- Email: [email protected]
Evidence for a free radical mechanism of N-demethylation of N,N-dimethylaniline and an analog by hemeprotein-H2O2 systems
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 190, No. 2, October, pp. 850-853, 1978
Evidence for a Free Radical Mechanism of A/-Demethylation of WV-Dimethylaniline an Analog by Hemeprotein-H202 Systems’
and
Horseradish peroxidase and metmyoglobin catalyxe the HtOa-supported N-demethylation of N,N-dimethylaniline and N,N-dimethyl-p-toluidine, The catalytic activities of horseradish peroxidase are more than 190-fold larger than those of metmyoglobin or those previously reported for liver microsomal cytochrome P-450. Distinct free radical species of these N-methyl substrates were detected with both catalysts. These findings establish the general validity of a recently proposed free radical mechanism of oxidative N-demethylation (Griffin, B. W., and Ting, P. L., Biochemistry (1978), 2206-2211), which is quite different from that previously suggested for the analogous cytochrome P-450-dependent reactions. Liver microsomal cytochrome P-450, as a microsomal preparation or as a purified protein, has been shown to catalyze the oxidation of several classes of substrates by OJNADPH’ or by hydroperoxides (l-4). Because the products of both the monooxygenation and the peroxidation reactions are presumably identical, it has been proposed that both reactions involve a common “active oxidixing” species of the hemeprotein, which functions as an hydroxylating agent (4). Although this enzyme species has been considered to be very similar to Compound I of horseradish peroxidase (HRP), the latter species oxidizes substrates by dehydrogenation (electron abstraction) rather than by direct oxygen insertion (5,6). It was recently reported from this laboratory (8) that HRP, metmyoglobin, and protohemin catalyze HxOz-supported N-demethylation of aminopyrine, considered to be a characteristic activity of cytochrome P-450 (1). These three heme catalysts were shown to have comparable or, in the case of HRP, much larger activities than those reported for cytochrome P-450. Moreover, each of these heme compounds catalyzed the oxidation of aminopyrine to the same free radical species (7, 8), which has been characterized extensively by room-temperature electron psramagnetic resonance (epr) techniques (7,8). Based on experimental evidence obtained for these enzyme systems (8), on previous studies of nonenxymatic Ndemethylation reactions (9, lo), and on the chemical properties of aminopyrine (ll), a reaction mechanism has been proposed (8): R~N-CHI
-1, RzN-CHs+ . --t: RzN+=CHz %? RzNHz+ + H&=0.
By contrast with the mechanism proposed for cytochrome P-450 catalysis of this reaction (4), the substrate is oxidized by sequential one-electron transfers, with formation of a free-radical intermediate; sign& cantly, the oxygen atom of formaldehyde arises from Hz0 and not from the peroxide oxidant. However, the rather unusual structural properties of aminopyrine are characteristic of violenes (dialkylaminoalkenes), which undergo facile one- and two-electron oxidation (12). Thus, it was necessary to test the generality of the proposed mechanism for other N-methyl substrates of cytochrome P-450. Because N,N-dimethylaniline is one of the best N-methyl substrates for both the monooxygenation and peroxidation reactions of the liver hemeprotein (4,13), this compound and an analog, N,N-dimethyl-p-toluidine, were selected for study. HRP (Sigma Type VI, with an RZ value of approximately 3.0) and metmyoglobin [purified as described previously (14)] catalyzed the N-demethylation of both compounds, as measured by the Nash spectrophotometric assay for formaldehyde (15). These rates are compared with those previously reported for aminopyrine N-demethylation in Table 1. For each substrate, HRP is a better catalyst than metmyoglobin, which has activities comparable to those of cytochrome P-450 (1,4). The epr signals arising from N,Ndimethylaniline in the presence of HRP and Hz02 are shown in Fig. 1. One signal detected approximately 1 min after initiating the reaction by addition of catalyst had a prominent component with considerable hyperfine structure (Fig. 1A); with time a minor component of this signal increased in intensity to a maximal value (Fig. 1C) and then decayed. With metmyoglobin as catalyst, identical EPR signals were generated from N,N-dimethylaniline, with qualitatively similar kinetic behavior. Both hemeprotein catalysts gave rise to a single epr signal from N,N-dimethyl-p-toluidine, which decayed rapidly (Fig. 2). For each substrate of Table 1, larger concentrations of metmyoglobin than of HRP were required to generate epr signals of equiv-
PI
’ Supported by NIH Grant No. AM 19627and Robert A. Welch Foundation Grant No. 1601. * Abbreviations used: NADPH, nicotinamide ade.?ine dinucleotide phosphate (reduced form); HRP, horseradish peroxidase; epr, electron paramagnetic resonance. 850
0003-9861/78/1902-0&50$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
N-DEMETHYLATION TABLE
I
RATES OF H202-D~~~~~~~~ FORMALDEHYDE PRODUCTION FROM N-DIMETHYL COMPOUNDS CATALYZED BY HRP AND METMYOGLOBIN~ Substrate
Catalyst HRP
N,N-Dimethylanihneb N,N-Dimethyl-ptoluidine* Aminopyrine
9.9 x lo3 (PH 6.0) 7.1 x lo4 (PH 6.5) 2.5 x 104= (pH 7.0)
851
OF N,N-DIMETHYLANILINE
Metymyoglobin 28.0 (PH 6.0) 49.0 (PH 6.0) 44.0* (PH 6%
(1Rates are expressed as moles of formaldehyde per minute per mole of heme at 37°C in 0.1 M potassium phosphate buffer at optimal pH. ’ Reactant concentrations employed were: amine, 3.0 mu; HzOx, 0.8 mu for N,N-dimethylanihne and 0.4 mru for the toluidine analog; HRP, 5-20 nM; and metmyoglobin, 0.5-1.2 PM. Because high concentrations of Hz02 interfered with the assay and also inhibited the HRP-catalyzed reactions, the true catalytic activities are probably somewhat higher than the values reported here. ’ Reference (7). * Reference (8). alent intensities (8); this is consistent with the relative catalytic activities of these two hemeproteins. Since it was possible that these epr signals arose from the products of the N-demethylation reaction, similar experiments were carried out with the N-monomethyl and unsubstituted analogs of both substrates. Both hemeprotein catalysts gave rise to the same epr signal from N-methylaniline, which was quite distinct from that observed with the parent dimethyl compound; similarly, with N-methyl-p-toluidine a single epr signal, obviously different from that of Fig. 2, was generated by both hemeproteins. Aniline and p-toluidine are well-characterized electron donor substrates of HRP (5), but the free radical intermediates presumably formed during enzymatic oxidation of these two compounds have not been detected by epr, as we confiied. However, p-toluidine very effectively inhibited the appearance of the HRP-dependent epr signals generated from the N-methyl-substituted toluidines, and aniline had a similar effect on free radical formation from N-methylaniline and N,N-dimethylaniline. As reported for aminopyrine (ll), N,N-dimethylaniline and its analogs undergo electrochemical oxidation in organic solvents (16-18) to free radical species, which in many cases, have been directly detected by epr. However, the one-electron oxidized form of N,Ndimethylaniline has not been detected because it is very unstable and undergoes a rapid coupling with
substrate to form NJ’-tetramethylbenzidine (16). This product is easily oxidized under the conditions of the electrochemical oxidation to an epr-detectable free radical species (16, 18). The mechanism of the HRPcatalyzed oxidation of N,N-dimethylaniline appears to be very similar, since N,N’-tetramethylbenzidine has previously been shown to be a significant product, which is also oxidized by HRP (19). We are attempting to obtain more stable, better-resolved epr signals of these several free radicals that can be positively identified by computer simulation. However, it appears that one of the signals detected with N,N-dimethylaniline is the radical cation of N,N’-tetramethylbenzidine
B
IO Gauss
/
------a’---4 Gauss D
g = 21003
FIG. 1. The epr signals generated enzymatically from N,N-dimethylaniline. (A) The reaction mixture contained 15 IIIM amine, 9.0 mM Hz02, and 1.9 p HRP in 0.1 M potassium acetate buffer, pH 4.1, 22°C; the spectrum was scanned approximately 1 min after mixing. Spectrometer settings: lo-mW microwave power, 9.160 GHz; 1.0-G modulation amplitude; 0.1~set time constant; 50-G/min scan rate; and 3.2 x lo3 gain. (B) Controls for (A) with either one substrate or the enzyme omitted, recorded with the same instrument settings. (C) The reaction mixture contained 15 mM amine, 5.0 mM Hz02 and 1.0 pM HRP in 0.1 M potassium acetate buffer, pH 5.0, 22’C; the spectrum was scanned approximately 3 min after mixing. Spectrometer settings: lo-mW microwave power; 9.160 GHz; 1.0-G modulation amplitude; O.l-set time constant; 20G/min scan rate; 1.5 X lo* gain. (D) Controls for (C), with either one substrate or the enzyme omitted, recorded with the same instrument settings. (Optimal conditions for detecting each radical were used.)
852
BRENDA
WALKER
GRIFFIN
I
g = 2.003 FIG. 2. The epr signal of a free radical species generated from N,N-dimethyl-p-toluidine. (A) The reaction mixture contained 15 mu amine, 15 mu HzOz, and 23.8 11~HRP in 0.1 M potassium acetate buffer, pH 5.0, 22’C. (B) AII controls for (A) with either one substrate or the enzyme omitted. Spectrometer settings for (A) and (B): lo-mW microwave power, 9.160 GHz; 1.0-G modulation amplitude; 0.1~set time constant; 50-G/min scan rate; 2.5 x lo3 gain.
and that the distinct signals generated from N,N-dimethyl-p-toluidine and from the N-monomethyl analogs are one-electron oxidized species of the respective substrates. These species are clearly not nitroxides, which have g-values near 2.006 (20), because ah of the free radicals reported herein have g-values of 2.003, identical to those of the free radical cations of aminopyrine (11) and other violenes (21). These experimental fmdings provide additional evidence that oxidative N-demethylation reactions catalyzed by HRP (7,8) and by metmyoglobin (8) proceed by the proposed free radical mechanism (Eq. [l]). The first reaction of this mechanism is consistent with the accepted mechanism of HRP-catalyzed oxidations (5); however, the fate of the free radical species formed depends critically on its chemical structure and reactivity. The free radicals generated from these Nmethyl compounds are unusuahy stable, since they can be readily detected in aqueous solution at room temperature without sophisticated mixing or flow systems. Although subsequent one-electron oxidation of the free radical is very probable (Eq. [l]), with N,Ndimethylanihne, coupling at the paru position to form the be&dine product is a significant competing reaction in both the electrochemical (16) and HRP-catalyzed (19) oxidations. However, if the coupling reaction is inhibited by apara substituent, as occurs with N,Ndimethyl-p-toluidine, there is considerable enhancement of the rate of N-demethylation relative to that of N,N-dimethyIanihne (Table 1). As was noted, in the proposed peroxidatic mechanism (Eq. [l]) the oxygen atom of formaldehyde arises from Hz0 and not directly from the peroxide oxidant (via a Compound I-like species), as has been proposed for cytochrome P-450-dependent N-demethylations supported by hydroperoxides (4). The experimental difficulties of determining the origin of the oxygen atom of the aldehyde product of these reactions have
been discussed in detail eLsewhere (8). These findings are significant because they have indicated that the free radical mechanism of oxidative N-demethylation may have more general validity, for other N-methylsubstrates, as well as other hemeprotein catalysts of these reactions. Clearly the monooxygenase and peroxidase N-demethylation reactions catalyzed by cytochrome P-450 must be re-examined in order to resolve the question of mechanism raised by this study. REFERENCES 1. CONNEY, A. H. (1967) Pharmacol. Rev. 19, 317-366. 2. Lu, A. Y. H., JUNK, K. W., AND COON, M. J. (1969) J. Bid. Chem. 244,3714-3721. 3. KADLUBAR, F. F., MORTON, K. C., AND ZIEGLER, D. M. (1973) Biochem. Biophys. Res. Commun. 64,1255-1261. 4. NORDBLOOM, G. D., AND COON, M. J. (1976) Arch. Biochem. Biophys. 180,343~347. 5. SAUNDERS, B. C., HOLMES-SIEDLE, A. G., AND STARK, B. P. (1964) Peroxidase, Butterworths, Washington, D.C. 6. SCHONBAUM, G., AND CHANCE, B. (1976) in The Enzymes, (Boyer, P., ed.), Vol. 13, Part C, pp. 363-408, Academic Press, New York. 7. GRIFFIN, B. W. (1977) FEBS Lett. 74, 139-143. 8. GRIFFIN, B. W., AND TINC, P. L. (1978) Biochemistry 2206-2211. 9. DE LA MARE, H. E. (1960) J. Org. Chem. 25, 2114-2126. 10. ROSENBLATT, D. H., HULL, L. A., DE LUCA, D. C., DAVIS, G. T., WEGLEIN, R. C., AND WILLIAMS, K. K. R. (1967) J. Amer. Chem. Sot. 89,
1158-1162. 11. SAYO, H, AND MASUI, M. (1973) J. Chem. Sot. Perkin Truns. 2, X40-1645.
A’-DEMETHYLATION
OF N,N-DIMETHYLANILINE
12. FRITSCH, J. M., WEINGARTEN, H., AND WILSON, J. D. (1970) J. Amer. Chem. Sot. 92,4038-4046. 13. MCMAHON, R. E. (1966) J. Pharm. Sci. 55, 457-466. 14. GOTOH, T., AND SHIKAMA, K. (1974) Arch. Biothem. Biophys. 163,476-481. 15. NASH, T. (1953) B&hem. J. 56,416-421. 16. MITZOGUCHI, T., AND ADAMS, R. N. (1961) J.
Amer. Chem. Sot. 84,2058-2061. 17. SEO, E. T., NELSON, R. F., FRITSCH, J. M., MARcoux, L. S., LEEDY, D. W., AND ADAMS, R. N. (1966), J. Amer. Chem. Sot. 88,3498-3503. 18. LATTA, B. M., AND TAFT, R. W. (1967) J. Amer.
Chem. Sot. 89,5172-5178. 19. NAYLOR, F. T., AND SAUNDERS, B. C. (1950) J.
853
Chem. Sot., 3519-3523. 20. FREED, J. H. (1976) in Spin Labeling. Theory and Applications (Berliner, L. J., ed.), pp. 73-75, Academic Press, New York. 21. FORRESTER, A. R., HAY, J. M., AND THOMSON, R. H. (1968) Organic Chemistry of Stable Free Radicals, pp. 254-258, Academic Press, New York. BRENDA WALKER GRIFFIN Biochemistry Department The University of Texas Health Science Center at Dallas 5323 Harry Hines Boulevard Dallas, Texas 75235 Received June 7, 1978
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 190, No. 2, October, pp. 850-853, 1978
Evidence for a Free Radical Mechanism of A/-Demethylation of WV-Dimethylaniline an Analog by Hemeprotein-H202 Systems’
and
Horseradish peroxidase and metmyoglobin catalyxe the HtOa-supported N-demethylation of N,N-dimethylaniline and N,N-dimethyl-p-toluidine, The catalytic activities of horseradish peroxidase are more than 190-fold larger than those of metmyoglobin or those previously reported for liver microsomal cytochrome P-450. Distinct free radical species of these N-methyl substrates were detected with both catalysts. These findings establish the general validity of a recently proposed free radical mechanism of oxidative N-demethylation (Griffin, B. W., and Ting, P. L., Biochemistry (1978), 2206-2211), which is quite different from that previously suggested for the analogous cytochrome P-450-dependent reactions. Liver microsomal cytochrome P-450, as a microsomal preparation or as a purified protein, has been shown to catalyze the oxidation of several classes of substrates by OJNADPH’ or by hydroperoxides (l-4). Because the products of both the monooxygenation and the peroxidation reactions are presumably identical, it has been proposed that both reactions involve a common “active oxidixing” species of the hemeprotein, which functions as an hydroxylating agent (4). Although this enzyme species has been considered to be very similar to Compound I of horseradish peroxidase (HRP), the latter species oxidizes substrates by dehydrogenation (electron abstraction) rather than by direct oxygen insertion (5,6). It was recently reported from this laboratory (8) that HRP, metmyoglobin, and protohemin catalyze HxOz-supported N-demethylation of aminopyrine, considered to be a characteristic activity of cytochrome P-450 (1). These three heme catalysts were shown to have comparable or, in the case of HRP, much larger activities than those reported for cytochrome P-450. Moreover, each of these heme compounds catalyzed the oxidation of aminopyrine to the same free radical species (7, 8), which has been characterized extensively by room-temperature electron psramagnetic resonance (epr) techniques (7,8). Based on experimental evidence obtained for these enzyme systems (8), on previous studies of nonenxymatic Ndemethylation reactions (9, lo), and on the chemical properties of aminopyrine (ll), a reaction mechanism has been proposed (8): R~N-CHI
-1, RzN-CHs+ . --t: RzN+=CHz %? RzNHz+ + H&=0.
By contrast with the mechanism proposed for cytochrome P-450 catalysis of this reaction (4), the substrate is oxidized by sequential one-electron transfers, with formation of a free-radical intermediate; sign& cantly, the oxygen atom of formaldehyde arises from Hz0 and not from the peroxide oxidant. However, the rather unusual structural properties of aminopyrine are characteristic of violenes (dialkylaminoalkenes), which undergo facile one- and two-electron oxidation (12). Thus, it was necessary to test the generality of the proposed mechanism for other N-methyl substrates of cytochrome P-450. Because N,N-dimethylaniline is one of the best N-methyl substrates for both the monooxygenation and peroxidation reactions of the liver hemeprotein (4,13), this compound and an analog, N,N-dimethyl-p-toluidine, were selected for study. HRP (Sigma Type VI, with an RZ value of approximately 3.0) and metmyoglobin [purified as described previously (14)] catalyzed the N-demethylation of both compounds, as measured by the Nash spectrophotometric assay for formaldehyde (15). These rates are compared with those previously reported for aminopyrine N-demethylation in Table 1. For each substrate, HRP is a better catalyst than metmyoglobin, which has activities comparable to those of cytochrome P-450 (1,4). The epr signals arising from N,Ndimethylaniline in the presence of HRP and Hz02 are shown in Fig. 1. One signal detected approximately 1 min after initiating the reaction by addition of catalyst had a prominent component with considerable hyperfine structure (Fig. 1A); with time a minor component of this signal increased in intensity to a maximal value (Fig. 1C) and then decayed. With metmyoglobin as catalyst, identical EPR signals were generated from N,N-dimethylaniline, with qualitatively similar kinetic behavior. Both hemeprotein catalysts gave rise to a single epr signal from N,N-dimethyl-p-toluidine, which decayed rapidly (Fig. 2). For each substrate of Table 1, larger concentrations of metmyoglobin than of HRP were required to generate epr signals of equiv-
PI
’ Supported by NIH Grant No. AM 19627and Robert A. Welch Foundation Grant No. 1601. * Abbreviations used: NADPH, nicotinamide ade.?ine dinucleotide phosphate (reduced form); HRP, horseradish peroxidase; epr, electron paramagnetic resonance. 850
0003-9861/78/1902-0&50$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
N-DEMETHYLATION TABLE
I
RATES OF H202-D~~~~~~~~ FORMALDEHYDE PRODUCTION FROM N-DIMETHYL COMPOUNDS CATALYZED BY HRP AND METMYOGLOBIN~ Substrate
Catalyst HRP
N,N-Dimethylanihneb N,N-Dimethyl-ptoluidine* Aminopyrine
9.9 x lo3 (PH 6.0) 7.1 x lo4 (PH 6.5) 2.5 x 104= (pH 7.0)
851
OF N,N-DIMETHYLANILINE
Metymyoglobin 28.0 (PH 6.0) 49.0 (PH 6.0) 44.0* (PH 6%
(1Rates are expressed as moles of formaldehyde per minute per mole of heme at 37°C in 0.1 M potassium phosphate buffer at optimal pH. ’ Reactant concentrations employed were: amine, 3.0 mu; HzOx, 0.8 mu for N,N-dimethylanihne and 0.4 mru for the toluidine analog; HRP, 5-20 nM; and metmyoglobin, 0.5-1.2 PM. Because high concentrations of Hz02 interfered with the assay and also inhibited the HRP-catalyzed reactions, the true catalytic activities are probably somewhat higher than the values reported here. ’ Reference (7). * Reference (8). alent intensities (8); this is consistent with the relative catalytic activities of these two hemeproteins. Since it was possible that these epr signals arose from the products of the N-demethylation reaction, similar experiments were carried out with the N-monomethyl and unsubstituted analogs of both substrates. Both hemeprotein catalysts gave rise to the same epr signal from N-methylaniline, which was quite distinct from that observed with the parent dimethyl compound; similarly, with N-methyl-p-toluidine a single epr signal, obviously different from that of Fig. 2, was generated by both hemeproteins. Aniline and p-toluidine are well-characterized electron donor substrates of HRP (5), but the free radical intermediates presumably formed during enzymatic oxidation of these two compounds have not been detected by epr, as we confiied. However, p-toluidine very effectively inhibited the appearance of the HRP-dependent epr signals generated from the N-methyl-substituted toluidines, and aniline had a similar effect on free radical formation from N-methylaniline and N,N-dimethylaniline. As reported for aminopyrine (ll), N,N-dimethylaniline and its analogs undergo electrochemical oxidation in organic solvents (16-18) to free radical species, which in many cases, have been directly detected by epr. However, the one-electron oxidized form of N,Ndimethylaniline has not been detected because it is very unstable and undergoes a rapid coupling with
substrate to form NJ’-tetramethylbenzidine (16). This product is easily oxidized under the conditions of the electrochemical oxidation to an epr-detectable free radical species (16, 18). The mechanism of the HRPcatalyzed oxidation of N,N-dimethylaniline appears to be very similar, since N,N’-tetramethylbenzidine has previously been shown to be a significant product, which is also oxidized by HRP (19). We are attempting to obtain more stable, better-resolved epr signals of these several free radicals that can be positively identified by computer simulation. However, it appears that one of the signals detected with N,N-dimethylaniline is the radical cation of N,N’-tetramethylbenzidine
B
IO Gauss
/
------a’---4 Gauss D
g = 21003
FIG. 1. The epr signals generated enzymatically from N,N-dimethylaniline. (A) The reaction mixture contained 15 IIIM amine, 9.0 mM Hz02, and 1.9 p HRP in 0.1 M potassium acetate buffer, pH 4.1, 22°C; the spectrum was scanned approximately 1 min after mixing. Spectrometer settings: lo-mW microwave power, 9.160 GHz; 1.0-G modulation amplitude; 0.1~set time constant; 50-G/min scan rate; and 3.2 x lo3 gain. (B) Controls for (A) with either one substrate or the enzyme omitted, recorded with the same instrument settings. (C) The reaction mixture contained 15 mM amine, 5.0 mM Hz02 and 1.0 pM HRP in 0.1 M potassium acetate buffer, pH 5.0, 22’C; the spectrum was scanned approximately 3 min after mixing. Spectrometer settings: lo-mW microwave power; 9.160 GHz; 1.0-G modulation amplitude; O.l-set time constant; 20G/min scan rate; 1.5 X lo* gain. (D) Controls for (C), with either one substrate or the enzyme omitted, recorded with the same instrument settings. (Optimal conditions for detecting each radical were used.)
852
BRENDA
WALKER
GRIFFIN
I
g = 2.003 FIG. 2. The epr signal of a free radical species generated from N,N-dimethyl-p-toluidine. (A) The reaction mixture contained 15 mu amine, 15 mu HzOz, and 23.8 11~HRP in 0.1 M potassium acetate buffer, pH 5.0, 22’C. (B) AII controls for (A) with either one substrate or the enzyme omitted. Spectrometer settings for (A) and (B): lo-mW microwave power, 9.160 GHz; 1.0-G modulation amplitude; 0.1~set time constant; 50-G/min scan rate; 2.5 x lo3 gain.
and that the distinct signals generated from N,N-dimethyl-p-toluidine and from the N-monomethyl analogs are one-electron oxidized species of the respective substrates. These species are clearly not nitroxides, which have g-values near 2.006 (20), because ah of the free radicals reported herein have g-values of 2.003, identical to those of the free radical cations of aminopyrine (11) and other violenes (21). These experimental fmdings provide additional evidence that oxidative N-demethylation reactions catalyzed by HRP (7,8) and by metmyoglobin (8) proceed by the proposed free radical mechanism (Eq. [l]). The first reaction of this mechanism is consistent with the accepted mechanism of HRP-catalyzed oxidations (5); however, the fate of the free radical species formed depends critically on its chemical structure and reactivity. The free radicals generated from these Nmethyl compounds are unusuahy stable, since they can be readily detected in aqueous solution at room temperature without sophisticated mixing or flow systems. Although subsequent one-electron oxidation of the free radical is very probable (Eq. [l]), with N,Ndimethylanihne, coupling at the paru position to form the be&dine product is a significant competing reaction in both the electrochemical (16) and HRP-catalyzed (19) oxidations. However, if the coupling reaction is inhibited by apara substituent, as occurs with N,Ndimethyl-p-toluidine, there is considerable enhancement of the rate of N-demethylation relative to that of N,N-dimethyIanihne (Table 1). As was noted, in the proposed peroxidatic mechanism (Eq. [l]) the oxygen atom of formaldehyde arises from Hz0 and not directly from the peroxide oxidant (via a Compound I-like species), as has been proposed for cytochrome P-450-dependent N-demethylations supported by hydroperoxides (4). The experimental difficulties of determining the origin of the oxygen atom of the aldehyde product of these reactions have
been discussed in detail eLsewhere (8). These findings are significant because they have indicated that the free radical mechanism of oxidative N-demethylation may have more general validity, for other N-methylsubstrates, as well as other hemeprotein catalysts of these reactions. Clearly the monooxygenase and peroxidase N-demethylation reactions catalyzed by cytochrome P-450 must be re-examined in order to resolve the question of mechanism raised by this study. REFERENCES 1. CONNEY, A. H. (1967) Pharmacol. Rev. 19, 317-366. 2. Lu, A. Y. H., JUNK, K. W., AND COON, M. J. (1969) J. Bid. Chem. 244,3714-3721. 3. KADLUBAR, F. F., MORTON, K. C., AND ZIEGLER, D. M. (1973) Biochem. Biophys. Res. Commun. 64,1255-1261. 4. NORDBLOOM, G. D., AND COON, M. J. (1976) Arch. Biochem. Biophys. 180,343~347. 5. SAUNDERS, B. C., HOLMES-SIEDLE, A. G., AND STARK, B. P. (1964) Peroxidase, Butterworths, Washington, D.C. 6. SCHONBAUM, G., AND CHANCE, B. (1976) in The Enzymes, (Boyer, P., ed.), Vol. 13, Part C, pp. 363-408, Academic Press, New York. 7. GRIFFIN, B. W. (1977) FEBS Lett. 74, 139-143. 8. GRIFFIN, B. W., AND TINC, P. L. (1978) Biochemistry 2206-2211. 9. DE LA MARE, H. E. (1960) J. Org. Chem. 25, 2114-2126. 10. ROSENBLATT, D. H., HULL, L. A., DE LUCA, D. C., DAVIS, G. T., WEGLEIN, R. C., AND WILLIAMS, K. K. R. (1967) J. Amer. Chem. Sot. 89,
1158-1162. 11. SAYO, H, AND MASUI, M. (1973) J. Chem. Sot. Perkin Truns. 2, X40-1645.
A’-DEMETHYLATION
OF N,N-DIMETHYLANILINE
12. FRITSCH, J. M., WEINGARTEN, H., AND WILSON, J. D. (1970) J. Amer. Chem. Sot. 92,4038-4046. 13. MCMAHON, R. E. (1966) J. Pharm. Sci. 55, 457-466. 14. GOTOH, T., AND SHIKAMA, K. (1974) Arch. Biothem. Biophys. 163,476-481. 15. NASH, T. (1953) B&hem. J. 56,416-421. 16. MITZOGUCHI, T., AND ADAMS, R. N. (1961) J.
Amer. Chem. Sot. 84,2058-2061. 17. SEO, E. T., NELSON, R. F., FRITSCH, J. M., MARcoux, L. S., LEEDY, D. W., AND ADAMS, R. N. (1966), J. Amer. Chem. Sot. 88,3498-3503. 18. LATTA, B. M., AND TAFT, R. W. (1967) J. Amer.
Chem. Sot. 89,5172-5178. 19. NAYLOR, F. T., AND SAUNDERS, B. C. (1950) J.
853
Chem. Sot., 3519-3523. 20. FREED, J. H. (1976) in Spin Labeling. Theory and Applications (Berliner, L. J., ed.), pp. 73-75, Academic Press, New York. 21. FORRESTER, A. R., HAY, J. M., AND THOMSON, R. H. (1968) Organic Chemistry of Stable Free Radicals, pp. 254-258, Academic Press, New York. BRENDA WALKER GRIFFIN Biochemistry Department The University of Texas Health Science Center at Dallas 5323 Harry Hines Boulevard Dallas, Texas 75235 Received June 7, 1978