Radical mechanism of aminopyrine oxidation by cumene hydroperoxide catalyzed by purified liver microsomal cytochrome P-450

ARCHIVES OF BIOCHEMISTRY Vol. 205, No. 2, December,

Radical

AND BIOPHYSICS pp. 543-553, 1980

Mechanism of Aminopyrine Oxidation Catalyzed by Purified Liver Microsomal

BRENDA

WALKER

GRIFFIN,2 BETTIE

AND Biochemistry

by Cumene Cytochrome

CHARLES MARTH, YUKIO SUE SILER MASTERS

Department, The University of Texas Health Science Center 5323 Harry Hines Boulevard, Dallas, Texas 75235 Received

April

Hydroperoxide P-4501 YASUKOCHI,3

at Dallas,

17, 1980

Under identical experimental conditions, purified preparations of rabbit liver microsomal cytochrome P-450 and beef heart metmyoglobin were equally effective at stimulating the oxidation of aminopyrine to a free radical species by cumene hydroperoxide. Mannitol had no effect on radical levels produced with either hemeprotein-hydroperoxide system; however, specific ligands of the two hemeproteins, substrates of cytochrome P-450, and phospholipid affected the two systems quite differently. Only the metmyoglobindependent oxidation of aminopyrine was significantly inhibited by fluoride and cyanide. Metyrapone, a specific ligand of cytochrome P-450, and benzphetamine, which was Ndemethylated by cumene hydroperoxide only in the presence of cytochrome P-450, inhibited only the cytochrome P-450-stimulated oxidation of aminopyrine. Moreover, only with the solubilized liver hemeprotein was aminopyrine radical generation markedly stimulated by phospholipid. Similar properties of aminopyrine N-demethylation and radical formation by the cytochrome P-450-cumene hydroperoxide system have strongly implicated the radical as a requisite intermediate in product formation. Micromolar concentrations of metyrapone caused parallel inhibition, by at least 50%, of both radical generation and formaldehyde production. These results support a radical pathway of Ndemethylation proposed for other hemeprotein-hydroperoxide systems (B. W. Griffin and P. L. Ting, 19’78, Biochemistry 17, 2206-2211), in which the substrate undergoes two successive one-electron abstractions, followed by hydrolysis of the iminium cation intermediate. Thus, for this class of substrates, the experimental data are consistent with the oxygen atom of the product arising from H,O and not directly from the hvdroDeroxide. mechanism for cytochrome P-456 which has been previously proposed as a general peroxidatic activities.

The detailed mechanism of monooxygenation reactions catalyzed by purified cytochrome P-450 from various sources is being investigated in many laboratories (l-3). The utilization of several hydroperoxides by liver microsomal cytochrome P-450 for oxidation of several classes of monooxygenase substrates of this enzyme has been taken

as evidence for similar mechanisms of the “peroxidase” and corresponding monooxygenase activities of cytochrome P-450 (4, 5). The proposed mechanism involves formation of a common “active” oxygenating species of the hemeprotein, presumed to be similar to Compound I of horseradish peroxidase (HRP)4 (6), which transfers an oxygen atom from the oxidant (either ROOH or 0,) to the electron donor substrate (5, 7). Thus, oxidation of the substrate is considered to proceed by concerted, two-electron reactions. However,

1 Supported by NIH Grants AM 19027 (B.W.G.), GM 16488 (B.S.S.M.), and HLBI 13619 (B.S.S.M.) and also by Grants I-601 (B.W.G.) and I-453 (B.S.S.M.) of the Robert A. Welch Foundation. * To whom correspondence should be directed. 3 Current address: 2nd Department of Biochemistry, Kyushu University School of Medicine, 3-1-1, Maedashi, Higashi-ku, Fuboka 812, Japan.

4 Abbreviations dilauroyl-GPC, 543

used: HRP, horseradish peroxidase; dilauroylglyceryl3-phosphorylcholine.

0003~9861/80/140543-11$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

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GRIFFIN

experimental evidence has been published recently from several laboratories (8- 12) for the one-electron oxidation of several distinct classes of substrates of liver microsomal cytochrome P-450 in various hemeprotein-hydroperoxide systems. For example, we have shown that the H,Ozdependent N-demethylation of aminopyrine and aromatic amines catalyzed by HRP proceeds via radical intermediates of these substrates, which are readily detectable by electron paramagnetic resonance (EPR) spectroscopy (8, 9). The mechanism of this enzymatic reaction appears to be quite analogous to the chemical N-demethylation of these compounds (13, 14), namely, dehydrogenation followed by hydrolysis of the iminium cation intermediate: R,N-CH,-eR,i+=CH

R,N-CH;2’

Hz0

R,NH,+

-H.

+ H&=0.

[I]

Significantly, this reaction sequence does not involve direct insertion of an oxygen atom from the hydroperoxide into the Nmethyl substrate. Since this mechanism of N-demethylation appears to have general validity for hemeproteinhydroperoxide systems (8, 9), the present study was undertaken with a purified preparation of liver microsomal cytochrome P-450 in order to determine if similar cumene hydroperoxide-supported reactions catalyzed by this hemeprotein (15, 16) involve one-electron oxidation of the substrate. Since the source (rabbit), purity, and catalytic properties of this enzyme preparation appear to be nearly identical to that employed by Nordblom et al. (17) in a similar study, these results can be directly compared with theirs. We have focused on aminopyrine, one of the Nmethyl substrates included in that study (17), because the purple aminopyrine radical can be detected very sensitively by spectrophotometry (18, 19). The .experimental results indicate that aminopyrine oxidation by cumene hydroperoxide, with purified liver microsomal cytochrome P-450 as catalyst, proceeds by the proposed radical mechanism (8).

ET AL. MATERIALS

AND METHODS

The cytochrome P-450 used in this study was purified from liver microsomes of phenobarbitol-induced rabbits by the procedure of Imai and Sato (20). The preparation had a specific content of 13.2 nmol cytochrome P-450/ mg protein: it contained a trace of NADPH-cytochrome b, reductase activity, but was free of NADPHcytochrome c (P-450) reductase activity and cytochrome b,. The metmyoglobin used in this study was a highly purified preparation isolated from beef heart (21); HRP (Type VI salt-free powder), with an RZ value of approximately 3.0, was purchased from Sigma. Aminopyrine was obtained from Aldrich; H,O, (AR grade, 30%), from Mallinckrodt; and dilauroylglyceryl 3-phosphorylcholine (dilauroyl-GPC), from Sigma. The sodium salt of cumene hydroperoxide was prepared (22) from a technical grade product supplied by Matheson Coleman and Bell; the purity of the salt was confirmed by high-performance liquid chromatography and by iodometric titration (22). In experiments with dilauroyl-GPC as a component, the phospholipid was added as a sonicated suspension at a concentration of 0.1 mg/ml, which Nordblom et al. (1’7) established as the minimal concentration required for maximal rates. The standard Nash assay (23) for formaldehyde was employed, in which the Nash reagent was incubated with the supernatant from the trichloroacetic acid-quenched reaction mixture for 8 min at 58°C. The rates of N-demethylation of aminopyrine, iV,N-dimethylaniline, N-methylaniline, and benzphetamine by cumene hydroperoxide, which were measured under experimental conditions identical to those reported by Nordblom et al. (IQ, were consistently 40-50% larger than their values. For example, the rate of aminopyrine oxidation in the present study was 17.3 nmoYminlnmo1 cytochrome P450 at 30°C in a reaction mixture containing 0.1 M potassium phosphate, pH 7.4, 10 mM aminopyrine, 3.3 mM cumene hydroperoxide, 1 pM cytochrome P450, and 0.1 mg/ml dilauroyl-GPC; the value reported in the study cited (17) was 12 nmol/min/nmol cytochrome P-450. We have previously reported that metmyoglobin exhibits a similarly low aminopyrine N-demethylase activity with cumene hydroperoxide, V = 8.1 nmol/min/nmol heme in 0.1 M potassium phosphate buffer, pH 7.5 at 37°C (24). The Nash assay was not significantly affected by the cumene hydroperoxide concentrations employed. However, we found that the high concentration of H,Oz (50 mM) used by Nordblom et al. (17) destroyed approximately 50% of the formaldehyde. An Aminco DW-2 spectrophotometer in the splitbeam mode was employed for spectrophotometric detection of the aminopyrine radical and for all spectrophotometric experiments with cytochrome P450; a Beckman 25 spectrophotometer was used for other absorbance measurements. The EPR experiments were performed and EPR signals were quanti-

RADICAL

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OF AMINOPYRINE

tated by use of a Digital Equipment Corporation PDP 11105 computer interfaced to a Varian E-4 spectrometer, as described in detail elsewhere (8, 25).

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f 0.2 n ,”

RESULTS

The identity of a purple species with the aminopyrine free radical, both of which were produced concomitantly during the electrochemical oxidation of this compound, has been previously established (18). Because the aminopyrine N-demethylase activity of cytochrome P-450 is very low compared to that of HRP (8), the aminopyrine radical was detected in this study by spectrophotometry, which is more sensitive than EPR. Moreover, this method of detection permits kinetic correlations of radical generation with formaldehyde production, under identical experimental conditions. Since the electrochemical oxidation of aminopyrine was carried out in a nonaqueous solvent (18), it was necessary to conclusively identify the purple intermediate of aminopyrine generated enzymatically in buffered aqueous solutions (8) as the radical species. The extinction coefficient of the aminopyrine radical was determined by relating the absorbance of the purple intermediate directly to the integrated intensity of the EPR signal, produced by enzymatic oxidation of aminopyrine with HRP-H20Z (8). The value of E (at 565 nm) so determined was 2.23 InM-’ -l, which agrees well with the reported tzue of 2.44 mM-* cm-’ in acetonitrile (18). In order to confirm the assumption that the colored species represents only the radical of aminopyrine, the stoichiometry of enzymatic formation of the purple intermediate with Hz02 was established. Under conditions where quantitative yields of this species could be obtained, the absorbance at 565 nm increased linearly with H,O, concentration, as shown in Fig. 1. From the slope of this line and the value of the extinction coefficient, the stoichiometry was calculated as 1.92 mol of purple species/m01 of H202, in agreement with the value of 2.0 predicted by: H,O, + 2 H+ + 2 aminopyrine 2 aminopyrinet

+ + 2 H,O

[2].

I of, , , ,I vYM IO

20

30

40

50

H202

FIG. 1. Dependence of the absorbance of the aminopyrine free radical on H,O, concentration in the HRP-catalyzed reaction. The absorbance at 565 nm was measured after adding H202 to the reaction mixture containing 0.1 M aminopyrine and 0.5 @M HRP in 0.1 M potassium phosphate buffer pH 6.2.

Thus, these data support the conclusion that the purple intermediate and the free radical arising from the oxidation of aminopyrine in aqueous solutions are the same species. In Fig. 2 the difference absorbance spectrum of the aminopyrine radical generated in the cytochrome P-450~cumene hydroperoxide system is compared with that in two other hemeprotein-hydroperoxide systems. The similarity of these spectra is apparent. Significantly, equivalent concentrations of metmyoglobin and cytochrome P-450 produced almost identical absorbance spectra under, the same experimental conditions; the small difference in spectral shape is attributed to measurable destruction of cytochrome P-450 heme by cumene hydroperoxide, as shown by the control experiment in which aminopyrine was omitted (Fig. 2A). This control shows clearly the inverse absolute spectrum of low-spin ferric cytochrome P-450, which has absorbance maxima at 535 nm (E = 10.4 1IIM-’ cm-‘) and at 568 nm (E = 11.2 rnM-’ cm-‘) (26). Although cumene hydroperoxide also destroys metmyoglobin (B. W. Griffin, unpublished results), the perturbation of the absorbance spectrum of the aminopyrine radical (Fig. 2B) is much less pronounced, because the extinction coefficient of metmyoglobin near 550 nm at this pH is only about 25% that of cytochrome P-450 (27). Destruction of cytochrome P-450 heme by cumene hydro-

546

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I

I 450

0.05

500

550

600

650

8

OC 450-w

500

550 WAVELENGTH,

650 nm

FIG. 2. Absorbance spectra arising from aminopyrine in three hemeprotein-hydroperoxide systems. For A and B, the baseline (- 0 -) was recorded with a solution of aminopyrine (0.09 M) and the hemeprotein in buffer (0.09 M potassium phosphate, pH 6.2) in both the sample and reference cuvettes; the reaction mixtures to which eumene hydroperoxide was added also contained 0.1 mg/ml dilauroyl-GPC. These spectra reflect the maximal absorbance which developed after adding the hydroperoxide to the sample cuvette. (A) (-) HRP, 0.16 pM and H,Oz, 15 pM; (-.-) cytochrome P-450, 1 pM and cumene hydroperoxide, 2 RIM; (- - -) control for the cytochrome P-456 experiment with aminopyrine omitted from the reaction mixture. (B) Each reaction mixture contained 2 mM cumene hydroperoxide and 1 pM of either metmyoglobin (-) or cytochrome P-450 (-+, in addition to the components stated above. Temperature, 22%.

peroxide was confirmed by measuring loss of the carbon monoxide complex of the dithionite-reduced hemeprotein. Aminopyrine inhibited both the rate and extent of this destruction; a similar protective effect of benzphetamine on cytochrome P-450 in the presence of cumene hydroperoxide was previously reported (17). In the absence of the hydroperoxide, cytochrome P-450 was stable over the time course, and under the conditions, of these experiments. The concentration of the aminopyrine radical generated with 1 PM cytochrome

ET AL.

P-450 (Fig. 2) is approximately 18 PM, neglecting the absorbance decrease due to heme destruction. This suggests that not all of the radical molecules are bound to the enzyme. A control experiment showed that the concentration of phospholipid employed in these experiments had no effect on the kinetics or extent of radical generation in the metmyoglobin-cumene hydroperoxide system; this result indicates that the phospholipid does not affect the extinction coefficient of the radical. However, omission of dilauroyl-GPC from the cytochrome P-450 system decreased the maximal radical yield by 60%; very similar effects of this phospholipid were noted by Nordblom et al. on the N-demethylase activities of .their cytochrome P-450 preparation (17). The stimulatory effect of phospholipid on the cytochrome P-450dependent reaction may involve dissociation of aggregates of the hydrophobic protein into monomers and/or facilitated interaction of the enzyme with the lipid-soluble hydroperoxide. Consistent with the failure of HRP to catalyze the oxidation of aminopyrine by cumene hydroperoxide, the aminopyrine radical was not detected in this system. Nonenzymatic oxidation of aminopyrine by cumene hydroperoxide produced an absorbance change only 5% of that observed in the presence of the hemeproteins, under the experimental conditions of Fig. 2. The effects of varying reactant and enzyme concentrations on the maximal absorbance measured in the cytochrome P450-cumene hydroperoxide system are shown in Fig. 3. The data of Fig. 4 show the marked effect of pH on the rate of change of the net radical concentration, which reflects both the rate of ‘generation and the rate of decay of this species. The general shape of these traces, in particular, the shift of the maximal absorbance to smaller values and shorter times at the larger pH values, indicates that the radical decays faster at more alkaline pH. This result is entirely consistent with previous observations on the pH stability of this radical (8). The pH dependence of generation of the radical under identical conditions in the metmyoglobin-cumene hydroperoxide sys-

RADICAL

MECHANISM

OF AMINOPYRINE

OXIDATION

547

aminopyrine radical produced with cytochrome P-450 .>and metmyoglobin result from a very small generation rate of the species, which is measurably larger than the decay rate only at acidic pH. By coni c trast, the rate of generation of the amino1 OK 0 n 0.5 1.0 15 2.0 pyrine radical in the HRP-H,O, system U-J CYTOCHROME P-450 , pm In in this pH range is so much larger than the decay rate that steady-state radical concentrations in the millimolar range have i ::;----_r been readily detected by EPR spectroscopy at room temperature (8). According to the proposed mechanism 0 20 40 60 SO m AMlNOPYRlNE, mm (cf. Eq. [l]), oxidation of the radical intercc mediate is required for the subsequent release of formaldehyde. Thus, when decay of the radical is rate limiting, a correlation between the steady-state radical concentration and the rate of product formation is 0 2.0 4.0 6.0 8.0 expected; this has indeed been observed for cUMENE HYDROPEROXIDE , mM the HRP-catalyzed reaction under various conditions (8, 28). However, FIG. 3. Dependence on reactant concentrations of experimental the maximal absorbance at 550 nm measured in reac- similar correlations over a range of experition mixtures containing aminopyrine, cumene hydromental conditions are less likely with the peroxide, and cytochrome P-450. All reaction mixtures cytochrome P-450-cumene hydroperoxide contained 0.1 mg/ml dilauroyl-GPC in 0.09 M potassium system because the rates of generation and phosphate buffer, pH 6.2, and the stated concentradecay of the aminopyrine radical appear to tion of substrates or enzyme. In A, aminopyrine was be the same order of magnitude. For ex0.08 M and cumene hydroperoxide was 2 mM; in B, ample, as shown in Fig. 5, the effect of cytochrome P-450 was 1 pM and cumene hydroincreasing temperature from 22 to 30°C was peroxide was 4 mM; in C, cytochrome P-450 was 1 pM to decrease the maximal value of the radical and aminopyrine was 0.08 M. Temperature, 22°C. concentration, which appears to result from an increase in the decay rate. Consistent tern was very similar to that shown in Fig. 4 with this explanation, the rate of formaldefor cytochrome P-450. These data suggest hyde production measured under identical that the very low concentrations of the conditions increased more than twofold,

FIG. 4. pH dependence of the kinetics of formation and decay of the aminopyrine radical in the cytochrome P-450-cumene hydroperoxide system. Equal volumes of a solution containing 0.1 M potassium phosphate buffer, 0.09 M aminopyrine, 0.1 mg/ml dilauroyl-GPC, and 1 pM cytochrome P-450 were added to both sample and reference cuvettes. The absorbance at 550 nm was monitored after adding 2 mM cumene hydroperoxide to the sample cuvette. The pH of these solutions was; (-) pH 6.25; (- .-) pH 6.55; (- A -) pH 7.05; (- - -) pH 7.55; (-- 0 -) pH 8.05. Temperature, 22°C.

548

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0' 0

' 100

j 200

' 300

400

j 500

600

,I 700

SECONDS

FIG. 5. Kinetics of formation and decay of the aminopyrine free radical generated with cytochrome P-450 and cumene hydroperoxide at two temperatures. The experimental conditions were identical to those described in the legend of Fig. 4 except that the pH was 6.2, and the temperature was 22°C (-) or 30°C (- .-).

from 7.1 to 17.3 nmol/min/nmol cytochrome P-450. However, the kinetic data of Fig. 5 do not exclude the possibility that the rate of radical generation also increased with temperature. The dependence of formaldehyde production in this system on other experimental variables was similar to the data in Fig. 3. The rate of product formation as a function of cumene hydroperoxide concentration exhibited similar behavior, with a sharper maximum near 1-2 mM; the data points at lower hydroperoxide concentrations were quite consistent with the apparent K, value of 0.68 InM reported by Nordblom et al. (17) for the cumene hydroperoxide-supported oxidation of Nmethylaniline. Over the pH range 6.0 to 8.0, formalydehyde production from aminopyrine in the cytochrome P-450-cumene hydroperoxide system was not significantly affected by pH, as Nordblom et al. observed for the oxidation of N-methylaniline by this system (17). Taken with the kinetic traces of Fig. 4, this result implies that the formation rate and decay rate of the aminopyrine radical are affected differently by pH. Concerning the possible routes of decay of the radical, oxidation leading to N-demethylation (rather than dimerization, for example) appears to be the only reaction of the species under these experimental conditions (8). Although several similarities of cyto-

ET AL.

chrome P-450 and metmyoglobin as catalysts of formation of the aminopyrine free radical with cumene hydroperoxide have been established, some important differences between these two hemeproteins are evident from the data in Table I. The observation of inhibition by fluoride, cyanide, or metyrapone on the extent of radical formation in these two systems was consistent with the known specificity of these ligands for coordination to the two hemeproteins (27, 29, 30). Neither system was affected by mannitol, a trap of hydroxyl radicals (31). Ethylmorphine was not Ndemethylated by either system, but benzphetamine, which inhibited aminopyrine radical generation in the cytochrome P-450containing system by more than 50%, was oxidized only by this system. The results obtained with cyclohexane, and the solvent employed for this hydrocarbon, are of interest since Nordblom et al. demonstrated hydroxylation of cyclohexane (dissolved in TABLE

I

EFFECTS OF VARIOUS COMPOUNDS ON MAXIMAL AMINOPYRINE RADICAL CONCENTRATIONS PRODUCED WITH CUMENE HYDROPEROXIDE AND EITHER CYTOCHROME P-450 OR METMYOGLOBIN" Radical concentration (% of control) produced with:

Addition None (control) KF (0.08 M) NaCN (2 mM) Metyrapone (400 ,uM) EDTA (2 ItIM) Mannitol (6.08 M) Ethylmorphine (20 mM) Benzphetamine (13 mM) Cyclohexane (10 mM in 1% acetone) Acetone (1%)

Cytochrome P-450

Metmyoglobin

100 100 80 33 100 100 80 48

100 48 18 100 100 100 83 90

228 228

90 98

” Experimental conditions were identical to those of Fig. 2B, which represents the respective control experiments; other compounds were added to the -reaction mixture at the stated concentrations.

RADICAL

MECHANISM

OF AMINOPYRINE

acetone) by the cytochrome P-450~cumene hydroperoxide system (17). The pronounced stimulator-y effect of acetone on the cytochrome P-450-dependent reaction cannot be readily explained. However, the role of solvent, possibly as a promoter of a radical process, in other cumene hydroperoxide-supported reactions of this hemeprotein warrants further investigation. Kinetic evidence for direct involvement of the aminopyrine radical in formaldehyde production from this substrate in the cytochrome P-450-cumene hydroperoxide system is presented in Fig. 6. The reaction product and the absorbance of the radical, measured under identical experimental conditions, were inhibited approximately 50% by a very low concentration of metyrapone (125 PM), and the inhibition was more pronounced at a higher metyrapone concentration. The absolute concentrations of both the radical intermediate and formaldehyde measured in these experiments provide additional support for a precursor-product relationship. Finally, we note that Nordblom et al. (17) were able to demonstrate the catalysis of the N-demethylation of benzphetamine by H,O, with their purified preparation of cytochrome P-450, but only at very high peroxide concentrations. Consistent with our previous findings that several hemeproteins catalyze the oxidation of aminopyrine in the presence of H,O, (8), we were able to generate the aminopyrine radical with cytochrome P-450 and H,O, under experimental conditions similar to those of Fig. 2. Although the absorbance change increased with H,O, concentration, this oxidant was considerably less effective than cumene hydroperoxide, as Nordblom et al. observed (17); for example, with 50 mM Hz02, the maximal radical concentration was approximately 40% that measured with 2 mM cumene hydroperoxide. However, the contribution of heme destruction to the net absorbance change measured at these high H,O, concentrations was relatively greater. Because of these properties and the destruction of formaldehyde by high H,O, concentrations noted under Materials and Methods, additional studies of this reaction were not undertaken.

100

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100)

100

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FIG. 6. Effect of metyrapone on the kinetics of radical formation and formaldehyde production from aminopyrine stimulated by cytochrome P-450 in the presence of cumene hydroperoxide. The experimental conditions were identical to those described in the legend of Fig. 4 except that the pH was 6.2. The metyrapone concentration was 0 for (-) and (- 0 -); 125 pM, for (-.--) and (- 0 -); and 400 p,M for (- - -) and (- - A - -). Temperature, 22°C. DISCUSSION

This investigation has clearly established that metmyoglobin is a valid model hemeprotein for studying the interaction of cytochrome P-450 with cumene hydroperoxide (19). Purified preparations of rabbit liver microsomal cytochrome P-450 and beef heart metmyoglobin were shown to be equally effective in stimulating the oxidation of aminopyrine to a radical species by cumene hydroperoxide. The experimental data have implicated this purple-colored radical as a requisite intermediate in formaldehyde production from aminopyrine, in both of these systems, as well as in other hemeprotein-hydroperoxide systems (8). The specific effects of various compounds on the maximal radical yields in these two systems were characteristic of the individual hemeproteins. Thus, hemeprotein ligands, such as fluoride and metyrapone,

550

GRIFFIN

inhibited the reaction to an extent qualitatively consistent with their relative affinities for metmyoglobin and ferric cytochrome P-450, respectively. It may be inferred that this inhibition results from competition of the ligand with cumene hydroperoxide for binding at the sixth coordination position of the heme iron (6, 27, 32). The mechanism by which benzphetamine specifically inhibits the cytochrome P-450dependent generation of the aminopyrine radical is less clear. Since benzphetamine undergoes N-demethylation by the cytochrome P-450~cumene hydroperoxide system, it seems likely that the two substrates compete for the same “active” oxidant generated in this system. Because this oxidant can clearly abstract a single electron from aminopyrine, it might, therefore, be concluded that benzphetamine is similarly oxidized to a radical species. Other Nmethyl substrates of cytochrome P450 which were included in the study of Nordblom et al. (17), namely, N,Ndimethylaniline and some structurally related analogs, are oxidized to EPR-detectable radical species by various hemeproteinhydroperoxide systems, including metmyoglobin-cumene hydroperoxide (9, 33). Although we have not been able to detect a radical species of benzphetamine spectrophotometrically in the cytochrome P-450cumene hydroperoxide system, we note that such a species is expected to be highly unstable, since it would lack the resonance stabilization of aminopyrine or the aromatic amines (34). However, the data do not exclude the possibility that aminopyrine and benzphetamine are oxidized by different mechanisms in this system. An alternate explanation of the inhibitory effect of benzphetamine on aminopyrine radical formation is that binding of this substrate near the heme group of cytochrome P-450 provides steric hindrance to binding of the hydroperoxide, which is presumed necessary for reaction to occur. Available evidence indicates that there is a binding site in close proximity to the heme of cytochrome P-450 which can accommodate one rather large substrate or ligand molecule (for example, benzo(a)pyrene or metyrapone) (32, 35). Therefore, it seems un-

ET AL.

likely that two large aromatic compounds, such as benzphetamine and cumene hydroperoxide, could bind simultaneously at this site. The ability of several N-methyl substrates, including those used in this study, to produce the absorbance spectrum characteristic of substrate-bound cytochrome P-450 (l-3) was examined. Under the conditions of Table I, the most effective substrate was benzphetamine, which converted approximately 15% of this preparation (a low-spin form (26)) to the high-spin form, as determined spectrophotometritally. It is of interest that specific and efficient binding of substrates by other solubilized preparations of liver microsomal cytochrome P-450 which are active monooxygenases is not well established. That benzphetamine did not significantly affect aminopyrine oxidation by the metmyoglobin-cumene hydroperoxide system is consistent with the failure of this system to oxidize benzphetamine. We note that benzphetamine is considerably more lipid soluble than aminopyrine and, thus, should be relatively more concentrated in the lipid phase of the cytochrome P-450-containing system. As a consequence, specific binding of benzphetamine to a fraction of the lipidassociated protein should be facilitated and, in addition, benzphetamine should react more readily with oxidizing species generated in the lipid phase. Both factors probably contribute to the effectiveness of benzphetamine as an inhibitor of aminopyrine radical formation in the cytochrome P-450~cumene hydroperoxide system. These findings raise the question of the identity of the one-electron oxidant of aminopyrine generated by metmyoglobin or cytochrome P-450 in the presence of cumene hydroperoxide. Although Hz02 oxidizes metmyoglobin to a form analogous to horseradish peroxidase Compound II (36), we have been unable to produce the same, or a similar, species of metmyoglobin with cumene hydroperoxide. The concentration of the hydroperoxide required to produce any measurable effect on the absorbance spectrum of metmyoglobin also caused irreversible loss of heme. We have recently reported the trapping, by nitrosobenzene, of identical radical species arising from

RADICAL

MECHANISM

OF AMINOPYRINE

cumene hydroperoxide in the presence of metmyoglobin or a microsomal fraction of cytochrome P-450 (16). These results indicated that both hemeproteins function like metal ions in initiating the decomposition of cumene hydroperoxide to the cumyloxy radical, which may then be reduced to cumenol or may decompose to the methyl radical and acetophenone (15). The latter two products have been positively identified in the metmyoglobin-cumene hydroperoxide system, and a role for the methyl radical in the oxidation of aminopyrine has been demonstrated (37). However, with more easily oxidized electron donors, e.g., N,N-dimethylaniline, the cumyloxy radical may be the more important one-electron oxidant (15), which would decrease proportionately the amount of acetophenone formed. Thus, one or both of the reactive radicals known to arise from cumene hydroperoxide may participate in the oxidation of the many compounds reported to undergo oxidation in cumene hydroperoxide-supported peroxidase reactions of cytochrome P-450 (4, 5, 17). These radicals are probably also involved in the cumene hydroperoxide-induced heme destruction observed for both hemeproteins (17). The finding that aminopyrine inhibits heme destruction suggests that the Nmethyl substrate and the electron-rich heme group compete directly for a common oxidant. The observation that mannitol did not inhibit aminopyrine oxidation in either hemeprotein-cumene hydroperoxide system indicates that hydroxyl radicals are not the one-electron oxidants in these systems (31), consistent with the known chemistry of the radical decomposition of cumene hydroperoxide (15). The possibility that a small fraction of the hemeprotein functions catalytically, i.e., undergoes more than one turnover, cannot be completely eliminated by these results. However, we have been unable to demonstrate the reversible formation of discrete states of metmyoglobin with cumene hydroperoxide, which might function catalytically in the oxidation of electron donors in this system. The experimental evidence, namely, the very low turnover numbers measured for both hemeproteins

OXIDATION

551

(4, 5, 17, 25), the substantial destruction of heme by the hydroperoxide observed previously (17) and in this study, and the evidence for formation of identical hydroperoxide-derived radicals in both systems (16), supports the interpretation that the apparent catalytic oxidation of aminopyrine by cumene hydroperoxide actually proceeds by a radical reaction initiated by the hemeprotein. Since only oneN-methyl substrate, which is oxidized to a resonance-stabilized radical species, was examined in detail in this study, the relevance of these results for other classes of cytochrome P-450 substrates may be questioned. Yang and Strickhart first reported several differences between the cumene hydroperoxideand O,/NADPH-supported oxidation of benzo(a)pyrene by microsomes from control and induced animals (38). Other research groups have recently reported results of detailed studies of the peroxidatic oxidation of benzo(a)pyrene: (i) by H,O, in a reaction catalyzed by horseradish peroxidase (11); (ii) by prostaglandin PGGz, the hydroperoxide-endoperoxide product of arachidonic acid oxygenation catalyzed by fatty acid cyclo-oxygenase (10); and (iii) by cumene hydroperoxide in the presence of liver microsomes (12). Significantly, phenols or dihydrodiols, which are the major products formed in the OJNADPHdependent oxidation of benzo(a)pyrene in liver microsomes (39), were not detected, but mixtures of quinone isomers, arising from one-electron oxidation pathways, were produced. It should be noted that the latter products are produced to some extent in the liver microsomal monooxygenation of benzo(a)pyrene (40). The proposal that cumene hydroperoxidesupported oxidation activities of microsomal cytochrome P-450 are appropriate models for corresponding monooxygenase activities of this hemeprotein has not been addressed in this study. It should be emphasized that these experimental findings cannot be directly extrapolated to 02/ NADPH-requiring N-demethylation reactions catalyzed by cytochrome P-450, as others have presumed, based on formation of the same reaction product in the peroxi-

552

GRIFFIN

datic and monooxygenase reactions. Direct detection of the aminopyrine radical during the microsomal monooxygenation of this substrate would appear to provide the best evidence for a common mechanism for this reaction and the cumene hydroperoxidesupported reaction. However, in the former case, electron transfer involving NADPHcytochrome P-450 reductase would place additional constraints on the rate of generation of the aminopyrine radical. The pH optima of most monooxygenation reactions catalyzed by liver mierosomal cytochrome P-450 are alkaline (l-3). However, it was shown in this study, and previously (8), that the aminopyrine radical is quite unstable under alkaline conditions. Finally, the problem of H,O, production during 02/ NADPH-dependent N-demethylation reactions of microsomal cytochrome P-450 (41, 42) has been discussed in detail elsewhere (8). Since aminopyrine could also be oxidized in an H,O,-consuming peroxidatic reaction catalyzed by this hemeprotein, actual detection of the substrate radical would not establish whether the monooxygenation reaction occurs by direct oxygen insertion or by dehydrogenation and hydrolysis. There is indirect evidence, namely, very large primary deuterium isotope effects, that the cytochrome P-450catalyzed hydroxylation of two different alkanes may involve radical intermediates of these electron donors (43, 44). There are many unresolved questions about the formation of substrate radicals during cytochrome P-450-catalyzed monooxygenation reactions which are now being investigated by several experimental techniques. REFERENCES 1. GRIFFIN, B. W., PETERSON, J. A., AND ESTABROOK, R. W. (1979) in The Porphyrins (Dolphin, D., ed.), Vol. VII, pp. 333-375, Academic Press, New York. 2. GUNSALUS, I. C., MEEKS, J. R., LIPSCOMB, J. D., DEBRUNNER, P., AND M~NCK, E. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.), pp. 559-613, Academic Press, New York. 3. SATO, R., AND OMURA, T. (eds.) (1978) Cytochrome P-450, Kodansha Ltd., Tokyo, and Academic Press, New York.

ET AL. 4. RAHIMTULA, A. D., AND O’BRIEN, P. J. (19’74) Biochem. Biophys. Res. Commun. 60,440-447. S. (1975) FEBS Lett. 5. ELLIN, A., AND ORRENIUS, 50,378-381. 6. SCHONBAUM, G., AND CHANCE, B. (1976) Enzymes 3rd ed. 13C, 363-408. 7. RAHIMTULA, A. D., AND O’BRIEN, P. J. (1975) Biochem. Biophys. Res. Commun. 62,268-275. 8. GRIFFIN, B. W., AND TING, P. L. (1978) Biochemistry 17,2206-2211. 9. GRIFFIN, B. W. (1978) Arch. B&hem. Biophys. 190,850-853. 10. MARNETT, L. J., AND REED, G. A. (1979) Biochemistry 18,2923-2929. 11. ROGAN, E. G., KATOMSKI, P. A., ROTH, R. W., AND CAVALIERI, E. L. (1979) J. Biol. Chem. 254, 7055-7059. 12. CAPDEVUA, J., ESTABROOK, R. W., AND PROUGH, R. A. (1980) Arch. B&hem. Biophys. 206, 186- 195. 13. ROSENBLATT, D. H., HULL, L. A., DELUCA, D. C., DAVIS, G. T., WEGLEIN, R. C., AND WILLIAMS, K. K. R. (1967) J. Amer. Ch.em. Sot. 89, 1158-1162. 14. FRITSCH, J. M., WEINGARTEN, H., AND WILSON, J. D. (1970) J. Amer. Chem. Sot. 92,4038-4046. 15. KHARASCH, A. F., AND NUDENBERG, W. (1950) J. Org. Chem. 15, 763-774. 16. GRIFFIN, B. W. (1980) in Microsomes, Drug Oxidations, and Chemical Carcinogenesis (Coon, M. J., Conney, A. H., Estabrook, R. W., Gelboin, H. V., Gillette, J. R., and O’Brien, P. J., eds.), pp. 319-322, Academic Press, New York. 17. NORDBLOM, G. D., WHITE, R. E., AND COON, M. J. (1976) Arch. Biochem. Biophys. 175, 524-533. 18. SAYO, H., AND MASUI, M. (1973) J. Chem. SOC. Perkin Trans. 2, 1640-1645. 19. GRIFFIN, B. W. (1979) in Fundamental Research in Homogeneous Catalysis (Tsutsui, M., ed.), Vol. 3, pp. 773-791, Plenum, New York. 20. IMAI, Y., AND SATO, R. (1974) Biochem. Biophys. Res. Commun. 60, 8-14. 21. GOTOH, T., AND SHIKAMA, K. (1974) Arch. Bio&em. Biophys. 163, 476-481. 22. MAIR, R. D., AND HALL, R. T. (1971) in Organic Peroxides (Swern, D., ed.), Vol. 2, p. 542, Wiley-Interscience, New York. 23. NASH, T. (1953) Biochem. J. 55,416-421. 24. GRIFFIN, B. W. (1976) in 10th Int. Congr. Biothem., July 1976, Hamburg, Abstr. 06-7-098. 25. EBEL, R. E., O’KEEFFE, D. H., AND PETERSON, J. A. (1975) FEBS Lett. 55, 198-201. 26. HAUGEN, D. A., AND COON, M. J. (1976) J. Biol. Chem. 251, 7929-7939. 27. ANTONINI, E., AND BRUNORI, M. (1971) Hemoglobin and Myoglobin in Their Reactions with Ligands, p. 46, American Elsevier, New York.

RADICAL

MECHANISM

OF AMINOPYRINE

28. GRIFFIN, B. W. (19’77),FEBS L&t. 74, 139-143. 29. JEFCOATE, C. R. E., GAYLOR, J. L., AND CALABRESE, R. L. (1969) Biochemistry 8, 3455-3463. 30. PETERSON, J. A., ULLRICH, V., AND HILDEBRANDT, A. G. (1971) Arch. Biochem. Biophys. 145, 531-542. 31. NETA, P., AND DORFMAN, L. M. (1968) Advan. Chem. Ser. 81,222-230. 32. GRIFFIN, B. W., SMITH, S. M., AND PETERSON, J. A. (1974) Arch. Biochem. Biophys. 160, 323-332. 33. GRIFFIN, B. W. (1979) Fed. Proc. 38, Abstr. 627. 34. LAWA, B. M., AND TAFT, R. W. (1967) J. Amer. Chem. Sot. 89, 5172-5178. 35. EBEL, R. E., O’KEEFE, D. H., AND PETERSON, J. A. (1978) J. Biol. Chem. 253, 3888-3897. 36. YONETANI, T., AND SCHLEYER, H. (1967) J. Biol. Chem. 242, 1974-1979. 37. GRIFFIN, B. W., AND TING, P. L. (1978) FEBS Let& 89, 196-200. 38. YANG, C. S., AND STRICKHART, F. S. (19’78) Biochem. Phurmacol. 27,2376-2378.

OXIDATION

553

39. HOLDER, G., YAGI, H., DANSETTE, P., JERINA, D. M., LEVIN, W., Lu, A. Y. H., AND CONNEY, A. H. (1974) Proc. Nat. Acad. Sci. USA 71, 4356-4360. 40. NAGATA, C., TAGASHIRA, Y., AND KODAMA, H. (1974) in Chemical Carcinogenesis (Ts’o, P. 0. P., and Di Paolo, J. A., eds.), Pt. A., pp. 87-111, Dekker, New York. 41. NORDBLOM, Biochem.

G. D., AND COON, M. J. (1977)Arch. Biophys. 180, 343-347.

42. ESTABROOK, R. W., AND WERRINGLOER, J. (1977) in Proceedings of the Third International Symposium on Microsomes and Drug Oxidations (Ullrich, V., Roots, I., Hildebrant, A. G., Estabrook, R. W., and Conney, A. H., eds.), pp. 748-757, Pergamon, Oxford. 43. GROVES, J. T., MCCLUSKY, G. A., WHITE, R. E., AND COON, M. J. (1978) Biochem. Biophys. Res. Commun. 81, 154-160. 44. HJELMELAND, L. M., ARONOW, L., AND TRUDELL, J. R. (1977) Biochem. Biophys. Res. Commun. 76,541-549.