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On the nature and reactivity towards substrates of the active oxygen hemoprotein complexes involved in the reaction of peroxidases and cytochrome P- 450 with two-electron oxidants
JoumuC uf Mole.cu&r GzECysis, 7 (1960) 215 - 226 0 Ekevfer Sequoia S.A.. Lausanne -Printed in the Netherlands
225
.:
owrm3Nmw~Eti~~~ti~crn& TOWARDS smsmms GF THE ACTWE OXYGEN EXEMOPRCWEXN COMPLEXES TNVOLVED IN THE REACEION OF PEROXEDASES AND t2YTQCHROME P- 450 WETH TW0-ELEmQN OXIDANTS
D. MANSW=,
J.-C_ CHOmARD,
Labom&oire de Chimie Cedex 05 (France)
M. LANGE
de 1’Ecole Nor~~le
and J.-P. BA’ITIONI
Supkieure
,24 rue Lhomorrd.
7523i
Pm-is
Two s&s of results are presented which contribute to the understanding of the nature and reactivity of the active oxygen complexes formed during reactions of catalase, horseradish peroxidase, and cytochrome P- 450 w-itb two-election oxidants. The oneelectron oxidation of vinylidene carbene complexes of iron porphyrins [Fe’ - C=CAr2] ++ [ FeIV=C= CArJ , by FeC13 or CuC12, leads to stable complexes which exhibit visible spectra very similar to those of catalase and horseradish peroxidase compounds I_ This suggests a common formulation [porphyrin Fe=X] - for the carbene camplexes, X = C=CAr,, and for the hemoprotein compounds I, X = 0. From model studies, evidence has been obtained in favor of the fonnation of a stable iron-carbene bond after oxidation of 1,Sbenzodioxole by the active oxygen cytochrome P-Q50 compiex. From these data, a stepwise mechanism for suhskak oxidation by twoelection oxidants, catalyzed by cytqchrome P-450, involving the possible formation of unstable iron-carbon G bond, is proposed. This mechanism exhibits a close similarity with that of hydrocarbon oxidations by peroxycompounds catalyzed by_ copper or iron salts. l
Cytochromes P-450, catalases and peroxidases catalyze the oxidation of various organic compounds by two-election oxidants such as hydroperoxides. Actually, cytachrome P-45O-dependeEt monaoxygenases catalyze
*Author
to whom correspondence should
he addressed.
216
the oxidation by dioxygen of a wide range of organic substrates, including inert aliphatic hydrocarbons, a reaction which needs the transfer of two electrons from NADPH to the cytochrome [l] _ The reactions of catalases and peroxidases with two-electron oxidants lead to intermediate iron complexes called compounds I and compounds II, which retain, respectively, two and one osidizing equivalents above the native Pem-hemoprotein (Scheme 1) M.
-
Scheme
-
-
C,r5krc~
P4yJ
1
Recently, it has been shown that a similar highly oxidized iron intermediate is formed during reaction of cytochrome P-450 with a number of oxidants such as hydroperoxides [S] _ The knowledge of the structures of the= reactive intermediates appears especially important since they play a key role in the catalytic functions of the hemoproteins. From their spectroscopic properties 141, compounds II of peroxidases and catalases are generally believed to be FerV (porphyrin) complexes with an exogenous axial ligand which could be an 0x0 or an OH ligand. The structures of compounds I, which are formally Fe’/ (porphyrin) complexes, are even less clear. ~~tochrome c peroxidase, compound I, has been depicted as an Fetv (porphyrin) complex with a free radical of an amino-acid residue in the vicinity of the heme iron [ 51. Horseradish peroxidase (HRP) and catalase compounds I have been described as Fen’ (porphyrin x-cation radical) complexes, hecause of their abnormal electronic spectra which resemble those of Zn-, Mg-, Ni- or Co- porphyrin ncation radicals [S] _ However, no preparation of a ircation radical of an iron-porphyrin of the natural type has yet been reported_ Very recently, [7] it has been shown that the chemical oxidation of the Fe(II)(benzoporphyrin)(CO)(pyridine) complex leads to the corresponding ferrous- benzoporphyrin ncation radical. This reaction occurs because of the remarkably low oxidation potential of the benzoporphyrinic ligand which is below that of iron (II). For other iron porphyrin complexes, Fe(IIE)(TPP)(Cl) or (Fe(III)(TPP)),O, electrochemical oxidation has given Fe(IV) complexes whose further oxidation has led to highly unstable species and irreversible modification of the porphyrin ring [6a] .
217
The ‘active oxygen’ cytochrome P-450 intermediate complex, formed upon reaction of this cytochrome either with two-electron oxidants or with diosygen and two electrons, is the equivalent of peroxidase compounds I since it has the Same formal iron oxidation state (Scheme 1). It has -been recently shown that this active oxygen cytochrome P-450 complex has a free radical-like reactivity since it abstracts hydrogens from hydrocarbons [ 8). Model reactions suggest that it could also add to aromatic substrates [ 9]_ The stepwise mechanism, prcposed by Groves et al. [8, lo] for alkanes hydroxylation by cytochrome P-450 is indicatedin Scheme 2. O-OH
P-450 -
02+2e-
Fern c
P-450-[heme]
P-450
+ H’ -_H
B em
2
P-450-FeV=O
‘--Fp=O I
P-450 Scheme
2 (from
-
I
Fe”
f ROH
-
+RH
1
R- f HO-Ferv+450
[ 8, lo] )
The two following results allow us to contribute to the understanding of the nature and reactivity of peroxidase or cytuchrome P-450 ‘compound I’.
Results and discussion A. Preparation of possible carbon analogs cataluse and peroxidase comp0und.s I The carbon analogs of the porphyrin-iron-ox0 species (P--FerV=O) possibly present in conpounds IT, are the porphyrin-iron-carbene complexes (P- FeiV = CRR’ ) : [PFerV=CRR’]
-
]PFe= -+ CRR’]
[PFeLV=O]
-
]PFes
+ 0]
Evidence has been presenti in favor of the involvement of such cytochrome P-450 carbene complexes 1111 and we have synthetized complexes of ironporphyrins with various carbene hgands [ 121. The latter exhibit electronic spectza similar to those of compounds II but are considerably more stable. For instanro, Fig. 1 show3 the great .simiIarity of the electzonic spectra of HRP compound II and of a carbene complex of deuteroporphyrin dimethylester (DPDME), Fe[DPDME] [C=C(pClC,~),] [N-metbyhmidazole] [W-c] _ In order to obtain possible carbon analogs of compounds I which derive from a one-electron oxidation of compounds II, we hawe undertaken the study of the oxidation of various ircn-porphyrirr-carbene complses by oneelectron oxidantssuch as ferric chloride. Addition of increasing amounts of
218
350 450 550 Fig. 1. Comparison OF the electronic spectra of HW ampound complex Fe [ DPDME 1 iC=C(pClC, J& )2 ] [Nmethylimidazole]
II (----) ( -)-
and of the
FeCIJ or CuC12 in CHJCN to a benzenic solution of the vinylidene carbene complex of meso-tetraphenylporphyti (TPP), Fe[TPP] [C= C(pClCsH,)2] 1 112 leads, with isobestic points at 377,430, 521 and 557 nm (Fig. 2(a)), to a new entity, 2, characterized by peaks at 428 (E = 5.4 X 104) and 669 run (7.8 X 103)_ Its formation is complete after addition of a stoichiometzic amount of CuC12. Then, the addition of a reducing agent (e.g., dithionite or iron powderj regenerates complex 1 quantitatively. Compls 2 has been isolated as a crystalline solid and puriZied by thin layer chromatrography. The following chzacteriskics of complex 2: (i) elementi analysis (C, H, N, Cl), corresponding to Fe(TPP)(C14HsC12)(Cl); (ii) mass spectroscopy, exhibiting an intense peak, with an isotopic cluster characteriskic of the presence of two chlorine atoms, at m/e 246 (for 3”Cl) corresponding to the vinylidene carbene (PC~C~H~)~C=C or a rezrranged product; (iii) its complete formation from complex 1 and one equivalent of FeCla or CuC12, which czm be totally reversed by the addition of a reducing agent. indicate that complex 2 derivfrom complex 1 only by a one-electron oxidation together with a chloride transfer, supporting a formula of the type FeiTPP] [C=C(C,H,Cl),] [Cl] _ The chloride anion may be present in complex 2 either as an iron ligand or as 2 counterion. The electronic structure of complex 2 remains to be determined. However, one of its main characteristic-s is its peculiar electronic spectrum with a weak and broad Soret peak and the presence of a peak around 670 nm, both distinctive features already reporkcl for some met.alIoporphyrin nation radicals [S]. As shown in Fig. 2(b), there is a great armlogy bet&en the electionic spectra of complex 2 and of catalase compound I. Both species also exhibit paramagnetism of the same magnitude at room temperature: p2 = 3.7 PB, P,~=] -I = 3.9 1131. Analogs of complex 2 with other porphyrins (octaethylporphyrin (0EP)r h (E) in benzene 382 (4.5 X lOa), 497 (9.8 X I@), 593 (5.8 X IO”), deuteroporphyrin-dimethyl ester (DPDME): 382 (3.5 X 104), 498 (9.3 X 103), 542 (6.9 X l@), 588 (5.3 X 103); protoporphyrin IX: z 500 (8 X 103) and 600 (5 X 10’) in DMF), can also be obtained by oxidation of the corresponding Fe[porphyrin] [C= C(pClC,H,),] complexes by FeC13 or CuC&. Figure 3 underlines the analogy between the ekctronic spectra of HRP compounds 11 and I and, respectively, of the complexes Fe[DPDME] [C=CAr2] [N-methylimidazole] and Fe[DPDME] [C=CAr2] [Cl] (Ar = pClC,I&).
c] ,
I 400
1
2a
‘,
‘\\ \
\
I
I
tm
I BOO
J\ 11111 3
1 _-
.I
Q!
4
M
lta
c,
6 8 IQ’!
Pig. 2, (a) Oxldntlon of complex 1 (O.lmM) In CaH” by incronslngumounts of CuCl2in CH$N, followed by viniblospectroscopy: -) complex 1 and succcaslvcaddltlons of 0,25 cquivalcnt of CuCl2ond racordlng of cnch tlpactra Afterabout 0,6 h; ----) complor: 2 obtninod nftor nddltion of 1 oqrlivnlcntof CuC12.(b) Comparisonof the spcctru of complex 2 (----) nnd I of catnlaeocompound I (- *- 0- a),
,O
-0 ERICI
220
1
0.5
7
0 e x Y)
1
1
I
I
I
0
I
650 Anm 450 550 Fig. 3. Comptison of the Plectronic spectra of HRP compounds II (3e---) and I and the of the complexes Fe[DPDME] [C=C(pC!lC,H,+),] [N-methylimi(3a -), dazole] (3b ----) and Fe[DPDME] [C=C@CICsH&] [Cl] (3b -)350
The above results suggest a common formulation [porphyrinFe=Y]‘with Y = C=CAra for complex 2 and its analogs with other porphyrins, or Y = 0 for catalase and HRP compounds I. More detailed studies are still necessary to determine the electronic structure of these complexes (possible structures are indicated in Scheme 3) which should depend, in the case of the hemoproteins, upon the nature of the sixth axial endogenous ligand and of the heme environment [SC] .
Whatever this structure may be, one should expect that the [porphyrinFe=O] - species would exhibit a free radical-like reactivity. Accordingly, HRP compound I acts as a one-electron acceptor towards various eleckonrich substrates [Z] _ It seems that, during the formation of cytochrome c peroxik compound I, one amino acid residue near the heme iron is oxidized to the corresponding free radical, leading to a structure [PerV f R- ] l
221
for this compound I [5] _ Moreover, it has been recently shown that the active oxygen cytochrome P-450 complex is able to abstract hydrogen atoms of hydrocarbons, leading to the corresponding free radicals [8] _ This hydrogen abstraction can be performed by ‘cytihrome P-450 compound I’ which may be suitabiy descri’bed by the oxyradical structure B (Scheme 3), favored, in the case of this cytochrome, by the presence of an axial endogenous thiolate ligand [I], and ‘the possible existence of the mesomeric forms: RS- Few-O-. A more precise understanding of this steep RSFeLV-Oam&s the determination of spectral characteristics of the active oxygen cytochrome P-450 complex. B. Fomiion of an iron-carbene bond during oxidation of l,3-benzodioxole denuatives by cyfoctrrome PG50 1141 The 1,3benzodioxole derivatives are oxidatively metabolized by cytochrome P-45Ckiependent monooxygenases with the formation of very stable complexes of this cytochrome in the.ferrous sake, characterized by a Soret peak at 455 nm [15] _ After aerobic removal of the reducing agent (NADPH), the Soret band shifts to 438 nm which has been explained by tie formation of the corresponding ferric complexes 1161 _ The latter are also obtained by oxidation of benzodioxole derivatives with cumene hydropcroxide and cytochrome P450 [17]. It has been proposed that the exogenous ligand present in these complexes is the 1,3-benzodioxol-2-carbene [IS] , formed by oxiclat,ion of the methylene group of 1,3benzodioxole:
O.\
a -ex=
CT& + P-450
Fern NO~D~&&o”
b [P45O-S
+
Fern -
X]
0'
_
[P45O-S
c
‘O ‘0
+
Fe”
+
Xl
m (--J
We have been able to prepare the carbene complex Fe[TPP] [l,3-benzodioxol-.Z-carbene] 3, by reduction of 2,2-dichloro-1,3benzodioxole by Fe[TPP] in the presence of an excess of a reducing agent (iron powder), according to the general preparation method of Fe[porphyrin] [carhene] complexes which we have recently described [12]_ The structure of complex 3, isolated in the crystalline state, has been estibkhed from its various speckal cbaractM.stics (uv-visible, rH and UC n.m_r. mass spectroscopy) [L4] _ Addition of n-butyl-thiolate in excess, to complex 3 in tohrene, in the presence of dibenzo-18-crown-6-ether, leads to an immediate appearance of 2 459 nm peak in visible spectroscopy. This peak tippears within a few minutes because of an irreversible reaction between the carbene iigand and thiolate in excess*_ Figure 4 compares the visible difference spectra of the microsomal *The iron-carbene nuckophiks [ 12]_
bond
of i?e(porphyrin)(carbene)
complexes
is easily destroyed
by
222
0.4
0.2
xnm
0
2 -0.2
-0.4
-0.6
Fig. 4. (----)
DiTTerence spectrum of rat liver microsomes (cytochrome P-450, 3~!) 01 lo-% 1.3.benzodioxole and lop3 M NADPH to the sample cuvette, 5 min incubation. and then addition of Na2SI,O.s to both cuvetks (A X l/10 for this difference spectrum). (p ) Difference spectrum corresponding to: 7.8 x lo-% cornplex 3 in toluene in the sample cuvette. 7.8 x lo-% Fe(TPP)(C!) in toluene in the reference cuvette, then addition of 150 pl of a DMF solution of nBuSX (lO_lM) and dibcnzo-lBcrown-6ether (5 x IO-%) to both 3 ml cuvettes, and immediate recording of
dter addition
the spectrum.
cytochrome P-450- Fe(II)benzodioxole derived metabolite complex us. microsomal cytochrome P-450, and of complex 3 us. Fe[TPP] [Cl], just after addition of excess n-RuSto both cuvettes. Their similarity indicates that complex 4 is a model for the cytochrome P-45aFe(II)-metabolite complex, strongly supporting the 1,3-benzodioxole-Zcabene nature of this metabolite: Fe[TPP]
+ R&Cl2
-0
Fe[TPP]
[CR,] 3
-0 R2 =
5
K
Fe[TPP]
[CR,] 4
i >8
The formation of the cytochrome P-45Obenzodioxolederived carbene complex is the f&t example of the involvement of an iron-carbon bond after oxidation of the methylene group of a substrate by the active oxygen cytochrome P-450 complex. if one admits that this active oxygen complex involves an 0x0 ligand, as previously proposed 1191, the formation of the carbene complex derived from 1,3-benzodioxole oxidation, corresponds formally to the replacement of this 0x0 ligand by the 1,3benzodioxole2-carbene : Fe=0
-+ Fe=C
/O 10
EnBuS
223
The mechanism of hydrocarbon hydroxylation catalyzed by q-tochrome P-450, which is indicated on Scheme 2, displays good analogies wi+h that generally accepted for the oxidation of some hydrocarbons by peroxycompounds catalyzed by copper or iron salts 1201 (Scheme 4a)**_ The last step of this mechanism is the oxidation of the free radical derived from the substrate by the metal ion in its higher oxidation state. For ‘the oxidations catalyzed by copper or iron salts, it is generally admitted that this step can involve the formation of a transient 0 metal-carbon ‘bond [2Oa, b] _ Accordingly, the reversible formation of a u-bonded complex between the benzyl radical CsH,CHz’ and Cr(If) has been described [21], and it has been shown that the reaction of C!u(IE) ions with the CHzCOOradical leads to a Cu”-CH&OOcomplex ]22] _ A possible evolution of the uns’able G complex is the transfer of a ligand inside the coordination sphere of the metal or the reaction of an external nucleophile (e.g., solvent) on the a-alkyl group c209 231From the above results and discussion, we propose the following mechanism for 1,3-benzodiosole oxidation catiyZed by cytochrome P-+50 (Scheme 4b): (i) hydrogen abstraction by an FeIV-Omoiety*+ (see resuits A); (ii) combination of the so formed benzodioxole radical with the FeW species leading to an unstable u iron-carbon bond; (iii) elimination of water, favored by the acidity of the hydrogen in the B-position relative to iron, and formation of the iron-carbene bond. This mechanism could more generally apply to any substrate R,R&Hz but for the last steps if the formation of the iron-carbene bond is not sufficiently favored, the G complex may lead to the elimination of R,R,CHOH. It should be noted that, as in the oxidation of hydrocarbons by peroxycompounds catiyzed by copper or iron salts, a direct oxidative ligand transfer ]2Oa, b] ‘to the intermediate free radical (R- or RIR&H-), without formation of a 0 complex, is also possible [S, lo] , especially in the case of a substrate too bulhy to come close enough to the iron atom surrounded by the porphyrin ring.
**JIn fact, the two mechanisms which are indicated in Scheme 4 differ by their fit step (the reaction of the metal ion with peroxycompound.s). This ste leads to a radical AO‘ and Fern (or Cum) in the cake of the reactions catalyzed by Fe If or CuI salts, and we have written the formaSon of a reactive Fe w-Oentity From reaction of cytochrome P-450 Fern and AOOH. However. as long as the structure of &jle active oxygen cytochrome P-%50 CTITIlex will not be precise1 - known, one cannot exclude the following El reaction I P-450 Fe f AOOH + P-450 Fe & -OR f AO‘. and tie involvement of AO’ as a reactive species, as in the reactions catalyzed by Fe” or Cu’ salts. Moreover, it has been proposed that the two reactive species, ‘OH and Few=O, involved in Fenton’s sy&m, are in equilibrium: Fen + HzOz
--f Fe =-OH
[ 10, 241. The two possible be in fast equilibrimnr P-S50
Few-OH
+ AO’
+ -OH -f Hz0
reactive *
c Fern-O-
intermediates
P-Z50
Few-O-
P-450 + AOH
f-t Few=0 Few-O-
and AO-
might
also
224
(a)
AOOA’
+ M” B
(AO-)M”+l
+ A’O-
_;:zHc
R- +M”+‘-OA A II
A, A’ = H, alkyl, acyl RH = substrate M” = Cu’, Fe”
r
lw+z OA
u
con
I fH@
Y‘\ M” f R-OA (b)
AOOH
+ P-450
Fern E
P-450
FelV-O
P-450
i Fe” =0
ROH
+ AOH
‘p-450*
R. - 1 \
C= Fe-P-450
J
(for R1 R2 =
I%; Scheme 4 (* see footnote) The above results, together with those of other laboratories Cl,8 - 10,191 point to a close analogy between the mechanisms of hydrocarbon hydroxylations by peroxycompounds, catalyzed either by cytuchrome P-450 or by copper and iron salts. These are free radical chain mechanisms, the high yields, and sometimes high selectivity, obtained, being due to the control of the intermediate radicals by the metal complexes_ *Dissociation P-450
Few-OH
of the OH ligand according *
P-%50
$eW
tc the equilibrium
+ -OH
is possible_ The combicxtion of tbc P-450 $eW entity with R,Rz-SH would then lead to the hexacoordinated P450 i?e-CHR,Rz complex. Such dissociation of a ligand in tbe intermediary reactive complexes involved in the oxidations catalyzed by Fen or Cui snlts, has been invoked [ ZOa, b] _
225 References 1
2
3
4
5 6 7 8 9 19 11
12
13 14 15
16 17 18 13 20
V. Ulhich, A. Hildebrandt, I. Rook, R. WY. Estabmok and A. H. Conney (eds.), &ficrosomes and D.-ug Osidotions, Peesmon Press, Oxford, 1977. (a) H_ B. Dunford and J. S. Stillman. Coot-d. Chem. Reu.. 19 (1976) 287; (ed.). (b) T. Yonetii, Adu. Enzymol.. 33 (1970) 309; (c) I. Yamasa ki. in O_ Hay&i lCfolecuIar fifechanisms of Oxygen Activation. Audemic Press, New York, 1974. p_ 535. (a) A. D. Rahimtula, P. J. O’Brien, E. G. Hrycay, J. A. Peterson and R. W. Estabrook. Biockem. Biophys. Res. Commun.. 60 (1974) 695; (b) F. Lichterabrger, W. Natainczyk and V. Ullrich, Btichem. Biophys. Res. Common., 70 (1976) 939; (c) G. D. Nordbloom and M. J. Coon, Arch. B:ocham. Btiphys.. 180 (1976) 1255 1261. (a) ‘I’. Moss, A. Ehrenberg and A. J. Ekarden, Biochemistry. 8 (1969) 4 152; (b) T. Haremi, Y_ Maeda, U. Morita, A. Trautaein and U. Gonser, J. Chem. Phys.. 67 (1977) 1 164; (c) G. Rakhit, T. G. Spiro and M. Weda, Biochem. Biophys. Res. Common., 71 (1976) 803; (d) R. I-i. Felton. A. Y. Remans, N. T. Yu and G. R. Schonbaum, Biochem Biophys. Acta, 434 (19’75) 82. (a) T. Yonetani. H. SchIeyer and A. Ehrenberg. J. Biol. Chem.. 241 (lS66) 3 240; (b) T. Yonetani and A. F. W. Coulson, Biochemistry. 14 (1975) 2 389_ (a) D_ Dolphin and R. H. Felton, Act_ Chem. Res.. 7 (1974) 26; (b) E. G: Job-n, 1 381; (c) R. K. Di Nello and T. Niem and D. Dolphin, Can. J. Chem., 56 (1978) D. Dolphin, Btichem. Biophys. Res. Commun.. 86 (1979) 190. A. Vogler, B_ Rethwisch, H. Kunkely and J. Hiitterman, Anger. Chem., fnt. Ed. Engl.. 17 (1978)952. .J. T_ Groves. G. A. McCYusky, R. E_ White and M. J_ Coon, BiochemBiophys. Res. Common., 70 (1976) 939. L. Castle and J. R. Lindsly Smith., Chem. Commun., (1978) 704. J. T. Groves and M. Van Der Puy. J. Am_ Chem. Sot.. 98 (1976) 5 290. (a) D. Mansuy, W. Nastinczyk and V. Ullrich, N.S. Arch. PharmakoZ.. 285 (1974) 315; (b) C. R. Wolf, D. Mansuy, W. Nastainczyk, G. Deut-schmann and V. Ullrich. dfol. Pharmacol., 13 (1977) 698. (a) D. Mansuy, M. Lange. J.-C_ Chottard. P_ Gu&in, P. Morlisre, D. Brault and M_ Rougee, J. Chem. Sot.. Chem. Commun. (1977) 648; (b) D. &buy, M. Lange, J.-C_ Chottard, J.-F. Rartoli. B. Chewier and R. Weiss. Angem_ Chem.. Int. Ed_ Engl., 17 (1978) 781; (c) D. Mansuy, M. Lange and J.-C. ChotLrd, 6. Am. Chem. Sot., 100 Chem.. 171 (1978) 3 214; (d) D. Maosuy, P. Gu&in and J.-C_ Chottard , J. Oqanomet. (1979) 195. A. S. Brill, in M. Florkin and E. H. Stiiitz (eds_), Comprehensiue Biochemistry. Elszvier, Am_sLerdam 14 (1966) 464_ D. Mansuy, J. P. Battioni, J.-C. Chottzud and V. Ulbich, J. Am. Chem. Sot.. 101 (1979) 3971. (a) R. M. Philpot and E. Hodgson, Chem. Eiol. Interact., 4 (1971) 185; (b) M. R. Frank;ir,Xen~bi~tico. L (1971)581. C. R. EIcoroSe. J. Bridges. R. H. Nimmo-Smith and K. J_ Netter. Biochem. Pharmocol.. 24 (1975) 1427. C. R. Elcorr-be, J. Bridges, R. H. Nimmo-Smith and J. Werringloer,Biochem. Sot. Trahs., 3 (:t!‘75) 967. V. Ullrich, in D. J. Jollow, J. J. Kocsis, R. Snyder and K-f. Vainio (eds.), Biological Reactiue Intermediates. Plenum E’ress. New York, 1977, p. 65. (a) E. G. Hrycay, J. Gustafsxm. M_ Lngemal-Sundberg and L. Ernster. Biochem. Biophys. Res_ Commun.. 66 (1975) 2Q9; (b> V. Ullricb. H. H. Ruf and P. Wende, Croat_ Chem Actu. 49 (1977) 213. (a) J. K. Kochi, Act. Che.m. Res.. Radicals. Vol. 1. Wiley, New York, 1973. Ch. 11; (c) D. J. Rawlinson and G. Sosnovsky. Synthesis, (1972) 1.
226 21 J. K. Kochi and J. W. Powers, J. Am. Chem. Sot.. 92 (1970) 137. 22 MM.Fribeg ar.d D. Meyerstein, Chem. Commun. (1977) 127. 23 C. Walling, Ace. Chem. Res.. 8 (1975) 125_ 24 A. E. Cahill and H. Taube,d. Am. Chem. Sot., 74 (1952) 2 312.
225
.:
owrm3Nmw~Eti~~~ti~crn& TOWARDS smsmms GF THE ACTWE OXYGEN EXEMOPRCWEXN COMPLEXES TNVOLVED IN THE REACEION OF PEROXEDASES AND t2YTQCHROME P- 450 WETH TW0-ELEmQN OXIDANTS
D. MANSW=,
J.-C_ CHOmARD,
Labom&oire de Chimie Cedex 05 (France)
M. LANGE
de 1’Ecole Nor~~le
and J.-P. BA’ITIONI
Supkieure
,24 rue Lhomorrd.
7523i
Pm-is
Two s&s of results are presented which contribute to the understanding of the nature and reactivity of the active oxygen complexes formed during reactions of catalase, horseradish peroxidase, and cytochrome P- 450 w-itb two-election oxidants. The oneelectron oxidation of vinylidene carbene complexes of iron porphyrins [Fe’ - C=CAr2] ++ [ FeIV=C= CArJ , by FeC13 or CuC12, leads to stable complexes which exhibit visible spectra very similar to those of catalase and horseradish peroxidase compounds I_ This suggests a common formulation [porphyrin Fe=X] - for the carbene camplexes, X = C=CAr,, and for the hemoprotein compounds I, X = 0. From model studies, evidence has been obtained in favor of the fonnation of a stable iron-carbene bond after oxidation of 1,Sbenzodioxole by the active oxygen cytochrome P-Q50 compiex. From these data, a stepwise mechanism for suhskak oxidation by twoelection oxidants, catalyzed by cytqchrome P-450, involving the possible formation of unstable iron-carbon G bond, is proposed. This mechanism exhibits a close similarity with that of hydrocarbon oxidations by peroxycompounds catalyzed by_ copper or iron salts. l
Cytochromes P-450, catalases and peroxidases catalyze the oxidation of various organic compounds by two-election oxidants such as hydroperoxides. Actually, cytachrome P-45O-dependeEt monaoxygenases catalyze
*Author
to whom correspondence should
he addressed.
216
the oxidation by dioxygen of a wide range of organic substrates, including inert aliphatic hydrocarbons, a reaction which needs the transfer of two electrons from NADPH to the cytochrome [l] _ The reactions of catalases and peroxidases with two-electron oxidants lead to intermediate iron complexes called compounds I and compounds II, which retain, respectively, two and one osidizing equivalents above the native Pem-hemoprotein (Scheme 1) M.
-
Scheme
-
-
C,r5krc~
P4yJ
1
Recently, it has been shown that a similar highly oxidized iron intermediate is formed during reaction of cytochrome P-450 with a number of oxidants such as hydroperoxides [S] _ The knowledge of the structures of the= reactive intermediates appears especially important since they play a key role in the catalytic functions of the hemoproteins. From their spectroscopic properties 141, compounds II of peroxidases and catalases are generally believed to be FerV (porphyrin) complexes with an exogenous axial ligand which could be an 0x0 or an OH ligand. The structures of compounds I, which are formally Fe’/ (porphyrin) complexes, are even less clear. ~~tochrome c peroxidase, compound I, has been depicted as an Fetv (porphyrin) complex with a free radical of an amino-acid residue in the vicinity of the heme iron [ 51. Horseradish peroxidase (HRP) and catalase compounds I have been described as Fen’ (porphyrin x-cation radical) complexes, hecause of their abnormal electronic spectra which resemble those of Zn-, Mg-, Ni- or Co- porphyrin ncation radicals [S] _ However, no preparation of a ircation radical of an iron-porphyrin of the natural type has yet been reported_ Very recently, [7] it has been shown that the chemical oxidation of the Fe(II)(benzoporphyrin)(CO)(pyridine) complex leads to the corresponding ferrous- benzoporphyrin ncation radical. This reaction occurs because of the remarkably low oxidation potential of the benzoporphyrinic ligand which is below that of iron (II). For other iron porphyrin complexes, Fe(IIE)(TPP)(Cl) or (Fe(III)(TPP)),O, electrochemical oxidation has given Fe(IV) complexes whose further oxidation has led to highly unstable species and irreversible modification of the porphyrin ring [6a] .
217
The ‘active oxygen’ cytochrome P-450 intermediate complex, formed upon reaction of this cytochrome either with two-electron oxidants or with diosygen and two electrons, is the equivalent of peroxidase compounds I since it has the Same formal iron oxidation state (Scheme 1). It has -been recently shown that this active oxygen cytochrome P-450 complex has a free radical-like reactivity since it abstracts hydrogens from hydrocarbons [ 8). Model reactions suggest that it could also add to aromatic substrates [ 9]_ The stepwise mechanism, prcposed by Groves et al. [8, lo] for alkanes hydroxylation by cytochrome P-450 is indicatedin Scheme 2. O-OH
P-450 -
02+2e-
Fern c
P-450-[heme]
P-450
+ H’ -_H
B em
2
P-450-FeV=O
‘--Fp=O I
P-450 Scheme
2 (from
-
I
Fe”
f ROH
-
+RH
1
R- f HO-Ferv+450
[ 8, lo] )
The two following results allow us to contribute to the understanding of the nature and reactivity of peroxidase or cytuchrome P-450 ‘compound I’.
Results and discussion A. Preparation of possible carbon analogs cataluse and peroxidase comp0und.s I The carbon analogs of the porphyrin-iron-ox0 species (P--FerV=O) possibly present in conpounds IT, are the porphyrin-iron-carbene complexes (P- FeiV = CRR’ ) : [PFerV=CRR’]
-
]PFe= -+ CRR’]
[PFeLV=O]
-
]PFes
+ 0]
Evidence has been presenti in favor of the involvement of such cytochrome P-450 carbene complexes 1111 and we have synthetized complexes of ironporphyrins with various carbene hgands [ 121. The latter exhibit electronic spectza similar to those of compounds II but are considerably more stable. For instanro, Fig. 1 show3 the great .simiIarity of the electzonic spectra of HRP compound II and of a carbene complex of deuteroporphyrin dimethylester (DPDME), Fe[DPDME] [C=C(pClC,~),] [N-metbyhmidazole] [W-c] _ In order to obtain possible carbon analogs of compounds I which derive from a one-electron oxidation of compounds II, we hawe undertaken the study of the oxidation of various ircn-porphyrirr-carbene complses by oneelectron oxidantssuch as ferric chloride. Addition of increasing amounts of
218
350 450 550 Fig. 1. Comparison OF the electronic spectra of HW ampound complex Fe [ DPDME 1 iC=C(pClC, J& )2 ] [Nmethylimidazole]
II (----) ( -)-
and of the
FeCIJ or CuC12 in CHJCN to a benzenic solution of the vinylidene carbene complex of meso-tetraphenylporphyti (TPP), Fe[TPP] [C= C(pClCsH,)2] 1 112 leads, with isobestic points at 377,430, 521 and 557 nm (Fig. 2(a)), to a new entity, 2, characterized by peaks at 428 (E = 5.4 X 104) and 669 run (7.8 X 103)_ Its formation is complete after addition of a stoichiometzic amount of CuC12. Then, the addition of a reducing agent (e.g., dithionite or iron powderj regenerates complex 1 quantitatively. Compls 2 has been isolated as a crystalline solid and puriZied by thin layer chromatrography. The following chzacteriskics of complex 2: (i) elementi analysis (C, H, N, Cl), corresponding to Fe(TPP)(C14HsC12)(Cl); (ii) mass spectroscopy, exhibiting an intense peak, with an isotopic cluster characteriskic of the presence of two chlorine atoms, at m/e 246 (for 3”Cl) corresponding to the vinylidene carbene (PC~C~H~)~C=C or a rezrranged product; (iii) its complete formation from complex 1 and one equivalent of FeCla or CuC12, which czm be totally reversed by the addition of a reducing agent. indicate that complex 2 derivfrom complex 1 only by a one-electron oxidation together with a chloride transfer, supporting a formula of the type FeiTPP] [C=C(C,H,Cl),] [Cl] _ The chloride anion may be present in complex 2 either as an iron ligand or as 2 counterion. The electronic structure of complex 2 remains to be determined. However, one of its main characteristic-s is its peculiar electronic spectrum with a weak and broad Soret peak and the presence of a peak around 670 nm, both distinctive features already reporkcl for some met.alIoporphyrin nation radicals [S]. As shown in Fig. 2(b), there is a great armlogy bet&en the electionic spectra of complex 2 and of catalase compound I. Both species also exhibit paramagnetism of the same magnitude at room temperature: p2 = 3.7 PB, P,~=] -I = 3.9 1131. Analogs of complex 2 with other porphyrins (octaethylporphyrin (0EP)r h (E) in benzene 382 (4.5 X lOa), 497 (9.8 X I@), 593 (5.8 X IO”), deuteroporphyrin-dimethyl ester (DPDME): 382 (3.5 X 104), 498 (9.3 X 103), 542 (6.9 X l@), 588 (5.3 X 103); protoporphyrin IX: z 500 (8 X 103) and 600 (5 X 10’) in DMF), can also be obtained by oxidation of the corresponding Fe[porphyrin] [C= C(pClC,H,),] complexes by FeC13 or CuC&. Figure 3 underlines the analogy between the ekctronic spectra of HRP compounds 11 and I and, respectively, of the complexes Fe[DPDME] [C=CAr2] [N-methylimidazole] and Fe[DPDME] [C=CAr2] [Cl] (Ar = pClC,I&).
c] ,
I 400
1
2a
‘,
‘\\ \
\
I
I
tm
I BOO
J\ 11111 3
1 _-
.I
Q!
4
M
lta
c,
6 8 IQ’!
Pig. 2, (a) Oxldntlon of complex 1 (O.lmM) In CaH” by incronslngumounts of CuCl2in CH$N, followed by viniblospectroscopy: -) complex 1 and succcaslvcaddltlons of 0,25 cquivalcnt of CuCl2ond racordlng of cnch tlpactra Afterabout 0,6 h; ----) complor: 2 obtninod nftor nddltion of 1 oqrlivnlcntof CuC12.(b) Comparisonof the spcctru of complex 2 (----) nnd I of catnlaeocompound I (- *- 0- a),
,O
-0 ERICI
220
1
0.5
7
0 e x Y)
1
1
I
I
I
0
I
650 Anm 450 550 Fig. 3. Comptison of the Plectronic spectra of HRP compounds II (3e---) and I and the of the complexes Fe[DPDME] [C=C(pC!lC,H,+),] [N-methylimi(3a -), dazole] (3b ----) and Fe[DPDME] [C=C@CICsH&] [Cl] (3b -)350
The above results suggest a common formulation [porphyrinFe=Y]‘with Y = C=CAra for complex 2 and its analogs with other porphyrins, or Y = 0 for catalase and HRP compounds I. More detailed studies are still necessary to determine the electronic structure of these complexes (possible structures are indicated in Scheme 3) which should depend, in the case of the hemoproteins, upon the nature of the sixth axial endogenous ligand and of the heme environment [SC] .
Whatever this structure may be, one should expect that the [porphyrinFe=O] - species would exhibit a free radical-like reactivity. Accordingly, HRP compound I acts as a one-electron acceptor towards various eleckonrich substrates [Z] _ It seems that, during the formation of cytochrome c peroxik compound I, one amino acid residue near the heme iron is oxidized to the corresponding free radical, leading to a structure [PerV f R- ] l
221
for this compound I [5] _ Moreover, it has been recently shown that the active oxygen cytochrome P-450 complex is able to abstract hydrogen atoms of hydrocarbons, leading to the corresponding free radicals [8] _ This hydrogen abstraction can be performed by ‘cytihrome P-450 compound I’ which may be suitabiy descri’bed by the oxyradical structure B (Scheme 3), favored, in the case of this cytochrome, by the presence of an axial endogenous thiolate ligand [I], and ‘the possible existence of the mesomeric forms: RS- Few-O-. A more precise understanding of this steep RSFeLV-Oam&s the determination of spectral characteristics of the active oxygen cytochrome P-450 complex. B. Fomiion of an iron-carbene bond during oxidation of l,3-benzodioxole denuatives by cyfoctrrome PG50 1141 The 1,3benzodioxole derivatives are oxidatively metabolized by cytochrome P-45Ckiependent monooxygenases with the formation of very stable complexes of this cytochrome in the.ferrous sake, characterized by a Soret peak at 455 nm [15] _ After aerobic removal of the reducing agent (NADPH), the Soret band shifts to 438 nm which has been explained by tie formation of the corresponding ferric complexes 1161 _ The latter are also obtained by oxidation of benzodioxole derivatives with cumene hydropcroxide and cytochrome P450 [17]. It has been proposed that the exogenous ligand present in these complexes is the 1,3-benzodioxol-2-carbene [IS] , formed by oxiclat,ion of the methylene group of 1,3benzodioxole:
O.\
a -ex=
CT& + P-450
Fern NO~D~&&o”
b [P45O-S
+
Fern -
X]
0'
_
[P45O-S
c
‘O ‘0
+
Fe”
+
Xl
m (--J
We have been able to prepare the carbene complex Fe[TPP] [l,3-benzodioxol-.Z-carbene] 3, by reduction of 2,2-dichloro-1,3benzodioxole by Fe[TPP] in the presence of an excess of a reducing agent (iron powder), according to the general preparation method of Fe[porphyrin] [carhene] complexes which we have recently described [12]_ The structure of complex 3, isolated in the crystalline state, has been estibkhed from its various speckal cbaractM.stics (uv-visible, rH and UC n.m_r. mass spectroscopy) [L4] _ Addition of n-butyl-thiolate in excess, to complex 3 in tohrene, in the presence of dibenzo-18-crown-6-ether, leads to an immediate appearance of 2 459 nm peak in visible spectroscopy. This peak tippears within a few minutes because of an irreversible reaction between the carbene iigand and thiolate in excess*_ Figure 4 compares the visible difference spectra of the microsomal *The iron-carbene nuckophiks [ 12]_
bond
of i?e(porphyrin)(carbene)
complexes
is easily destroyed
by
222
0.4
0.2
xnm
0
2 -0.2
-0.4
-0.6
Fig. 4. (----)
DiTTerence spectrum of rat liver microsomes (cytochrome P-450, 3~!) 01 lo-% 1.3.benzodioxole and lop3 M NADPH to the sample cuvette, 5 min incubation. and then addition of Na2SI,O.s to both cuvetks (A X l/10 for this difference spectrum). (p ) Difference spectrum corresponding to: 7.8 x lo-% cornplex 3 in toluene in the sample cuvette. 7.8 x lo-% Fe(TPP)(C!) in toluene in the reference cuvette, then addition of 150 pl of a DMF solution of nBuSX (lO_lM) and dibcnzo-lBcrown-6ether (5 x IO-%) to both 3 ml cuvettes, and immediate recording of
dter addition
the spectrum.
cytochrome P-450- Fe(II)benzodioxole derived metabolite complex us. microsomal cytochrome P-450, and of complex 3 us. Fe[TPP] [Cl], just after addition of excess n-RuSto both cuvettes. Their similarity indicates that complex 4 is a model for the cytochrome P-45aFe(II)-metabolite complex, strongly supporting the 1,3-benzodioxole-Zcabene nature of this metabolite: Fe[TPP]
+ R&Cl2
-0
Fe[TPP]
[CR,] 3
-0 R2 =
5
K
Fe[TPP]
[CR,] 4
i >8
The formation of the cytochrome P-45Obenzodioxolederived carbene complex is the f&t example of the involvement of an iron-carbon bond after oxidation of the methylene group of a substrate by the active oxygen cytochrome P-450 complex. if one admits that this active oxygen complex involves an 0x0 ligand, as previously proposed 1191, the formation of the carbene complex derived from 1,3-benzodioxole oxidation, corresponds formally to the replacement of this 0x0 ligand by the 1,3benzodioxole2-carbene : Fe=0
-+ Fe=C
/O 10
EnBuS
223
The mechanism of hydrocarbon hydroxylation catalyzed by q-tochrome P-450, which is indicated on Scheme 2, displays good analogies wi+h that generally accepted for the oxidation of some hydrocarbons by peroxycompounds catalyzed by copper or iron salts 1201 (Scheme 4a)**_ The last step of this mechanism is the oxidation of the free radical derived from the substrate by the metal ion in its higher oxidation state. For ‘the oxidations catalyzed by copper or iron salts, it is generally admitted that this step can involve the formation of a transient 0 metal-carbon ‘bond [2Oa, b] _ Accordingly, the reversible formation of a u-bonded complex between the benzyl radical CsH,CHz’ and Cr(If) has been described [21], and it has been shown that the reaction of C!u(IE) ions with the CHzCOOradical leads to a Cu”-CH&OOcomplex ]22] _ A possible evolution of the uns’able G complex is the transfer of a ligand inside the coordination sphere of the metal or the reaction of an external nucleophile (e.g., solvent) on the a-alkyl group c209 231From the above results and discussion, we propose the following mechanism for 1,3-benzodiosole oxidation catiyZed by cytochrome P-+50 (Scheme 4b): (i) hydrogen abstraction by an FeIV-Omoiety*+ (see resuits A); (ii) combination of the so formed benzodioxole radical with the FeW species leading to an unstable u iron-carbon bond; (iii) elimination of water, favored by the acidity of the hydrogen in the B-position relative to iron, and formation of the iron-carbene bond. This mechanism could more generally apply to any substrate R,R&Hz but for the last steps if the formation of the iron-carbene bond is not sufficiently favored, the G complex may lead to the elimination of R,R,CHOH. It should be noted that, as in the oxidation of hydrocarbons by peroxycompounds catiyzed by copper or iron salts, a direct oxidative ligand transfer ]2Oa, b] ‘to the intermediate free radical (R- or RIR&H-), without formation of a 0 complex, is also possible [S, lo] , especially in the case of a substrate too bulhy to come close enough to the iron atom surrounded by the porphyrin ring.
**JIn fact, the two mechanisms which are indicated in Scheme 4 differ by their fit step (the reaction of the metal ion with peroxycompound.s). This ste leads to a radical AO‘ and Fern (or Cum) in the cake of the reactions catalyzed by Fe If or CuI salts, and we have written the formaSon of a reactive Fe w-Oentity From reaction of cytochrome P-450 Fern and AOOH. However. as long as the structure of &jle active oxygen cytochrome P-%50 CTITIlex will not be precise1 - known, one cannot exclude the following El reaction I P-450 Fe f AOOH + P-450 Fe & -OR f AO‘. and tie involvement of AO’ as a reactive species, as in the reactions catalyzed by Fe” or Cu’ salts. Moreover, it has been proposed that the two reactive species, ‘OH and Few=O, involved in Fenton’s sy&m, are in equilibrium: Fen + HzOz
--f Fe =-OH
[ 10, 241. The two possible be in fast equilibrimnr P-S50
Few-OH
+ AO’
+ -OH -f Hz0
reactive *
c Fern-O-
intermediates
P-Z50
Few-O-
P-450 + AOH
f-t Few=0 Few-O-
and AO-
might
also
224
(a)
AOOA’
+ M” B
(AO-)M”+l
+ A’O-
_;:zHc
R- +M”+‘-OA A II
A, A’ = H, alkyl, acyl RH = substrate M” = Cu’, Fe”
r
lw+z OA
u
con
I fH@
Y‘\ M” f R-OA (b)
AOOH
+ P-450
Fern E
P-450
FelV-O
P-450
i Fe” =0
ROH
+ AOH
‘p-450*
R. - 1 \
C= Fe-P-450
J
(for R1 R2 =
I%; Scheme 4 (* see footnote) The above results, together with those of other laboratories Cl,8 - 10,191 point to a close analogy between the mechanisms of hydrocarbon hydroxylations by peroxycompounds, catalyzed either by cytuchrome P-450 or by copper and iron salts. These are free radical chain mechanisms, the high yields, and sometimes high selectivity, obtained, being due to the control of the intermediate radicals by the metal complexes_ *Dissociation P-450
Few-OH
of the OH ligand according *
P-%50
$eW
tc the equilibrium
+ -OH
is possible_ The combicxtion of tbc P-450 $eW entity with R,Rz-SH would then lead to the hexacoordinated P450 i?e-CHR,Rz complex. Such dissociation of a ligand in tbe intermediary reactive complexes involved in the oxidations catalyzed by Fen or Cui snlts, has been invoked [ ZOa, b] _
225 References 1
2
3
4
5 6 7 8 9 19 11
12
13 14 15
16 17 18 13 20
V. Ulhich, A. Hildebrandt, I. Rook, R. WY. Estabmok and A. H. Conney (eds.), &ficrosomes and D.-ug Osidotions, Peesmon Press, Oxford, 1977. (a) H_ B. Dunford and J. S. Stillman. Coot-d. Chem. Reu.. 19 (1976) 287; (ed.). (b) T. Yonetii, Adu. Enzymol.. 33 (1970) 309; (c) I. Yamasa ki. in O_ Hay&i lCfolecuIar fifechanisms of Oxygen Activation. Audemic Press, New York, 1974. p_ 535. (a) A. D. Rahimtula, P. J. O’Brien, E. G. Hrycay, J. A. Peterson and R. W. Estabrook. Biockem. Biophys. Res. Commun.. 60 (1974) 695; (b) F. Lichterabrger, W. Natainczyk and V. Ullrich, Btichem. Biophys. Res. Common., 70 (1976) 939; (c) G. D. Nordbloom and M. J. Coon, Arch. B:ocham. Btiphys.. 180 (1976) 1255 1261. (a) ‘I’. Moss, A. Ehrenberg and A. J. Ekarden, Biochemistry. 8 (1969) 4 152; (b) T. Haremi, Y_ Maeda, U. Morita, A. Trautaein and U. Gonser, J. Chem. Phys.. 67 (1977) 1 164; (c) G. Rakhit, T. G. Spiro and M. Weda, Biochem. Biophys. Res. Common., 71 (1976) 803; (d) R. I-i. Felton. A. Y. Remans, N. T. Yu and G. R. Schonbaum, Biochem Biophys. Acta, 434 (19’75) 82. (a) T. Yonetani. H. SchIeyer and A. Ehrenberg. J. Biol. Chem.. 241 (lS66) 3 240; (b) T. Yonetani and A. F. W. Coulson, Biochemistry. 14 (1975) 2 389_ (a) D_ Dolphin and R. H. Felton, Act_ Chem. Res.. 7 (1974) 26; (b) E. G: Job-n, 1 381; (c) R. K. Di Nello and T. Niem and D. Dolphin, Can. J. Chem., 56 (1978) D. Dolphin, Btichem. Biophys. Res. Commun.. 86 (1979) 190. A. Vogler, B_ Rethwisch, H. Kunkely and J. Hiitterman, Anger. Chem., fnt. Ed. Engl.. 17 (1978)952. .J. T_ Groves. G. A. McCYusky, R. E_ White and M. J_ Coon, BiochemBiophys. Res. Common., 70 (1976) 939. L. Castle and J. R. Lindsly Smith., Chem. Commun., (1978) 704. J. T. Groves and M. Van Der Puy. J. Am_ Chem. Sot.. 98 (1976) 5 290. (a) D. Mansuy, W. Nastinczyk and V. Ullrich, N.S. Arch. PharmakoZ.. 285 (1974) 315; (b) C. R. Wolf, D. Mansuy, W. Nastainczyk, G. Deut-schmann and V. Ullrich. dfol. Pharmacol., 13 (1977) 698. (a) D. Mansuy, M. Lange. J.-C_ Chottard. P_ Gu&in, P. Morlisre, D. Brault and M_ Rougee, J. Chem. Sot.. Chem. Commun. (1977) 648; (b) D. &buy, M. Lange, J.-C_ Chottard, J.-F. Rartoli. B. Chewier and R. Weiss. Angem_ Chem.. Int. Ed_ Engl., 17 (1978) 781; (c) D. Mansuy, M. Lange and J.-C. ChotLrd, 6. Am. Chem. Sot., 100 Chem.. 171 (1978) 3 214; (d) D. Maosuy, P. Gu&in and J.-C_ Chottard , J. Oqanomet. (1979) 195. A. S. Brill, in M. Florkin and E. H. Stiiitz (eds_), Comprehensiue Biochemistry. Elszvier, Am_sLerdam 14 (1966) 464_ D. Mansuy, J. P. Battioni, J.-C. Chottzud and V. Ulbich, J. Am. Chem. Sot.. 101 (1979) 3971. (a) R. M. Philpot and E. Hodgson, Chem. Eiol. Interact., 4 (1971) 185; (b) M. R. Frank;ir,Xen~bi~tico. L (1971)581. C. R. EIcoroSe. J. Bridges. R. H. Nimmo-Smith and K. J_ Netter. Biochem. Pharmocol.. 24 (1975) 1427. C. R. Elcorr-be, J. Bridges, R. H. Nimmo-Smith and J. Werringloer,Biochem. Sot. Trahs., 3 (:t!‘75) 967. V. Ullrich, in D. J. Jollow, J. J. Kocsis, R. Snyder and K-f. Vainio (eds.), Biological Reactiue Intermediates. Plenum E’ress. New York, 1977, p. 65. (a) E. G. Hrycay, J. Gustafsxm. M_ Lngemal-Sundberg and L. Ernster. Biochem. Biophys. Res_ Commun.. 66 (1975) 2Q9; (b> V. Ullricb. H. H. Ruf and P. Wende, Croat_ Chem Actu. 49 (1977) 213. (a) J. K. Kochi, Act. Che.m. Res.. Radicals. Vol. 1. Wiley, New York, 1973. Ch. 11; (c) D. J. Rawlinson and G. Sosnovsky. Synthesis, (1972) 1.
226 21 J. K. Kochi and J. W. Powers, J. Am. Chem. Sot.. 92 (1970) 137. 22 MM.Fribeg ar.d D. Meyerstein, Chem. Commun. (1977) 127. 23 C. Walling, Ace. Chem. Res.. 8 (1975) 125_ 24 A. E. Cahill and H. Taube,d. Am. Chem. Sot., 74 (1952) 2 312.