Nov 26, 1991 - Computer graphics analyses were carried out at the University of California Computer Graphics facility (R. Langridge, Director) supported by ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY C 3 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 267, No. 13,Issue of May 5, pp. 8936-8942,1992 Printed in U.S. A.
Monooxygenase Activity of Cytochrome c Peroxidase* (Received for publication, November 26, 1991)
Vaughn P. Miller$, Gia D. DePillisS, Juan C. Ferrerj, A. Grant Maukj, and Paul R. Ortiz deMontellano$II From the $Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446and the SDeDartment of Biochemistrv. - _ university of British Columbia, Vancouver, British Columbia V6T lZ3, Canada
Recombinant cytochrome c peroxidase (CcP) and a W61A mutant of CcP, in contrast to other classical peroxidases, react with phenylhydrazine to give abonded phenyl-iron complexes. The conclusion that the heme iron is accessible to substrates is supported by the observation that CcP and W61ACcP oxidize thioanisole to the racemic sulfoxide with quantitative incorporation of oxygen from HzOz.Definitive evidence for an open active site is provided by stereoselective epoxidation by both enzymes of styrene, cis-& methylstyrene,and trans-B-methylstyrene. trans-& methylstyrene yields exclusively thetrans-epoxide, but styreneyields the epoxide and phenylacetaldehyde, and cis-g-methylstyrene yields both the cis- and transepoxides and 1-phenyl-2-propanone. The sulfoxide, stereoretentive epoxides, and1-phenyl-2-propanone are formed by ferryl oxygen transfer mechanisms because their oxygen atom derives from H2O2.In contrast, the oxygen in the trans-epoxide from the cisolefin derives primarily frommolecular oxygen and is probably introduced by a protein cooxidation mechais oxidized to nism. ~is-[1,2-~H]-l-Phenyl-l-propene [ l,l-2H]-l-phenyl-2-propanone without a detectable isotope effect on the epoxide:ketone product ratio. The phenyl-iron complex is not formed and substrate oxidation is not observed when the prosthetic group is replaced by 6-meso-ethylheme. CcP thus has a sufficiently open active siteto form a phenyl-iron complex, to oxidize thioanisole to thesulfoxide, and toepoxidize styrene and b-methylstyrene. The results indicatethat a ferryl (Fe(IV)=O)/proteinradical pair can be coupled to achieve two-electron oxidations. The unique ability of CcP to catalyze monooxygenation reactions does not conflict with its peroxidase function because cyto(DePillis, chrome c is oxidized at a distinct surface site G . D., Sishta, B. P., Mauk, A. G., and Ortiz deMontellano, P. R. (1991) J. Biol. Chern. 266, 19334-19341).
The catalytic action of cytochrome P450 monooxygenases and hemoprotein peroxidases is thought to be mediated by
* This work wassupported by National Institutes of Health (NIH) Grant GM32488 (to P. R. 0. M.) and Medical Research Council of Canada Grant MT-7182 (to A. G. M.). Computer graphics analyses were carried out at the University of California Computer Graphics facility (R. Langridge, Director) supported by NIH Grant RR-1081. Mass spectra were obtained in the University of California, San Francisco, Bio-Organic, Biomedical Mass Spectrometry Facility (A. Burlingame, Director) supported by NIH Grants RR-01614 and P-50 DK-26743. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. )I To whom correspondence should be addressed.
closely related two-electron oxidized speciesin which the iron is oxidized by one electron to a ferryl (Fe(IV)=O) complex and a second electron is removed from either the porphyrin or the protein (1-5). Abstraction of the second electron from the porphyrin in HRP’ and most other peroxidases yields a porphyrin radical cation (3, 5, 6), whereas abstraction of the electron from the protein,as in CcP, produces a protein radical (4,5,7). Theelectron may initially come, even in CcP, from the porphyrin, butthe resulting porphyrin radical cation is not detectable and, if formed, rapidly oxidizes a protein residue to give the protein radical. The protein radical in the two-electron oxidized formof CcP (“compound I”) is centered on Trp-191 (8-lo), but it appears that approximately 10% of the radical spin density is located on other (probably tyrosine) residues (11).The only evidence for initial removal of the electron from the porphyrin in CcP stems from a kinetic analysis of the reaction which suggests that cytochrome c is partially oxidized by a pFecursor of the compound I species with a protein radical (12). In contrast to the solid evidence on the electronic structures of the oxidized forms of HRP and CcP, little is known about the electron distribution in the catalytically active form of cytochrome P450 because it has never been directly observed (1,2). HRP (13-151, CcP (16), and other peroxidases (17,18) are inactivated during their catalytic turnover of phenylhydrazine, alkylhydrazines, or azide due, at least in part,to addition of substrate-derived radicals to the6-meso-carbon of the prosthetic heme group. In contrast, the inactivation of cytochrome P450 (19))catalase (20), and myoglobin (21) by phenylhydrazine involves addition of the phenyl group to the iron atom to give a phenyl-iron complex that rearranges, upon acidic, aerobic work-up, to the four regioisomers of N-phenylprotoporphyrin IX. The phenyl-iron complex and N-phenylprotoporphyrin IX adducts are thus formed with all the proteins with active sites known from x-ray data tobe relatively open and substrate-accessible. As a result, we have proposed that classical peroxidases (those with a histidine as the proximal iron ligand) differ from the cytochrome P450 monooxygenases in that they have closed active sites that force substrates to interact with the &heme edge rather than with the ferryl oxygen. The finding that HRP does not catalyze the epoxidation of styrene (22) or butadiene (23) agrees with this hypothesis, but the observation that HRP, lactoperoxidase, and thyroid peroxidase catalyze the sulfoxidation of thioanisoles by a mechanism that results in incorporation of oxygen from the peroxide appears to conflict with the hypothesis and The abbreviations and trivial names used are: HRP, horseradish peroxidase; heme, iron protoporphyrin IX regardless of the oxidation and ligation state; CcP, cytochrome c peroxidase; W51A CCP, cytochrome c peroxidase with a Trp-51- Ala mutation; 0-methylstyrene epoxide, 1-phenylpropylene oxide; HPLC, high-pressure liquid chromatography; GLC, gas-liquid chromatography.
8936
Monooxygenme Activity of Cytochrome c Peroxidase
8937
The final CcP concentration was typically 75nM. The rate of formation of tetraguaiacol, the primary oxidation product, was monitored at 470 nm (c = 26,600 M" cm"). The background rate for the oxidation of guaiacol in the absence of the enzyme was negligible. Horse heart ferrocytochrome c was prepared from commercial cytochrome c by reduction with dithionite according to the method of Yonetani (29). The concentration of the reduced protein thus obtained, generally in the 0.5-1.0 mM range, was determined from the Routine assays were absorption at 550 nm (c = 27,600 M"cm"). performed by adding H202 (160 p ~ to) a 25-30 p~ solution of ferrocytochrome c in 50 mM sodium acetate buffer (pH 6.0; pm = 0.05) at 25 "C. The background rate of cytochrome c oxidation, measured for 20-30 s before CcP (1 nM) was added, was subtracted from the final rate. The rate of cytochrome c oxidation was deter. ~ ~ M" mined from the loss of absorbance at 550 nm ( ~ ~ =d 19,600 cm"). Identification of the Metabolites of Styrene and cis- and trans-@Methylstyrene-Cytochrome c peroxidase (18p M ) in 0.5 ml of sodium phosphate buffer (50 mM, pH 7.0) was incubated with styrene (10 mM, added neat in small aliquots), or cis- or trans-j3-methylstyrene (5 mM, delivered as a concentratedmethanol solution) in the presence or absence of hydrogen peroxide. The hydrogen peroxide was added in 0 . 5 - m ~aliquots to yield a final nominal concentration of 10 mM. EXPERIMENTALPROCEDURES The reactions were conducted at 25 "C for a total of 30 (styrene) or Materials-Horseradish peroxidase (type VI), horse heart cyto- 60 min (@-methylstyrenes)before the internal standard (trans-@chrome c, bovine liver catalase, 30% HzOz, guaiacol, l-phenyl-2- methylstyrene in styrene incubations, styrene oxide in @-methylstyrpropanone, and sodium ascorbate were obtained from Sigma. ["O] ene incubations) was added. The incubation mixtures were extracted H 2 0 (97% lag)and ['aO]HzOz(98% lag)were purchased from Icon with 0.2 ml of diethyl ether (styrene) or CHzClz (8-methylstyrenes) (Summit, NJ). trans-@-Methylstyrene,1-phenyl-1-propyne, (1S,2S)- and analyzed by GLC on a 0.5 mm X 30-m DB-5 column. For the (-)-1-phenylpropylene oxide, and (lR,2R)-(+)-l-phenylpropylene styrene products, the column was programmed to rise from 35 to oxide were purchased from Aldrich. cis-@-Methylstyrenewas from K 80 "C a t 70 "C/min, then to 150 "C a t 4 "C/min after 1 min a t 80 "C. & K laboratories (Cleveland, OH). Deuterium gas wasfrom Matheson The retention timesof styrene, phenylacetaldehyde, and styrene oxide Gas Products (Newark, CA). Ethylhydrazine oxalate was from Fluka under these conditions were 7.49, 12.28, and 13.13 min, respectively. (Ronkonkoma, NY). Buffers were made with glass-distilled, deionized For the @-methylstyreneproducts, the column was programmed to water and were batch-treated with Chelex 100 (Bio-Rad). The recom- run at 80 "C for 3 min and then to rise at 2 "C/min to 150 "C. The binant wild-type CcP and itsW51A variant were obtained as reported retention times for &-@-methylstyrene, trans-@-methylstyrene, cis(28). 6-meso-Ethylheme, obtained as previously described (14, 16), @-methylstyreneoxide, trans-B-methylstyrene oxide, and l-phenyl-2was purified by reversed-phase HPLC on a 9 X 250-mm Whatman propanone were 14.9, 17.0, 22.2, 23.2, and 24.0 min, respectively. Partisil50DS-3 semipreparative column eluted isocratically a t 4 ml/ Determination of Turnouer Numbers-CcP (15 pM) in 1.5 ml of min with MeOH:H20:HOAc (6832:lO). Apo.CcP was constituted sodium phosphate buffer (50 mM, pH 7.0) was mixed with cis- or trans-@-methylstyrene(5 mM) followed by addition of Hz02 (trans, with 6-meso-ethylhemeas reported earlier (16). Synthesis of cis-@-MethylstyreneEpoxide-To a mixture of meta- 0.1 mM; cis, 1 mM). Aliquots (250 pl) taken a t 30-8 to 20-min time chloroperbenzoic acid (1.4 g, 8.1 mmol) and potassium fluoride (450 points were transferred to vials containing 250 pl of CH2C12, 250 pl mg, 8.1 mmol) was added &-@-methylstyrene (864 pl, 6.7 mmol). The of 50 mM sodium phosphate buffer, and 2.5 nmol of styrene oxide reaction was stirred overnight, filtered through Celite, and concen- (internal standard). The mixtures were vortexed and briefly centritrated by distillation. The residue was purified by flash chromatog- fuged before 5-pl aliquots of the CHzC12phase were analyzed by GLC. raphy (33% CHZC12/hexane)in a yield of 75%. The purity of the Product formation was quantitated relative to astandard curve product was established by GLC. constructed by carrying the authentic metabolites through the same (1,2-Dideuterated cis- extraction and quantitation procedure. Synthesis of cis-[1,2-2H]-l-Phenyl-l-propene @-Methylstyrene)-The air in a flask containing 1-phenyl-1-propyne Stereochemistry of Olefin Epoxidation-The CHzC12 epoxide ex(1.39 mg, 12 mmol), palladium on barium sulfate (60 mg; palladium tracts from standard incubations (see above) were concentrated 2-3content, 5%), quinoline (66 mg, 0.5 mmol), and hexane (30 ml) was fold prior to GLC analysis a t 120 "C on a 0.25 mm X 30-m Chiraldex removed by evacuating and flushing with deuterium three times. The G-TA capillary column (Advanced Separation Technologies, Inc., mixture was then stirred at 1atm and25 "C with monitoring by thin Whippany, NJ). The retention times of the 1S,2R and 1R,2S cis-@layer chromatography until the reaction was approximately 75% methylstyrene epoxide and 1S,2S and 1R,2R trans-@-methylstyrene complete (40 min). The palladium catalyst was removed byfiltration epoxide enantiomers were, respectively, 13.9,17.4,14.1, and 16.2 min. through Celite, andthe filtrate was concentrated by distillation. Identification of S-Oxygenation Products-CcP (18 pM) in 0.5 ml Purification by flash chromatography (hexane) afforded the desired of sodium phosphate buffer (50 mM, pH 7.0)was incubated with deuterated product in 70% yield 'H NMR (CDC13) 7.75-7.57 (5H, m, thioanisole (5 mM, delivered as a concentrated methanol solution) in aryl H),2.29 (3H, s, Me); 13CNMR (CDCL) 137.5, 128.8, 128.0, 126.3 the presence or absence of hydrogen peroxide. The hydrogen peroxide (aryl carbons), 129.9, 126.5 (vinyl carbons), and 14.4 ppm (Me). The was added in 0 . 5 - m ~aliquots to a final nominal concentration of 10 sample is >95% dideuterated, as shown by the I3C shifts in the NMR mM. The reactions were conducted at 25 "C for a total of 60 min.The of the vinyl carbons on deuteration (129.9 + 129.4 and 126.5-+ 125.8 incubation mixtures were extracted with 0.2 ml of CH2C12, the solvent ppm) andby mass spectrometry. was removed with a stream of argon, and the residue was dissolved Spectroscopic and Analytical Methods-Absorption spectra were in 100 p1 of hexane and analyzed by chiral HPLC on a Chiralcel OB recorded on aHewlett-Packard8450A diode array spectrophotometer. column (0.46 X 25 cm). Isocratic elution was performed using 9:l Analytical HPLC was performed on a Hewlett-Packard Model 1090 hexane:2-propanol at 0.5 ml/min. Retention times for the S(-) and system equipped with a diode array detector. Semipreparative HPLC R(+) sulfoxides were 40.3 and 66.1 min, respectively. Control incuwas done on a system consisting of a Varian 9010 pumping system bations were carried out in the absence of CcP. and a Hewlett-Packard 1040A diode array detector. GLC was done Incubation with [laO]HzOz-T~ a solution of W51A CcP (18 PM) on a Hewlett-Packard Model 5890instrument equipped with a flame and cis-@-methylstyrene, trans-@-methylstyrene,or thioanisole (7 ionization detector and a Hewlett-Packard 3390A recording integra- mM) in 0.5 ml sodium phosphate (pH 7.0) buffer was added [180] tor. GLC/mass spectrometry was performed on a Hewlett-Packard Hz02 (10 p l , 0.27 M) in 1-pl aliquots over 10 min. The mixture was Model 5890 gas liquid chromatograph interfaced with a VG-70-250/ incubated for another 50 min a t 25 "C followed byextraction with 0.2 SE mass spectrometer. ml of diethyl ether. The ether extracts were analyzed by GLC/mass Activity Assays-Guaiacol oxidation was routinely assayed by add- spectrometry for "0 enrichment. ing a 5-pl aliquot of CcP solution to 1 ml of a solution of guaiacol Incubation with ~aO]HzO-W51A CcP (18 pM) in 0.5 mM sodium (100mM) and H202 (0.6 mM) in 50 mM phosphate buffer (pH 7.0). phosphate (pH 7.0) buffer was lyophilized to dryness, and the result-
remains to be explained (24-26). We recently demonstrated that cytochrome c and small substrates bindto, and are oxidized at, different sites of CcP (16). The oxidation of small substrates is inhibited by 6-mesoheme substitution, whereas cytochrome c oxidation is relatively insensitive to such substitution (16). We also found that inactivation of guaiacol peroxidation by phenylhydrazine correlates with covalent binding of one equivalent of the inhibitor to the protein, whereas inactivation of cytochrome c oxidation occurs more slowly and correlates with the appearance of a long wavelength absorption maximum indicative of a phenyl-iron complex (16). This implies that CcP may have a more open active site than suggested by inspection of its crystal structure (27). We confirm here that CcP forms a phenyl-iron complex with phenylhydrazine and, more importantly, demonstrate that it not only catalyzes the sulfoxidation of thioanisoles but the more diagnostic and revealing epoxidation of styrene and cis- and tramts-/3-methylstyrene.
8938
Monooxygenme Activity of Cytochrome c Peroxidase
ing solid was dissolved in 0.5 ml of ['sO]H20. All other methods were the same as previously described.
myoglobin (30). HPLC analysis of the prosthetic group from phenylhydrazine-inactivated CcP extracted under different conditions indicates that small amounts of two additional RESULTS products are formed that co-elute with the 6-meso-phenyl and Formation of a +Bonded Phenyl-Iron Complex-In earlier 8-hydroxymethyl derivatives of heme isolated from phenylwork we demonstrated that theinactivation of CcP-catalyzed hydrazine-treated HRP (not shown) (13), but these products cytochrome c oxidation by phenylhydrazine correlates with have not been fully characterized. The two peaks preceding formation of a complex with a red-shifted absorption maxi- the four N-phenylprotoporphyrin IX isomers in Fig. 1 may mum (16). The similarity in the spectroscopic change with correspond to these products. The formation of a phenyl-iron that observed in the myoglobin-phenylhydrazine reaction, in complex is a characteristic of hemoproteins with open active which a phenyl-iron complex is formed (21,30), suggests that sites (19-21) and is not observed with other classical peroxia similar complex is formed in the CcP reaction. To examine dases (13, 17, 18, 32). this point, we have exploited the fact that phenyl-iron comOxidation of Thioanisole-The observation of a phenyl-iron plexes extracted from hemoproteins undergo an intramolec- complex suggests that theheme iron of CcP is more accessible ular iron-to-nitrogen migration of the phenyl group when to substrates than itis in other imidazole-ligated peroxidases. exposed to acid and oxygen (30, 31). To minimize H202- TOtest this possibility, we first examined the oxidation of dependent degradation of the products, experiments were thioanisoles by CcP. Thioanisoles are oxidized to the correcarried out with phenyldiazene and relatively high concentra- sponding sulfoxides by several peroxidases, including HRP tions of CcP or its W51A mutant. Phenyldiazene, the two- (24), lactoperoxidase (26), and thyroid peroxidase (26), by a electron oxidation product of phenylhydrazine, is thought to mechanism that incorporates an atom of oxygen from HzOz be the species that actually reacts with the heme group to into the sulfoxide. The oxygen-labeling result is consistent give the phenyl-iron complex (30,31). The regioisomers of N- with a ferry1 oxygentransfer mechanism analogous to thatof phenylprotoporphyrin IX areunfortunately formed in too low cytochrome P450 monooxygenases (1). CcP similarly oxidizes a yield with CcP itself to be readily characterized, but they thioanisole tothe sulfoxide. Furthermore, incubation of can be isolated and identified from incubations with the thioanisole with CcP and ['80]H202, followed by mass specW51A mutant (Fig. 1). The four possible regioisomers, ob- trometric analysis of the sulfoxide, indicates that all of the tained in approximately equal amounts, were identified by sulfoxide oxygen derives from the peroxide (Table I). The their spectroscopic and chromatographic identity with the sulfoxidation occurs with little enantioselectivity because authentic isomers isolated from phenylhydrazine-treated bothCcPandits W51A mutant produce nearly racemic mixtures of the S(-) and R ( + ) sulfoxides (Table I). Oxidation of Styrene and cis- and trans-&MethylstyreneThe finding that the sulfoxidation of thioanisole occurs with incorporation of oxygen from the peroxide is not surprising, in view of the fact that the same observation has been made with other peroxidases (24-26). It is therefore not a discriminating test of the active site structural features that differentiate peroxidases from monooxygenases. A much more definitive test is provided by olefin epoxidation, a reaction not mediated by hemoproteins with relatively closed active sites. As already noted, neither styrene (22)nor butadiene (23) is oxidized by HRP? Somewhat to our surprise, incubation of styrene with CcP or W51A CcP produces styrene oxide in an enzyme-, time-, and H202-dependent manner. GLC analysis indicates that two products are formed that co-elute with authentic styrene oxide and phenylacetaldehyde. The styrene oxide and phenylacetaldehyde are produced in approximately equal amounts by CcP and a 3:l ratio by the W51A mutant (Table 11). trans-/3-Methylstyrene is oxidizedby both CcP 0 10 20 30 and W51A CcP to a single product that co-elutes by GLC with trans-P-methylstyrene oxide (Table 11). The turnover Time (min) number for the oxidation of trans-p-methylstyrene, as deterFIG. 1. HPLC of the N-phenylprotoporphyrin IX isomers mined from a plot of product formation versus time, is -12 obtained by aerobic, acidic extraction of the phenyl-iron com- pmol/min/nmol enzyme. cis-@-Methylstyreneis oxidized by plex from the W61A CcP (bottom). and phenylhydrazine- both CcP and the W51A mutant to at least three products treated myoglobin (top). The four possibleN-phenylprotopor(Fig. 2, Table 11). The threeproducts have been identified by phyrin IX isomers are numbered from 1 to 4. The peaks labeled A direct chromatographic and mass spectrometric comparison and B may be due to 6-meso-edge-modified heme groups (see text). with authentic standards ascis-P-methylstyrene oxide, transThe chromatographic conditions are given in the text. 8-methylstyrene oxide, and 1-phenyl-2-propanone. The turnover numbers for the formation of these products are, respecTABLEI tively, 7 , 3 , and 7 pmol/min/nmol CcP. Benzaldehyde was not Stereochemistrv of thwanisole sulfoxidation detected among the incubation products. Amount of sulfoxide Enzyme Amount of "0 from Absolute Stereochemistry of cis-P-Methyktyrene Epoxida['801H@2 S(-) R(+) tion-The cis-epoxide is stereoselectively produced from cisP-methylstyrene by CcP and W51A CcP (Table 111). It is of CCP 49 ND" 51 W51A 98 45 55 Incubation of cis-0-methylstyrene with HRP also produces no more than a trace of the epoxide (unpublished). a ND, not done.
Monooxygenase Activity of Cytochrome c Peroxidase
8939
TABLE I1 W51A CcP-catalyzedoxidation of styrene and cis- and trans-@-methylstyrene Substrate
Styrene
Styrene trans-@-Methylstyrene cis-@-Methylstyrene
a
Amount of "0 from F"IHzO,
Product %
%
oxide (67) Phenylacetaldehyde (33) trans-@-Methylstyreneoxide (100) cis-@-Methylstyreneoxide (52) tram-@-Methylstyreneoxide (16) 1-Phenyl-2-propanone (32)
ND" ND 85' 99 14' 61 (100)'
ND, not done.
'Incubations with ['80]H20 show that none of the epoxide oxygen derives from water.
e Rapid exchange of the ketone oxygen with water results in a low value for the ''0 incorporation. The value in parenthesis is that corrected from control experiments (see text) in which the exchange reaction was monitored.
TABLE111 Stereochemistry of epoxidation of cis-B-methrlstyrene
a
Metabolite
CCP
W51A
cis epoxide
37
0
20
10
30
TIME (min)
FIG. 2. GLC of the products from the oxidation of cis-& methylstyrene by W51A CcP. The peaks correspond to: a, cis-@methylstyrene; b, cis-@-methylstyreneoxide; c, tram-@-methylstyrene oxide; and d, 1-phenyl-2-propanone. The unlabeled peak at 17.0 min is due to a trace of thetram isomer in the substrate. Control experiments show that the trans-epoxide does not derive from the trace of trans-olefin. This is confirmed by the observation that 85% of the oxygen in the trans-epoxide from the trans-olefin derives from HpO, (Table 11),whereas only 15%of the oxygen in the trans-epoxide from the cis-olefin does so. The GLC conditions are given in the text.
particular interest that the lR,2S-cis-p-rnethylstyreneoxide isomer is favored in the CcP-catalyzed oxidation, whereas the 1S,2R isomer is favored in the oxidation catalyzed by the W51A mutant. Much lower oxidation stereoselectivity is observed for the trans-epoxide produced from cis-P-methylstyrene (Table 111). Mechanisms ofOlefin Metabolite Formation-In order to examine the mechanism(s) of the epoxidation reactions, we have determined the source of the oxygen incorporated into the epoxide products. As shown in Table 11,85%of the oxygen in the trans-epoxide formed from trans-p-methylstyrene derives from the peroxide (Table 11). Analogous incubations with ['80]Hz0 show that none of the oxygen incorporated into the epoxide is from the medium, so the remaining 15% of the oxygen must derive from molecular oxygen. It has not been possible to verify this due to the current unavailability of "02. Analysis of the cis-epoxide produced from cis-8-methylstyrene shows that its oxygen derives quantitatively from H202(Table 11). In contrast, only 14% of the oxygen in the trans-epoxide from cis-p-methylstyrene derives from H2O2 and none from water. The oxygen in the trans product from the cis-olefin thus derives primarily from molecular oxygen. Mass spectrometric analysis of the 1-phenyl-2-propanone obtained in experiments with ['80]H202indicated that it contained approximately 63% labeled oxygen aftera 40-min incubation with CcP and59% after a 60-min incubation with W51A CcP (Table11).However, control experiments in which 1-phenyl-%propanone was incubated in ['80]Hz0 for 70 min
90e e
34
63
32
26
48
45
trans epoxide
55
90ee
04
In
showed that approximately 45% of the ketone oxygen exchanges with oxygen from the medium during the indicated period. This corresponds quite closely to the 37 and 41% nonperoxide oxygen in the 1-phenyl-2-propanone from the enzymatic incubations. The 59 and 63% peroxide oxygen incorporation observed in the enzymatic experiments therefore reflects approximately quantitative incorporation of peroxide oxygen followed byexchange with the medium. cis-P-Methylstyrene with a deuterium at each of the two vinylic positions was synthesized by catalytic reduction of 1-
Monooxygenme Activity
8940
of Cytochrome c Peroxidase
phenyl-1-propyne. Oxidation of this compound by W51ACcP yields the same ratio of products asoxidation of the unlabeled substrate (not shown). The molecular ion in the mass spectrum of the l-phenyl-2-propanone, however, is two mass units higher than in the spectrum of the undeuterated compound, indicating that itretains bothdeuterium atoms. Furthermore, the fragmentation pattern, particularly the ion due to the tropylium (CTH:) cation, shows that thetwo deuterium atoms are located at the benzylic position. 1-Phenyl-2-propanone formation therefore involves migration of the deuterium from the methyl-substituted vinylic carbon to theadjacent benzylic carbon (Fig. 3). 6-meso-EthylhemeSubstitution-Previous work on the peroxidase activity of CcP demonstrated that small substrates such as guaiacol and ferrocyanide are oxidized at a different site thancytochrome c, the normal substrate, and specifically identified the site of their oxidation as the6-meso-heme edge (16). Key evidence in this regard was provided by the observation that peroxidase activity is blocked, whereas cytochrome c oxidation activity is not, when the heme of CcP is replaced by a 6-meso-alkylheme. In fact, when CcP is reconstituted with 6-meso-ethylheme it is inactive with respect to both formation of the phenyl-iron complex and epoxidation of cis-(3-methylstyrene (not shown). Substitution of the 6meso edge thus blocks both peroxidation and monooxygenation of small substrates. DISCUSSION
Previous studies have shown that peroxidases with an imidazole fifth ligand, including horseradish (13), Coprinus macrorhizus (17)) manganese (18))and lignin (32) peroxidases, do not react with phenylhydrazine to give phenyl-iron complexes comparable to those obtained with myoglobin (21,30), hemoglobin (31), catalase (33), and cytochrome P450 (19,34). This observation, and thefact that thecatalytically generated radicals from phenylhydrazine, alkylhydrazines, and azide add to the 6-meso-heme carbon of HRP (13, 14, 15), led us to propose that peroxidative reactions are mediated by electron transfer to the 6-meso edge rather than directly to the ferryl oxygen (5, 13). As a corollary to thishypothesis, we proposed
R-00'
kI '
FIG. 3. Mechanismsproposed for the oxidation of cis-& methylstyrene to cis-B-methylstyrene oxide, trans-&methylstyrene oxide, and 1-phenyl-2-propanone. Fey= 0 is a formalism for the ferryl plus porphyrin or protein radical. R stands for a protein residue.
that the well defined functional differentiation of monooxygenases and peroxidases is due to the fact that theheme iron (and therefore ferryl oxygen) is readily accessibleto substrates in the former but not the latterclass of enzymes (13,14). The present finding that CcP reacts with phenyldiazene to give a phenyl-iron complex, demonstrated by isolation of N-phenylprotoporphyrin IX adducts after aerobic rearrangement of the W51A CcP complex (Fig.l ) , clearly suggests that theiron in the CcP active site is more accessible to small organic substrates than it is in the active sites of other classical peroxidases. This is true of CcP itself, but even more so of the W51A mutant, in which the bulky tryptophan side chain is replaced in the active site by an alanine methyl group. Examination of the crystal structure of CcP by computer graphics suggests that its active site is very congested, but it is apparently not too congested to accommodate a phenyl group by small displacements of active site residues. A precedent for this is provided by the formation of a phenyl-iron complex with myoglobin, a reaction shown by x-ray crystallography to require displacement of several active site residues (21). The inference that the active site of CcP might be sufficiently open or malleable to allow small substrates to interact with the iron atom is borne out by the finding that CcP and W51A CcP not only catalyze sulfoxide oxidation, a reaction also mediated by other classical peroxidases (24-26, 35), but also olefin epoxidation, a diagnostic monooxygenase reaction that is not catalyzed by most classical peroxidases (22, 23). Thioanisole sulfoxidation, as observed with the other peroxidases, results in high yield incorporation of peroxide oxygen into thesulfoxide (Table I), presumably via direct transfer of the ferryl oxygen to thesulfur. The facility with which sulfur undergoes what is nominally a peroxidase-catalyzed cytochrome P450-like ferryl oxygen transfer is under investigation in this andother laboratories but is notyet understood. The epoxidation of styrene and cis- and trans-&methylstyrene convincingly demonstrates the unique ability of CcP (unique for an imidazole-ligatedperoxidase)to catalyze monooxygenation reactions, albeit at a rate ten times slower than cytochrome P450. CcP and W51A CcP oxidize styrene to styrene oxide and phenylacetaldehyde, trans-P-methylstyrene exclusively to the trans-epoxide, and cis-P-methylstyrene to the cis-epoxide, l-phenyl-2-propanone, and a small amount of the trans-epoxide (Fig. 2, Table 11). The stereoretentive epoxidation reactions are primarily or exclusively mediated by ferryl oxygen transfer mechanisms not only because the stereochemistry is retained, but also because the epoxide oxygen derives primarily or exclusively from HzOz(Fig.3, Table 11). Support for an active site-mediated process is provided by the fact that CcP produces the 1R,2S diastereomer of the cis-epoxide from the cis-olefin with 32% enantiomeric excess, whereas the 1S,2R diastereomer is obtained in 26% enantiomeric excess with the W51A mutant. Computer graphics docking experiments do not immediately provide an obvious rationale for the difference in the preferred stereochemistry of the CcP- and W51A CcP-catalyzed reactions. Olefin oxidation by CcP and W51A CcP does not exclusively produce epoxides. The epoxidations of styrene and cisP-methylstyrene, but not trans-B-methylstyrene, are accompanied by a 1,2-hydrogen shift to give phenylacetaldehyde and l-phenyl-2-propanone, respectively (Table 11). We have explicitly demonstrated the 1,2-hydrogen shift by deuterium labeling only in the case of cis-P-methylstyrene, but there is little doubt that thesame mechanism holds for the formation of phenylacetaldehyde from styrene. Phenylacetaldehyde is obtained in the oxidations of styrene catalyzed by cytochrome
Monooxygenase
8941
Activity of Cytochrome c Peroxidase
Me cis
H
Me
trans FIG. 4. Conformational lyzed oxidation of cis- but
H rationalization of the not trana-&methylstyrene.
formation
P450 (as a very minor pathway) (36), chloroperoxidase (22), and metalloporphyrins (36-39), and has been shown in those systems to involve a 1,2-hydrogen shift (22, 38). Unexpected, in this context, is the fact that no trace of l-phenyl-2-propanone, the 1,2-hydrogen rearrangement product, could be detected in incubations of CcP or W51A CcP with trans-flmethylstyrene (Table II). The difference between the ci.s- and trans-olefins is readily rationalized by conformational differences in the putative reaction intermediates. Regardless of the details of the mechanism that results in addition of the ferry1 oxygen to the double bond, the 1,2-hydrogen shift undoubtedly occurs in an intermediate with the ferry1 oxygen bound to the methyl-substituted carbon and a carbocation at the benzylic position (Fig. 3). Binding of the ferry1 oxygen to the nonbenzylic carbon is supported by nearly quantitative incorporation of “0 from the peroxide into the carbonyl group of the rearranged product (Table II). The two carbons of the original double bond must rotate by approximately 60” relative to one another to align the hydrogen that is to migrate with the acceptor p-orbital of the benzylic cation (Fig. 4). This rotation, in the case of trans-P-methylstyrene, increases the energy of the steric interaction between the phenyl group and either the terminal methyl group or the ferry1 oxygen/heme group, but it decreases the steric interaction between the phenyl and methyl groups in the case of the cis isomer. Rotation of the intermediate to the conformation required for 1,2-hydrogen shift is therefore disfavored for the tram isomer but favored for the cis isomer. Oxidation of cis-&methylstyrene results not only in formation of the cis-epoxide and l-phenyl-2-propanone but also of a minor amount of the trans-epoxide (Table II). A cytochrome P450-like mechanism is highly unlikely for this epoxidation because the olefin stereochemistry is invariably retained in cytochrome P450 epoxidations (1). Furthermore, 86% of the oxygen incorporated into the trans epoxide is not from hydrogen peroxide or water and therefore must come from molecular oxygen (Table II). This argues strongly against an oxidation mechanism triggered by addition of the ferry1 oxygen to the K bond. We previously reported that the peroxide-dependent epoxidation of styrene by myoglobin and hemoglobin proceeds with partial loss of the oletin stereochemistry and incorporation of an atom of molecular oxygen (40, 41). To rationalize these observations, we proposed that
of 1-phenyl-2-propanone
in the
CcP-cata-
The double-headed arrows indicate steric interactions.
styrene is cooxidized by the protein peroxy radical generated when oxygen binds to the peroxide-engendered protein radical (40, 41). Support for this mechanism is provided by the observation that 4-methylphenol mediates the HRP-dependent cooxidative epoxidation of styrene (42). The same mechanism can be invoked for the CcP-catalyzed oxidation of the cis-olefm to the trans-epoxide. EPR studies show that the CcP protein radical is not located exclusively on Trp-191, a residue that has little apparent contact with the protein exterior or the putative substrate binding site. Approximately 10% of the unpaired electron density resides on residues, tentatively identified as tyrosines (111, that may be located closer to the surface or substrate binding site. The observation that the trans-epoxide is produced from the cis-olefin with little stereospecificity (Table III) is consistent with such a mechanism. Furthermore, the 15% of the trans-epoxide from the trans-olefin that incorporates an oxygen atom from O2 rather than H202 can be rationalized by the same mechanism. Thioanisole sulfoxidation and olefin epoxidation are well established reactions catalyzed by iron porphyrins and hemoproteins. The two oxidation equivalents required for these reactions are stored in the well characterized model systems as an Fe(IV) = O/porphyrin radical cation (e.g. 43-45). Use of the two oxidation equivalents to promote nominally concerted, two-electron oxidations is readily envisioned when they are part of a single functional group. However, the two oxidation equivalents are located on distinct, spatially separated functionalities in compound I of CcP because the ferry1 species is paired with a protein rather than porphyrin radical (8-10). The two oxidation equivalents therefore must be recruited into a common reaction process in order to catalyze the sulfoxidation and epoxidation reactions. Furthermore, epoxidation of &s-/3-methylstyrene with retention of the olefin stereochemistry requires utilization of the two oxidation equivalents without the formation of a long-lived intermediate. One possibility is that the protein radical is in equilibrium with the porphyrin radical cation and the latter species is responsible for the epoxidation. However, if the ferryl/protein radical is the actual catalytic species, the two oxidation equivalents required for the epoxidation are likely to be employed in a nonconcerted manner. This argues against a concerted epoxidation mechanism, such as formation of a metallaoxetane intermediate (46), and for a mechanism, such as initial
8942
Monooxygenase Activity of Cytochrome c Peroxidase
formation of a charge transfer complex (47), that is consistent with stepwise utilization of the oxidation equivalents. The oxidations of thioanisole, styrene, and cis- and trans-P-methylstyrene by CcP and W51A CcP demonstrate, in any case, that Fe(1V) = 0 and a protein radical can be coupled to promote cytochrome P450-like two-electron oxidations. The heme creviceof CcP is connected to theprotein surface by a 6-A-wide, 11-A-longchannel that enters thecrevice near the 6-meso-carbon of the heme group (27). We have recently demonstrated that the peroxidation of guaiacol and other small substrates is blocked by 6-meso-heme substitution and have concluded that these substrates migrate up the channel to the b-meso edge, where they are oxidized (16). The same channel appears to provide access to theactive site for monooxygenase substrates because phenyl-iron complex formation and olefin oxidation are blocked when CcP is reconstituted with 6-meso-ethylheme. The inability to catalyze monooxygenase reactions is not due to a functionally disabled enzyme because compound I is still formed and the enzyme retains full cytochrome c oxidizing activity (16). It is therefore likely that the 6-meso substituent simply blocks the access channel. The monooxygenase activity of CcP does not conflict with the primary function of this enzyme as a cytochrome c peroxidase because the latterreaction takes place at a surface site remote from the heme iron that is insulated from potential monooxygenative reactions with the ferryl oxygen. In summary, we have ( a ) identified the first olefin epoxidation reactions catalyzed by a classical peroxidase, ( b )established that a peroxidase can catalyze monooxygenations if the heme iron is accessible, strengthening the hypothesis that the iron is relatively inaccessible in most peroxidases, and (c) established that monooxygenase reactions can be mediated by species in which the ferryl oxygen and second oxidation equivalent are located on independent sites.
12. 13. 14. 15. 16.
17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
Acknowledgment-We thank Julie Fruetel for obtaining the mass spectrometric data.
33.
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34.
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