504A1.The rate of hydroxylation of lauric acid by the electrolysis

method is dependent on the concentration of rFP4504A1 andthe concentration of the mediator, Co(sep)3+. Increasing theconcentration of rFP4504A1 increases the rate of lauric acidhydroxylation with a maximal turnover number of 0.65 nmol of12-hydroxydodecanoic acid formed per min/nmol ofrFP4504A1 observed when using 0.5 ,uM rFP4504A1. Like-wise, increasing the concentration of the electrochemicalmediator, Co(sep)3+, increases the rate of lauric acid hydroxy-lation with a maximal rate obtained using 1 mM Co(sep)3+.Varying the polarizing voltage from -250mV to -650mV (vs.NHE) showed that the maximal rate of lauric acid hydroxy-lation was obtained when using -450mV (vs. NHE). When therate of lauric acid metabolism driven by the electrolyticreaction was measured with the reaction mixture equilibratedat temperatures ranging from 4°C to 36°C, the rate at 22°C wasfound to be optimal. Monitoring the temperature during thereactions revealed no significant temperature increase during.a 60-min electrolysis experiment.Other compounds were tested for their ability to mediate

electron transfer between the working electrode and the P450fusion protein. One millimolar solutions of methyl viologen,Ru(acac)3, phenosafranine, flavin mononucleotide, and meth-ylene blue were tested. When using the experimental condi-tions described above, only Co(sep)3+ was capable of support-ing the catalytic activity of rFP4504A1.Requirement for the Fusion Protein, rFP4504Al. A series

of experiments was carried out using conditions similar tothose described above to test the ability of rP4504Al(His)6alone or reconstituted with purified NADPH-P450 reduc-tase to catalyze the hydroxylation of lauric acid by theelectrolysis method. As summarized in Table 1, no hydroxy-lation of lauric acid was observed when the mediator, 1 mMCo(sep)3+, was used alone or together with purified fla-voprotein, NADPH-P450 reductase. Likewise, no hydroxy-lation of lauric acid was observed when the mediator andpurified rP4504Al(His)6 were used. However, whenrP4504A1(His)6 and an equivalent concentration of NAD-PH-P450 reductase were present together, using preincuba-tion conditions for reconstitution of lauric acid o-hydroxy-lase activity, a rate of formation of 12-hydroxydodecanoicacid that was about one-half of that seen with the purified

Table 1. Hydroxylation of lauric acid

fusion protein, rFP4504A1 was observed. These results showthe need for P450 and the NADPH-P450 reductase toachieve an electrocatalytically functional reaction system.Table 1 summarizes the results obtained using various

experimental conditions. The presence of a 5-fold excess ofpurified cytochrome b5 slightly inhibited the rate of lauric acidc-hydroxylation catalyzed by rFP4504A1. The addition of an

excess of NADPH-P450 reductase to the reaction system, witheither rFP4504A1 or the reconstituted system withrP4504A1(His)6, stimulated the rate of lauric acid hydroxyla-tion (data not shown). These results agree with our earlierstudies of the enzymatic properties of rFP4504A1 whenNADPH was used as a source of electrons for the hydroxyla-tion reaction (2). The hydroxylation of lauric acid, using eitherNADPH or the electrolysis method, was inhibited by imida-zolyldecanoic acid, a specific inhibitor of the P4504A1 enzyme(15).

Stability of the Bioreactor System. The hydroxylation oflauric acid by rFP4504A1, using the electrolysis method with1 mM Co(sep)3+, was influenced by catalase. Omission ofcatalase from the reaction system resulted in a time-dependentdecrease in the rate of formation of 12-hydroxydodecanoicacid. Measurement of the overall yield of product formation,as determined after a reaction time of 1 hr of electrolysis,showed that there was a 90% conversion of 17 ,uM lauric acidin the presence of catalase and only 50% when the catalase wasomitted. This result suggests that H202 is being formed,influencing the stability of rFP4504A1. When the rate of H202production was measured during the electrolytic reaction usinga stirred solution equilibrated with air, it was observed thatH202 was produced at an initial rate of about 80 ,uM/min,which decreased to a rate of almost zero by 5 min. Thisproduction of H202 was independent of the presence ofrFP4504A1 and increased with increasing concentrations ofCo(sep)3+. The addition of catalase significantly decreasedthis rate of H202 production to about 9 ,uM H202 per min.Incorporation of an oxygen electrode in the reaction systemshowed the rapid uptake of oxygen during the first 10 min ofthe electrolysis reaction, which leveled off at a steady-stateconcentration of about 40 AM 02-

Studies were carried out to measure the stability ofrFP4504A1 during 30 min of electrolysis. Aliquots of thereaction mixture were removed at designated times and theconcentration of the CO complex of reduced P450 was deter-

Activity, nmol of lauric acid metabolizedCondition per min/ml

NADPH*0.5 ,M rP4504Al(His)6 + 0.5 ALM NADPH-P450 reductase 0.70.5 ,uM rFP4504A1 1.10.5 ALM rFP4504A1 + 1 ,uM IDA 0.1

Electrolysist1 mM Co(sep)3+ 0.00.5 ZM rP4504A1(His)6 + 1 mM Co(sep)3+ 0.00.5 ,uM NADPH-P450 reductase + 1 mM Co(sep)3+ 0.00.5 ,LM rP4504A1(His)6 + 0.5 ,LM NADPH-P450 reductase

+ 1 mM Co(sep)3+ 0.30.5 ,iM rFP4504A1 + 1 mM Co(sep)3+ 0.650.5 AM rFP4504A1 + 1 mM Co(sep)3+* 0.50.5 AM rFP4504A1 + 2.5 ,LM b5 + 1 mM Co(sep)3+ 0.50.5 ,uM rFP4504A1 + 5 ,uM IDA + 1 mM Co(sep)3+ 0.0

All experiments were carried out in a reaction volume of 3.5 ml, stirred in air at 22°C, using a buffermixture containing 50mM Tris HCl (pH 7.5), 10mM MgCl2, 0.1 M KCI, and 17 t.M [14C]lauric acid. IDA,co-imidazolyldecanoic acid.*For NADPH experiments, the reaction was started by adding 1 mM NADPH.tFor electrolysis experiments, the working electrode was poised at -450 mV (vs. NHE) and the reactionmixture was supplemented by the addition of 2000 units of catalase.*Surface gassed with nitrogen.

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Pt gauze

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FIG. 2. Degradation of P450 during elecimolar rFP4504A1 was incubated with 1 mIlauric acid in 3.5 ml of a reaction mixture corbuffer (pH 7.5), 10 mM MgCl2, and 0.1 M Iof catalase were added where indicated. In onof the stirred reaction mixture was gassed by a

electrodes were polarized at -450 mV (vs. Nreaction mixture were removed at the timestrophotometric analysis of P450 content.

mined spectrophotometrically. As shoabsence of catalase, <20% of rFP4504binding CO after 30 min of electrolysicatalase, about 70% of the rFP4504A1 j30 min. It is interesting to note that afteat least 90% of the P450 immediatelysample was mixed with CO-saturated Imetric measurement of the electrocatalyCo(sep)3+ showed an initial rate of 20steady-state concentration of 0.35 mM CThe stability of the rFP4504A1 s

present, was also studied under conditiand these conditions appear to be thstabilizing rFP4504A1 (Fig. 2) with a miurate of lauric acid hydroxylation (Table

DISCUSSIONThe present study demonstrates the felectromotive force to supply the electfunctioning of a cytochrome P450 asdroxylation of the fatty acid, lauric acpermits the application of electrochemmany different forms of P450s for thstereo- and regio-specific hydroxylated pconditions at room temperature andNADPH. One factor contributing to thiis the use of the recombinant fusion pheme domain of P4504A1 linked to tNADPH-P450 reductase. The abilitylarge quantities of this catalyst, togethfurther engineer unique binding domain;the P450-containing fusion protein, makcandidate for future applications (16,been determined for optimizing thecatalyst, permitting use of the electrolysiincubation periods.The cyclic function of P450s requir

electron is needed for reduction of ti(complexed with a molecule of substrat

FIG. 3. Schematic representation of reactions occurring at theplatinum working electrode during an electrolysis experiment asdescribed in the text.

20 25 30 and a second electron is needed for the reduction of the ferroushemeprotein (complexed with substrate and oxygen) to gen-erate the "activated oxygen" state of P450 (18, 19). The success

trolysis. One-half micro- of the electrolysis method described here is due to the ability of4 Co(sep)3+ and 17 ,uM the proteins to accumulate two or more electrons donated by theitaining 50 mM Tris-HCl electrochemical mediator. This can be achieved by using eitherKCl. Two thousand units the fusion protein which contains a heme domain and flavin~e experiment the surface.. . 'stepreamonitrOgen. The domains in a single functional unit where intramolecular electronaHE) and aliquots of the transfer reactions are facilitated, or the reconstituted P450 systemindicated for the spece containing the P450 protein and purified NADPH-P450 reduc-

tase brought together by preincubation conditions.The reactions occurring during an electrolysis experiment

iwn in Fig. 2, in the are depicted in Fig. 3. There is a small amount of oxygenkl was still capable of reduced at the platinum electrode surface (reaction A) result-is. In the presence of ing in the formation of superoxide, which dismutates to H202.remains unchanged at The electroactive mediator, Co(sep)3+, is reduced to Co-r 5 min of electrolysis, (sep)2+ (reaction B), which either can react with oxygenbound CO when the (reaction C) to form superoxide (and H202) or reduces thebuffer. Spectrophoto- cytochrome P450 complex with the flavoprotein NADPH-(tic reduction of 1 mM P450 reductase (reaction D). The H202 generated by reactions,uM/min, attaining a A and C can be decomposed by the addition of catalase or'o(sep)2+ after 20 min. markedly reduced by decreasing the oxygen tension in theystem, with catalase reaction mixture. The ability of P450s to function at low oxygenons of low 02 tension tension permits the hydroxylation of lauric acid to occur underie most favorable for conditions where the destructive effect of H202 is minimized.nimal influence on the

1). We thank Dr. Robert Hanzlik, University of Kansas, for a generousgift of to-imidazolyldecanoic acid. This work was supported by grantsfrom the U.S. Public Health Service, National Institutes of Health(NIGMS 16488), and the Robert A. Welch Foundation (I-0959).

easibility of using an K.M.F. is a Research Fellow of the Welch Foundation.trons required for theit catalyzes the ca-hy- 1. Nelson, D. R., Kamataki, T., Waxman, D. J., Guengerich, F. P.,-id. This method now Estabrook, R. W., Feyereisen, R., Gonzalez, F. J., Coon, M. J.,iical techniques to the Gunsalus, I. C., Gotoh, O., Okuda, K & Nebert, D. W. (1993)e synthesis of unique DNA Cell Biol. 12, 1-51.)roducts using reaction 2. Fisher, C. W., Shet, M. S., Caudle, D. L., Martin-Wixtrom, C. A.I without the use of & Estabrook, R.W. (1992) Proc. Natl. Acad. Sci. USA 89,

t succes of ths study10817-10821.e success of this study 3. Shet, M. S., Fisher, C. W., Holmans, P. L. & Estabrook, R. W.-rotein containing the (1993) Proc. Natl. Acad. Sci. USA 90, 11748-11752.:he flavin domains of 4. Shet, M. S., Fisher, C. W., Arlotto, M. P., Shackleton, C. H. L.,to isolate and purify Holmans, P. L., Martin-Wixtrom, C. A., Saeki, Y. & Estabrook,er with the ability to R. W. (1994) Archiv. Biochem. Biophys. 311, 402-417.s for immobilization of 5. Jenkins, C. M. & Waterman, M. R. (1994) J. Biol. Chem. 269,-es this catalyst an ideal 27401-27408.17). Conditions have 6. Omura, T. & Sato, R. (1964) J. Biol. Chem. 239, 2370-2378.stability of this P450 7. Holmans, P. L., Shet, M. S., Martin-Wixtrom, C. A., Fisher,method for extended C. W. & Estabrook, R. W. (1994) Arch. Biochem. Biophys. 312,

554-565.8. Shen, A., Porter, T. D., Wilson, T. E. & Kasper, C. B. (1989) J.

es two electrons: one Biol. Chem. 264, 7584-7589.ie ferric hemeprotein 9. Allen, P. M., Hill, H. A. 0. & Walton, N. J. (1984) J. Electroanal.e) to the ferrous form, Chem. 178, 69-86.

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11. Shet, M. S., Faulkner, K M., Holmans, P. L., Fisher, C. W. &Estabrook, R. W. (1995) Arch. Biochem. Biophys. 318, 314-321.

12. Fultz, M. L. & Durst, R. A. (1982) Anal. Chim. Acta 140, 1-18.13. Richardson, D. E. (1990) Inorg. Chem. 29, 3213-3217.14. Geselowitz, D. A. (1981) Inorg. Chem. 20, 4457-4459.15. Alterman, M. A. & Hanzlik, R. P. (1994) FASEB J. 8, A1244.16. Fisher, C. W., Shet, M. S., Holmans, P. L. & Estabrook, R. W.

(1994) in Proceedings of the 8th International Conference on

Proc. Natl. Acad. Sci. USA 92 (1995) 7709

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17. Achtnich, U. R., Tiefenauer, L. X. & Andres, R. Y. (1992)Biosens. Bioelectron. 7, 279-290.

18. Schenkman, J. B., Cammer, W., Remmer, H., Cooper, D. Y.,Narasimhulu, S. & Rosenthal, 0. (1966) in Biological and Chem-ical Aspects of Oxygenases, eds. Bloch, K. & Hayaishi, 0. (Ma-ruzen, Tokyo), pp. 153-170.

19. White, R. E. & Coon, M. J. (1980) Annu. Rev. Biochem. 49,315-356.

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