ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 180, 343-347 (1977)

Hydrogen Peroxide Formation and Stoichiometry of Hydroxylation Reactions Catalyzed by Highly Purified Liver Microsomal Cytochrome P-450’

GERALD D. NORDBLOM AND MINOR J. COON

Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, Michigan 48109 Received July 23, 1976

The stoichiometry of hydroxylation reactions catalyzed by cytochrome P-450 was studied in a reconstituted enzyme system containing the highly purified cytochrome from phenobarbital-induced rabbit liver microsomes. Hydrogen peroxide was shown to be formed in the reconstituted system in the presence of NADPH and oxygen; the amount of peroxide produced varied with the substrated added. NADPH oxidation, oxygen consumption, and total product formation (sum of hydroxylated compound and hydrogen peroxide) were shown to be equimolar when cyclohexane, benzphetamine, or dimethylaniline served as the substrate. The stoichiometry observed represents the sum of two activities associated with cytochrome P-450. These are (1) hydroxylase activity: NADPH + H+ + O2 + RH + NADP+ + H,O + ROH; and (2) oxidase activity: NADPH + H+ + O2 + NADP+ + H,O,. Benzylamphetamine (desmethylbenzphetamine) acts as a pseudosubstrate in that it stimulates peroxide formation to the same extent as the parent compound (benzphetamine), but does not undergo hydroxylation. Accordingly, when benzylamphetamine alone is added in control experiments to correct for the NADPH and O2 consumption not associated with benzphetamine hydroxylation, the expected 1:l:l stoichiometry for NADPH oxidation, O2 consumption, and formaldehyde formation in the hydroxylation reaction is observed.

Cytochrome P-450, the terminal oxidase purified enzyme fractions, the addition of of the liver microsomal mixed-function ox- catalase reduced the oxygen uptake to the idase system, catalyzes the NADPH-de- expected value (9). pendent hydroxylation of a wide variety of In the present paper, the stoichiometry drugs, steroids, fatty acids, and alkanes. of the hydroxylation of several substrates According to the general equation for a was studied with a reconstituted system mixed-function oxidation reaction (2), the containing highly purified P-45RMz and stoichiometry of NADPH oxidation, oxy- NADPH-cytochrome P-450 reductase. Hy- gen consumption, and product formation is drogen peroxide was shown to be produced 1:l:l. Experimental values obtained with in varying amounts depending upon the liver microsomal suspensions have varied substrate present. The expected stoichiom- with the conditions employed, and various etry was obtained for NADPH and oxygen procedures have been used to correct for utilization and for product formation, with the endogenous rates of NADPH and oxy- several substrates, provided that hydrogen gen utilization (3-8). In our earlier investi- peroxide formation was also taken into ac- gation of the stoichiometry of drug de- count. The NADPH-dependent production methylation using resolved and partially of hydrogen peroxide by liver microsomes 1 This research was supported by Grant BMSi’l- was first reported by Gillette et al. (10) and 01195 from the National Science Foundation and * Abbreviations used: P-450,,, liver microsomal Grant AM-10339 from the United States Public cytochromeP-450;P-45&,,, phenobarbital-inducible Health Service. A preliminary report of part of this form ofP-45&,; dilauroyl-GPC, dilauroylglyceryl-3- investigation has been presented (1). phosphorylcholine; DEAE, diethylaminoethyl. 343 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 0003-9861 344 NORDBLOM AND COON

has also been described by others in more containing P-45&, (0.1 PM), NADPH-cytochrome recent studies (11-14). P-450 reductase preparation (36 pg of protein/ml), dilauroyl-GPC (30 fig/ml), 0.1 M potassium phos- phate buffer, pH 7.4, substrate (10 mM cyclohexane EXPERIMENTAL PROCEDURES or N,N-dimethylaniline, added in 0.01 ml of metha- P-4%&, was isolated from liver microsomes of nol, or 1.0 mM benzphetamine), and 0.1 mM phenobarbital-induced rabbits as described previ- NADPH. In some experiments, benzylamphetamine ously (15, 16). The procedure involves fractionation was added at a final 1.0 mM concentration in place of of cholate-solubilized microsomes with polyethylene the usual substrates. In all such assays P-45kM was glycol, followed by DEAE-cellulose and hydroxylap- the rate-limiting component, and the molar ratio of atite column chromatography. The final prepara- reductase to P-45RM was about 5:l. In microsomes, tion, which is electrophoretically homogeneous, con- in contrast, P-450,,, is in excess. The order of addi- tains the form of P-45RM induced by phenobarbital. tion and other conditions were described earlier (16). This form has been designated by its relative elec- Oxygen consumption was measured under the same trophoretic mobility as P-45RMZ (17). The prepara- conditions, but in a 3-ml reaction mixture, with a tions used in the present study had the following Clark-type electrode used in conjunction with a specific content, given as nanomoles per milligram Heath EU-BOB recorder. NADPH was the last addi- of protein: microsomes, 3.4; fraction precipitated by tion, and its consumption was measured at 340 nm polyethylene glycol, 6.0; DEAE-cellulose column with a spectrophotometer equipped with a multiple eluate, 8.6; and hydroxylapatite column eluate, sample absorbance recorder. Formaldehyde forma- 16.9. In all cases, except with microsomes, the en- tion from N,N-dimethylaniline was measured by the zyme system was reconstituted with rabbit liver method of Nash (19) as modified by Cochin and microsomal NADPH-cytochrome P-150 reductase Axelrod (20). The N-demethylation of radioactive (specific activity, 19.2 pmol of cytochrome c reduced/ benzphetamine (1.2 x lo5 cpm/pmol) was deter- min/mg of protein at 30°C) purified by a modification mined by the method of Poland and Nebert (21) and of methods described eariler (16, 18) and dilauroyl- the hydroxylation of radioactive cyclohexane (1.8 x GPC. lo4 cpm/pmol) was determined by isolation of the d-Benzphetamine and d-benzylamphetamine resulting cyclohexanol according to the chromato- (desmethylbenzphetamine) were generously pro- graphic procedure of Gholson et al. (22). Hydrogen vided by Dr. P. W. McConnell of the Upjohn Com- peroxide production was measured by the change in pany and d-[N-methyl-‘%lbenzphetamine was pro- rate of oxygen consumption upon the addition of 3 vided by Dr. A. Y. H. Lu, Hoffmann-La Roche, Inc.; pg (108 units) of catalase to the reaction mixture. lU-‘4C1cyclohexane was obtained from Amersham/ Catalase had no effect on the rates of NADPH oxida- Searle, dilauroyl-GPC was from Serdary Research tion or of product formation from the added sub- Laboratories, London, Ontario, and bovine liver cat- strates. For comparison, rates of peroxide formation alase (twice recrystallized) and NADPH were from were also determined in the same experiments by Sigma. N,N-Dimethylaniline was purchased from the ferrithiocyanate method described by Thurman Aldrich and redistilled, while cyclohexane from et al. Cl), and the values obtained by the two meth- Eastman Chemical (Spectra ACS grade) was used ods were found to be in excellent agreement. without further purification. RESULTS Assay of components in hydroxylation reactions. Rates of NADPH oxidation and product formation Stoichiometry of substrate hydroxyl- were determined at 30°C in l.O-ml reaction mixtures ation. As shown in Table I, highly purified

TABLE I ST~ICHIOMETRY OF CYTOCHROME P-450-CATALYZED REACTIONS IN RECONSTITUTED SYSTEM” Substrate Change in compone;~~(;mol/min/nmol of P- NADPH/O,/ rod- M uct + H, 8 2 -NADPH -0, Producti H,Oz Benzphetamine 77 75 33 42 1.0/1.0/1.0 Cyclohexane 66 62 50 17 1.0/0.9/1.0 N.N-Dimethylaniline 53 56 30 25 1.0/1.1/1.0 None 21 22 - 21 1.0/1.0/1.0 n In this and subsequent tables, the data presented are average values of three experiments. TheP-45RM, had a specific content of 16.9 nmol/mg of protein, and the P-450,,M,:reductase molar ratio was 1:5. b Formaldehyde was measured as the product of benzphetamine and dimethylaniline hydroxylation, and, cyclohexanol was measured as the product formed from cyclohexane. H202 FORMATION BY PURIFIED CYTOCHROME P-450 345 P450r,M catalyzes the formation of H,O, purification of P-45%, and with a homoge- from NADPH and oxygen in the reconsti- neous preparation of the cytochrome. The tuted enzyme system in either the pres- low efficiency of benzphetamine hydroxyl- ence or the absence of substrate. Peroxide ation relative to NADPH oxidation is seen formation was much greater with benz- to be due to the effect of this particular phetamine than with the other substrates substrate and not to changes occurring in tested. Therefore, the oxygen and NADPH the enzyme during solubilization from the consumed in peroxide formation must be membrane and subsequent purification. taken into account in studying the stoichi- The increased turnover number (moles of ometry of each P-45%,-mediated hydrox- benzphetamine demethylated per minute ylation reaction. The results presented per mole of P-450& during purification is show that, when this is done, NADPH oxi- undoubtedly due to removal of other forms dation, oxygen consumption, and total of P-45RM known to have less activity to- product formation are equimolar. Control ward this substrate (17). experiments established that the NADPH Effect of benzphetamine and benzylam- oxidase activity observed in the absence of phetamine on hydrogen peroxide forma- substrate is due almost entirely to the cy- tion. Results already presented (Table I) tochrome P-450, rather than the reduc- indicate that the amount of hydrogen per- tase. In other experiments with the recon- oxide formed varies with the substrate stituted system, cyclohexanol was shown present. Rates of hydrogen peroxide for- by gas-liquid chromatography to be the mation in the presence of dimethylaniline only product formed from cyclohexane, and cyclohexane were similar to those ob- and benzylamphetamine and N-methylan- served in the absence of substrate; in con- iline were shown to be the only products trast, benzphetamine caused a twofold (other than formaldehyde) formed from stimulation of peroxide formation. The benzphetamine and dimethylaniline, re- data in Table III show that the product spectively. of benzphetamine demethylation, benzyl- Effect of P-450,,purification on relative amphetamine, has a similar effect to that amounts of product and hydrogen perox- of the parent compound. Benzylamphetam- ide formed. Because of the relatively large ine acts as a pseudosubstrate in that it amount of peroxide formed, particularly does not undergo hydroxylation by P- during the hydroxylation of benzpheta- 45NM2 (as shown by gas-liquid chromatog- mine (see Table I), the possibility was con- raphy in this laboratory), but stimulates sidered that the cytochrome becomes more electron transfer to yield hydrogen perox- readily autoxidizable, and therefore pro- ide to the same extent as that seen with duces more hydrogen peroxide, upon solu- benzphetamine. It should be noted that, bilization from microsomes and purifica- with the desmethyl compound present, hy- tion. The data in Table II compare the drogen peroxide formation, NADPH oxi- changes in reaction components during dation, and 0, consumption are equimo- benzphetamine demethylation in micro- lar. Accordingly, benzylamphetamine somes at two intermediate stages in the may be added in control experiments (in

TABLE II STOICHIOMETRY OF BENZPHETAMINE HYDROXYLATION BY P-45RM AT VARIOUS STAGES OF PURIFICATION Preparation Change in compone;~~(;mol/min/nmol of P- Efficii;cy M 0 -NADPH -0, CH,O H,O, Microsomes 9 10 4 6 44 Polyethylene glycol fraction 34 35 11 16 44 DEAE-cellulose column eluate 6’7 64 31 33 47 Hydroxylapatite column eluate 77 75 33 42 43 n Efficiency is expressed as the amount of substrate hydroxylated (determined by formaldehyde forma- tion) relative to NADPH oxidized. NORDBLOM AND COON

TABLE III STOICHIOMETRY WITH BENZPHETAMINE OR BENZYLAMPHETAMINE PRESENTI Expt Compound added Change in compone;~ts(;mol/min/nmol of P- T$ II$- ?I c! + W:) -NADPH -0, CH,O H,O, A Benzphetamine 77 75 33 42 75 (1.0) (1.0) (9.4) (0.6) (1.0) B Benzylampheta- 45 44 42 - mine (1.0) (1.0) - (0.9) A minus B 32 - - (1.0) n Rates relative to that of NADPH oxidation are given in parentheses. TheP-45Rn, had a specific content of 16.9 nmol/mg of protein, and the cytochrome:reductase molar ratio was 1:5. the absence of benzphetamine) to correct peared to decrease the oxygen uptake dur- for the NADPH and 0, consumption not ing benzphetamine demethylation to a associated with benzphetamine hydroxyl- value corresponding to the amount of ation, and the expected 1:l:l stoichiometry NADPH oxidized and formaldehyde of the hydroxylation reaction is then ob- formed. The significance of that earlier tained, as shown in Table III. finding is questionable, since the present results with a system containing highly DISCUSSION purified P45%M and reductase establish The results presented show that two re- that added catalase has the expected effect actions, each involving a two-electron of generating 0.5 mol of oxygen per mole of transfer to molecular oxygen, contribute to hydrogen peroxide present. the stoichiometry observed with purified Our results indicate that the nature of P4EQM in the reconstituted system: hy- the substrate alters the efficiency of the droxylase activity, where RH represents enzyme, i.e., the relative contributions of substrate, hydroxylase and oxidase activities in the overall consumption of NADPH and oxy- NADPH + H+ + 0, + RH PI gen. Hydroxylase activity exceeds oxidase + NADP + H,O + ROH; activity when cyclohexane or dimethylani- line is present, whereas the reverse is true NADPH oxidase activity, with benzphetamine. Since benzylam- NADPH + H+ + 0, PI phetamine acts as a pseudosubstrate with + NADP + H,O,. an effect like that of benzphetamine, it appears that certain structural features of The latter reaction is apparently due to the the substrate help determine the fate of autoxidation of the reduced cytochrome in the oxygen molecule undergoing reduction the presence of oxygen. The finding that in this enzyme system. Thus, the sub- hydrogen peroxide is produced by the puri- strate alters the relative extent of oxygen fied cytochrome is particularly interest- reduction to hydrogen peroxide, as com- ing, in view of the fact that such enzyme pared to oxygen reduction to water accom- preparations are known to utilize peroxide panied by substrate hydroxylation. These for the hydroxylation of a variety of sub- two processes may involve a common spe- strates in the absence of NADPH and mo- cies of activated oxygen, as proposed in a lecular oxygen (23, 24). It is possible, previous mechanistic study (23). Staudt et therefore, that Reaction [2] may contrib- al. (8) found that perfluoro-n-hexane acts ute to the overall hydroxylation capability as an uncoupler when added to rabbit liver of this enzyme system. In an earlier study microsomal suspensions; no product was (9) carried out under somewhat different formed, and 2 mol of NADPH were oxi- conditions with a partially purified P- dized per mole of oxygen. Their findings 45RM preparation, added catalase ap- suggest that the oxygen was reduced to H,O, FORMATION BY PURIFIED CYTOCHROME P-450 347 water, which would be expected since mi- 10. GILLETTE, J. R., BRODIE, B. B., AND LADu, B. N. crosomes generally contain adsorbed cata- (1957) J. Pharmacol. Exp. Thu. 119, 532-540. lase. Their work agrees well with our own 11. THURMAN, R. G., LEY, H. G., AND SCHOLZ, R. when catalase is added to our reaction (1972) Eur. J. Biochem. 25, 420430. mixtures. 12. BOVERIS, A., OSHINO, N., AND CHANCE, B. (1972) Biochem. J. 128, 617-630. 13. HILDEBRANDT, A. G., SPECK, M., AND ROOTS, I. ACKNOWLEDGMENTS (1973) Biochem. Biophys. Res. Commun. 54, We are grateful to Barbara M. Michniewicz for 968-975. skillful assistance in the research. 14. HILDEBRANDT, A. G., AND ROOTS, I. (1975)Arch. Biochem. Biophys. 171, 385-392. REFERENCES 15. VAN DER HOEVEN, T. A., HAUGEN, D. A., AND 1. NORDBLOM, G. D., AND COON, M. J. (1976) Fed. COON, M. J. (1974) Biochem. Biophys. Res. Proc. 35, 81. Commun. 60, 569-575. 2. MASON, H. S. (1957) Science 125, 1185-1188. 16. VAN DER HOEVEN, T. A., AND COON, M. J. (1974) 3. ORRENIUS, S. (1965) J. Cell Biol. 26, 713-723. J. Biol. Chem. 249, 6302-6310. 4. ESTABROOK, R. W., AND COHEN, B. (1969) in 17. HAUGEN, D. A., VAN DER HOEVEN, T. A., AND Microsomes and Drug Oxidations (Gillette, J. COON, M. J. (1975) J. Biol. Chem. 250, 3567- R., Conney, A. H., Cosmides, G. J., Esta- 3570. brook, R. W., Fouts, J. R., and Mannering, G. 18. VERMILION, J. L., AND COON, M. J. 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