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Canadian Journal Journal canadien de biochirnie of Biochemistry Published by THENATIONAL RESEARCH COUNCIL OF CANADA
P ~ t b l i kpar LE CONSEIL NA'I'IONAL
Vol~ln~e 53
Volur~te53
N ~ ~ l n b e6 r June 1975
DE RECKERCHES DU
CANADA
numCro 6 juin 1975
Titration Study of Guaiacol Oxidation by Horseradish Peroxidase
Received October 17, 1974 Santimone, ha. (1975) Titration Study of Guaiacol Oxidation by Horseradish Peroxidahe. Ctul. 9. Bioclzent. 53, 649-657 Titration of guaiacol by hydrogen peroxide in the presence of a catalytic amount of horseradish peroxidase shows that the reduction of hydrogen peroxide proceeds by the abstraction of two electrons from a guaiacol molecule. In the same way, it can be demonstrated that 8.5 mol of guaiacol can reduce, at low temperature, 1 mol of peroxidase compound I to compound 11. Moreover, the reaction between equal amounts of compound I and guaiacol at low temperature produces the native enzyme. A reaction scheme is proposed which postulates that two electrons are transferred from guaiacol to compound I giving ferriperoxidase and oxidized guaiacol with the intermediary formation of compound 11. The direct two-electron transfer from guaiacol to compound I witl~duta dismutation of product free radicals must be considered as an exception to the general mechanism involving a single-electron transfer. Santimone, hl. (1975) Titration Study of Guaiacol Oxidation by Horseradish Peroxidase. Cart. 9. Biochern. 53, 649-657
Le titrage du guaiacol par le peroxyde d'hydrogkne en prksence d'une quantitC catalytique de peroxydase du raifort montre que-la rCduction du peroxyde d'hydrogkne se fait par abstraction de deux Clectrons de la molkule de gua'iacol. De la mCme f a ~ o nil, peut Ctre dCmontrC qu'une demi-molCcule de guai'acol peut rkduire, a basse tempkrature, une molCcule du compost2 peroxydasique I en composd II. De plus, la reaction entre des quantitks Cgales de csmposC I et de guaiacol B basse tempdrature produit l'enzyme native. Le schCma rdactionnel proposC postule que deux Clectrons sont transfkres du guaiacol au composC I donnant la ferriperoxydase et le guaiacol oxydC avec formation intermkdiaire du compose 11. Le transfert direct de deux electrons du guaiacolau composC I sans une dismutation des radicaux libres du produit doit Ctre considdrk comme une exception au mdcanisme gknCral impliquant le transfert d'un seul electron. [Traduit par le journal]
Introdhetion It is currently believed that most of the oxidation processes involving the electron exchange between an electron donor and the peroxideperoxidase system occur through the classical mechanism of Chance (1-6) and George (7-10). The first step in this mechanism involves the transfer of the two oxidizing equivalents of H 2 0 2to the ferric enzyme. The compound I thus obtained then exchanges one oxidizing equivalent with 1 mol of the electron donor. This oxidoreduction reaction leads to compound I1 and to
the oxidized donor. Compound 11 then reacts with a second mole of donor and is reduced to ferric peroxidase. Thus in this classical reaction scheme, two molecules of an electron donor are needed to reduce one molecule of compound H to ferriperoxidase i.e., only one of the two electrons of a molecule of the donor is exchanged with peroxidase. Various experimental results have shown that peroxidases can catalyze the oxidation of two electron donors such as sulfite (Il), iodide (12), and ethanol (13). The oxidation of these donor
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650
CAN. J.
BIOCHEM.
n~oleculesoccurs with a two-electron transfer in one step and does not involve compound 11. Moreover, the stoichiometry observed for the reaction of H z 0 2 or con~pound H and these donors is 1:l. 'The direct two-electron transfer fr~m these substrates to compound I, thus forming peroxidase without the intermediate formation of compound 11, is generally considered as an exception to the mechanism of ChanceG e ~ r g e(1-10). It is of interest to determine whether other two-electron donors could be oxidized by peroxidase in a two-electron process, either with the intermediate formation of compound HH or not. Since guaiacol, as we shall demonstrate, is a two-electron donor, this substrate has been chosen in the present study. Materials and Methods The horseradish peroxidase isoenzyme used in the present work, designated P2 (14), is very similar to isoenzyme C of horseradish peroxidase previously described by Shannon et al. (15). It is isolated and purified from the commercial enzyme preparation obtained from Fluka. The purification method (14) was derived from that of Maaaa st al. (16). The hemoprotein thus obtained was homogeneous on analytical centrifugation and electrophoresis. Its Reinheitsaahl (R.2.)was close to 3. Some sf the physico-chemical characteristics of this enzyme preparation have already been reported 4 14). The kinetics of compound I reduction was recorded with a Spectralux 1800 spectrophotometer equipped with a Sefram recorder. This apparatus was modified so that it was possible to inject rapidly a reagent into the medium and record the absorbance change at a fixed wavelength. Mixing was effected by a mechanical stirrer. The stirring and injection apparatus was positioned outside the path of the light beam. The equipment for low-temperature spectrophotometry was derived from that designed by Douzou (17). Its characteristics have already been described (18). The antifreeze solvent was methanol - aqueous phosphate buffer, rased in a 58-50 volume ratio. Denaturation of the protein during the preparation of the solutions at low temperature was avoided by an appropriate balance between the temperature and the volume ratio of the solvent. The theoretical titration curves of an electron donor by hydrogen peroxide were obtained by computer (Siris 10070) sin~ulation.Depending on the number of transferred electrons and the degree of polymerization of the product, one has to solve equations of the second, third, fourth, sixth, and eighth degrees. This has been done with the so-called Polrt (19) Fortran program.
Results It is well known, that during the oxidation of guaiacol, the enzymatic reaction is followed by a
VOL. 53. 1975
non-enzymatic polymerization process leading to a brown product, P. The structure of this product is still unknown. The polymerization process is certainly rapid with regard to the enzymatic reaction, for the overall rate is proportional to the peroxidase concentration, at least for a Barge range of substrate concentratior~s. The titration curve of guaiacol oxidation by hydrogen peroxide with a catalytic concentration of peroxidase (Fig. 1) shows a change of slope for a 1:1 ratio of peroxide to guaiacol. This result could be interpreted by a 1:1 stoichiometry in the overall process. However, deviation from the strict titration curve is observed, which could be attributed to an eEect of reversibility, to the nature of the reaction product, or to secondary reactions competing for hydrogen peroxide. This observed deviation casts doubt on the conclusion that the stoichiometry of the reaction is 1:l. Therefore, theoretical titration curves have been simulated assuming that either a one-electron donor (Fig. 2) or a two-electron donor (Fig. 3) is oxidized by hydrogen peroxide. Moreover, calculations were effected with differeizt equilibrium constant values supposing that the reaction product is a monomer (scheme A of Figs. 2 and 3), a dimer (scheme B of Figs. 2 and 3), or a tetrainer (scheme C of Figs. 2 and 3). The curves obtained clearly indicate that, for an equilibrium constant not lower than 100, a change of slope is observed that indicates the number of transferred electrons. When the change of slope is observed for a 1:2 ratio of peroxide to donor. this implies a one-electron
FIG. 1. Titration of guaiacol by hydrogen peroxide in the presence of horseradish peroxidase P2. Absorbance changes are plotted against the ratio [I-1202]j[guaiacoBl. The concentrations of peroxidase and of guaiacol are 0.94 X M and 1.56 X 10-8 M, respectively. The pH is 7.0 (0.2 M phosphate buffer).
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SANTIMONE : GUAIACOL OXIDATION
65 1
FIG.2. Theoretical titration curves of a one-electron donor, AH2, by hydrogen peroxide. The oxidation product is supposed to be either a monomer (A), a dimer (B), or a tetramer (C).The overall equations corresponding to the various situations are presented in the same figure. The initial concentrations of the donor are supposed to be either 1 (A), 2 (B), or 4 (C) in arbitrary units. Curves are computed for different values of the equilibrium constant K. Concentrations of the product are given in arbitrary units.
oxidation of the donor molecule. When it is obtained for a 1 :1 ratio, then two electrons are abstracted from the same donor molecule. It is noteworthy that this conclusion can be drawn whatever the reason for the deviation observed on the theoretical titration curves. Then, the titration curve of guaiacol oxidation by hydrogen peroxide (Fig. 1) is only compatible with the
view that an exchange of two electrons occurs between one molecule of guaiacol and PI2O2. Indeed, a change of slope can be observed for a 1 :1 ratio of peroxide to guaiacol. Though this conclusion seems unquestionable, it was interesting to obtain information about the molecular weight of the reaction product. The brown product, B, of guaiacol oxidation was
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652
CAN. 9 . BIOCHEM. VBPL. 53, 1975
FIG.3. Theoretical titration curves of a two-electron donor, AMs, by hydrogen peroxide. The oxidation product is again supposed to be either a monomer (A), a darner (B), or a tetramer (C). The overall equations corresponding to the various situations are presented in the same figure. The initial concentrations of the donor are supposed to be either 1 (A), 2 dB), or 4 ( C ) in arbitrary units. Curves are computed for different values of the equilibrium constant K . Concentrations of the product are given in arbitrary units.
thought at first to be tetraguaiacol(20), then dihydroxydimethoxydipheny1(2 I). As was pointed out by Saunders (21), the structure of tetraguaiacol cannot account for the visible spectrum of P. Recently, the n~olecular weight of this product has been determined by mass speetrometry (22) and the value obtained was 458, thus obviously incompatible with the view that guaiacol has been oxidized into dihydroxydi-
~netboxydiphenyl.However, in the light, P is slowly reduced to a new product (23) with a molecular weight of 246 and that seenas very likely to be dihydroxydimethoxydiphenyl (2 1). Since the mcsleeular weight of guaiacol and of the main reaction product are 124 and 458, respectively, it appears that the non-enzymatic polymerization process leads to the formation of a tetramer and that. during this reaction, part of
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SANTIMONE: GUAIACOL OXIDATION
I
Compound H /
I
I
FIG.4. Spectra of peroxidase, compound 1, and compound II in the Soret and in the visible regions. Compound 1 is obtained by the addition of 26.6 X 10-3 pmol of H202to the same amount of horseradish peroxidase P2. Compound E l is obtained by the addition of 13.3 X ctmolof guaiacol. The final volume of the reaction medium is 3 ml. The pH is 7 (8.08 IM phosphate buffer). The antifreeze solvent is methanol - aqueous buffer (volume ratio 58-50). The temperature is -58 "C.
the oxidized guaiacol molecule has been lost. That guaiacol is a two-electron donor is verified in Fig. 4, which represents the spectrum in the Soret and in the visible regions of compound I1 obtained by the addition of 0.5 mol of guaiacol to B mol of compound I. It is also possible to confirm the fact that guaiacol is a two-electron donor molecule. by a titration of compound I at low temperature. At -50 "C the rate of spontaneous reduction of compound I to compound IT is relatively slow and can be recorded by an increase in absorbance at 410 nm, which is the isosbestic point between peroxidase and compound I1 (curve b of Fig. 5A). However, when small amounts of guaiacol are introduced, into the medium, the transformation t o coimpound 11 is much more rapid (curve a of Fig. 5A). The absorbance changes measured upon successive additions of guaiacol allow one to determine the percentage of compound I reduced. The exact titration curve (curve b of Fig. 5B) is calculated subtracting spontaneous reduction of this compound to the total reduction (curve a of Fig. 5B). It is very important t o note that the slope of the straight line obtained shows that I mol of compound I is fully reduced t o compound I1 by only 0.5 mol of guaiacol. Since the transition from
compound 1 to compound Il needs one reducing equivalent, this confirms the idea that guaiacol is a two-electron donor nlolecule. The titration of compound I by an equimolar amount of guaiacol, giving rise to the appearance of ferriperoxidase, can be followed (curve I of Fig. 6) at -58 "C and 394 nm. which is the isosbestic point of compound I and compound II. Under the same experimental conditions, mixing of compound 1 and a one-electron donor, such as ferrocyanide, in equimolar concentrations, must give rise to the compound TI and not to the ferriperoxidase, so that no absorbance change is observed (curve 2 of Fig. 6). A similar result has been obtained by Bouzou using luminol as an electron donor (14). Since conlpound I is a twoelectron acceptor, while guaiacol is a twoelectron donor, it seems reasonable t o assume that the transfer of the two oxidizing equivalents of compound I is effected on a single guaiacol inole without a free radical dismutation (24, 25). As will be seen in the discussion, this point is very important. Discussion The classical Chance-George (1-10) mechanism corresponds to the following reactions:
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CAN. J. BIOCHEM. VOL. 53, 1975
Time [min]
FIG.5. Titration of compound I of horseradish peroxidase P2 by guaiacol. A. Curve a shows the kinetics of the ompound I formation followed by its reduction by guaiacoI. Absorbance changes are recorded at 410 nm (isoskstic paint fferriperoxidase- compound 11). Compound I is formed by injection of 27.8 X 10-3 rmol sf H202 (arrow 1) into t k same amount of petoxidax. The positive absorbance increments ob:ained upon successive additions of 2.32 x 10-3 pmol of guaiacol (mows 2) correspond to the reduction of compound I by both the guaiacol and the endogenous &nor, Curve b r e p m s the spontaneous reduction of compound 1 by the endogenous donor. B. Percentage of corrmpou~.ctI reduced by both the guaiacsl and the endogenous donor (curve a) and by only the guaiacol (curve la). The final volume of the reaction d u r n is 3 ml. The pH is 7 (0.01 M phosphate bufler). The antifreeze solvent is methand - aqueous buffer (volume ratis 543-50). The temperature is - 50 "C.
PI
E+HzOz+CpdI accounts for the stoichiornetry of the oxidation of single-electron donors by hydrogen peroxide Cpd I +-AH2 4 Cpd I1 f AH [2]! in the presence of perpxidase, in which only one PI. CpdI'I A)-IZ-+E + A M electron of the doppr is exchanged with the where E, Cpd 1, and Cpd I1 are ferriperoxidase, peroxidase. The overall reaction cpmpound I, and cornpound IT, respectively, andi.FM2is a one-electron donor. This scheme [4] H 2 0 2 2AH2-+ 2H20 2AH
+
+
,
is +
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SANTIMONE: GUAIACOL. OXIDATION
0.03 pmol ferrecyanide
FIG.6. Reaction of peroxidase compound I with guaiacol at low terflgerature. Equimolar amounts (0.03 #md)of peroxidase and H z 0 2are mixed in the antifreeze solvent at - 50 "C. The absorbance changes are recorded at 397 i m (isoskstic point compound I - compound 11). The maximum a A value corresponds to the transformlion of fmigeroxidase into compound I. After several minutes (arrows), 0.03 pmol of either guaiacol (curve I ) or ferrscyzuri& (curve 2) are introduced into the reaction medium. For the one-electron donor (ferrocyanide), no spectral change is detected at 397 nm. This is due to the accumulation of compound 11. When guaiacol is added, the return to fmiperoxidase is observed (curve 2). The final volumes of the reaction media are 3 mI each. The pH is 7 (0.81 M phosphate buffer). The antifreeze solvent is methanol - aqueous buffer (volume ratio 50-58).
George (8), and Yamazaki and Souzu (25) have suggested that free radicals (AH') would be formed from substrates as intermediates in peroxidatic oxidation, and one of the possibilities of their disappearance is a dismutation reaction : In this case, the overall process of the oxidation of a two-electron donor by hydrogen peroxide would be the sum of the two following reactions: [ti] H202+2AH2-i2H2O+2AHS
+
171 2AH' -+ AH2 A Thus, titration results obtained with a catalytic amount of peroxidase (Figs. 1-3) could be interpreted by the one-electron exchange between compound I and compound 11, and this electron donor through the Chance-George mechanism (Eqs. 1-3), followed by the radical dismutation (Eq. 5). The two-electron transfer between one molecule of compound I and one molecule of guaiacol giving ferriperoxidase (Fig. 6) could be explained by the same reactions.
On the other hand, the radical dismutation cannot account for the fact that compound I is fully reduced to compound I1 by half a mole of guaiacol (Figs. 4 and 5). As previously reported (1 1, 12), when a two-electron donor is oxidized by compound I. it is more likely that the two oxidizing equivalents of compound I are transferred to a single mole of the two-electron danm rather than separately transferred on two distiw moles of donor. This may not apply to the guaiacol reaction, however. According to Yarnszaki and Piette (261, the free radicals formed in the enzymic oxidation seem to be slow substrata for the enzymes, decaying principally by dismutation or dimerization. However, to take into account the results of the reducing titration of compound I to compound 11 at low temperature (Fig. 51, it is possible that free radicctls formed reduce compound I, at least in the case of guaiacol. The two electrons sf guaiacol are thus transferred on compound 1 by the following proposed mechanism, suggested by the ChanceGeorge mechanism (1- 10).
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656
[$I PI
CAN. 4.
BIOCHEM. VQE. 53,
1945
+ AH2 Cpd 11 + AH' Cpd 1 + AH' >- Cpd 11 + A
in this case. On the other hand, when 2 mol of guaiacol are added, the 2 11101of the compound I1 formed with 1 mol of gaiaiacol react with the Overall, only 0.5 mol of guaiacol is necessary for two reducing equivalents of the second nlole of the reduction of 1 moI of compound I to com- guaiacol, giving 2 mol of ferriperoxidase through pound HI. The same mechanism can be applied the reactions of Eqs. 10 and 1I . The comparison for the reduction of compound II to ferric of Figs. 6 and 5 clearly shows that the overall compound I reduction to ferriperoxidase (Fig. 6) peroxidase. as slower than the transition of compound I to cornpound HI (Fig. 5). The two-phased kinetics of the compound I reduction to ferriperoxidase, Cpd I1 AH' + E A 11 predicted by the above considerations, cannot be Thus when 0.5 naol of ganaiacol is added to 1 mol seen in Fig. 6 because, at the wavelength used of compound I, compound I1 is formed quanti- (397 nm), the transition of conzpound I to comtatively; when 1 11101 of guaiacol is added, ferri- pound I1 cannot be observed. peroxidase is formed. Though the stoichion~etry Although in the proposed reaction scheme of the proposed mechaizisin is difTere~atfrona that (Eqs. 1, 9, 10, 12) two electrons are transferred of the Chance-George mechanism (1-1 01, these Bi0n.a the gbiaiacol molecule to the enzyme, it two nzschanisms are not inconsistent, since in should be noted that this scheme is probably both cases the reduction of the electron donor over-sinaplified. Indeed, little is known about the AH2 involves the participation of compound J number of internzediate steps and the reversibiiity and compound HI. When peroxidase is obtained of these reactions. For instance, it is possible from compound I through reactions of Eqs. 8 that the radical reactions of Eqs. 9 and/or 11 and 11, the free radical AH' may be released from might be mediated by the reaction of Eq. 13. the compound TI - radical intermediate complex followed by the reaction of Eqs. 84 or 15: which must be formed (27). Then, this radical AH' 0 2 + 0 2 A binds to compound 11, forming again a com- U33 pound I1 - radical intermediate complex before e>2> Cpd B + 0 2 Cpd t l giving ferriperoxidase. These reactions can prob- 1141 0 2 " Cpd II - 3O 2 E ably be written as follows: [I51 Cpd I
+
+
+
+
+ +
el21 Cpd 1
+ AH2
?:-
Cpd 11-AH t .Cpd I %
.k
+ AH'
E + A However, the direct formation of ferriperoxidase in Eq. 1%is incompatible with the observation of quantitative compound II formation when 0.5 mol of guaiacol is added to 1 mol of compound I. This incompatibility can be explained when one remembers that the transition of compouizd B to conzpound HI is much more rapid than the transition of compound HI to ferriperoxidase (28, 29). Therefore, when I rnol of guaiacol is .added to 2 mol of compound 1, one reducing equivalent of guaiacol is necessarily utilized by 1 mol of compound I through the reaction of Eq. 8, which is rapid. Then, the second reducing equivalent, which has the possibility to react through the reaction of Eqs. 9 or B B , proceeds through the reaction of Eq. 9, which is more rapid. Thus 2 rnol of compound 11 are obtained
+
+
+
Therefore, the sum of the reaction of Eqs. 13 and 14 or 15 is the reaction of Eqs. 9 or % 1. It is noteworthy that, in contrast to the mechanisms previously proposed for other two-electron donors (11-19, the oxidation of guaiacol occurs with the intermediate formation of compound II. Probably the most obvious conclusion that can be drawn from the present results is that the reaction mechanism of the oxidation process is not unique, as was thought at first, but largely dependent on the nature of the electron donor. We wish to express our gratitude to Professor J. Rimrd, in whose laboratory this work was carried out. We thank Professor H. B. Dunford for carefully reading the manuscript and for helpful criticism. We are grateful to Dr. P. Penon for stimulating discussions. The skilful technical assistance of Mrs. M. Wsudstra is also gratefully acknowledged. 1. 2. 3. 4.
Chance, B. (1940) Science 92,455-46 1 Chance, B. (1943) J . B i d . Chew. 151, 553-577 Chance, B. ( 1 949) Arcla. Biochern. 22, 224-252 Chance, B. (1949) Arch. Biocbtern. 21, 416-430
SANTIMONE: GUAIACBL BXIDAT ION
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5. Chance, B. (1951) Adv. Enzymol. 12, 153-190 6 . Chance, B. (1949) Arch. BiocBaem. 24, 389-409 7. George, P. (1952) Adv. Gaful. 4, 367-428 8. George, P. (1953) BiocBrem. J. 54, 267-276 9. George, P. (1953) Biochem. J. 55, 228-230 10. George, P. (1952) Biocinem. J. 52, 51 1-517 11. Roman, R. & Dunford, H. B. (1973) Carl. J. Ghem. 51, 588-596 12. Roman, R. & Bunford, H. B. (1972) Bioclremisbq. 11, 2076-2082 13. Thomas, J. A., Morris, D. R. & Hagger, L. P. (1970) J . Biol. Chem. 245, 3 135-3 142 14. Santimone, M. (1973) Thkse, Universitk d'AixMarseille 11, Centre de Luminy, France 15. Shannon, L. M.. Kay, E. & Lew, J. Y. (1966) J . Biol. Chem. 241, 2166-2172 16. Mazza, G., Charles, Cl., Bouchet, M., Rimrd, J . & Reynaud, J. (1968) Biochim. Biopitys. Acta 167, 89-98 17. Bouzou, P., Sireix, W. & Travers, F. (1970) Pro@. ~Vabl.Acad. Sci. U.S. 66, 787-792 18. Ricard, J. & Job, D. (1974) Eur. J. Biochem. 44, 359-374
657
19. I.lW.B. Scientijc Subroutine P ~ c A N ~360 P ,A. C M . 03X Version 111. Programmer's Manual, pp. 181-183 20. Bertrand, C, R. (1903) C . R . Acad. Sci. Paris 137, 3 269-1 272 31. Both, H. & Saunders. B. C. (1956) J. CBlem. SOC. (part X), 940-948 22. Santimone, M. & Dou, PI. J. M. (1974) C . R . Acad. Sci. Puris 278, (series C), 1469-1471 23. Santimone, M. (1974) C . R . Acad. Sci. Puris 279, (series D), 951-954 24. Yamazaki, I., Mason, H. S. & Piette, L. M. (1960) J . Biol. C i ~ e m235, . 2444-2452 25. Yamazaki, I. & Souzu, H. (1960) Arch. Biocioent. Biopktys. 86, 294-381 26. Yamazaki, I. & Piette. E. H. (1961) Biochim. Biopizys. Actn 50, 62-69 27. Hosoya, T. (1960) J. Biochem. Tokj'e, 47, 794-803 28. Chance, B. (1952) Arch. Biochem. Biophys. 44. 40441 1 29. Yonetani, T. (1966) J . Biol. Ckem. 241, 2562-257 1
Canadian Journal Journal canadien de biochirnie of Biochemistry Published by THENATIONAL RESEARCH COUNCIL OF CANADA
P ~ t b l i kpar LE CONSEIL NA'I'IONAL
Vol~ln~e 53
Volur~te53
N ~ ~ l n b e6 r June 1975
DE RECKERCHES DU
CANADA
numCro 6 juin 1975
Titration Study of Guaiacol Oxidation by Horseradish Peroxidase
Received October 17, 1974 Santimone, ha. (1975) Titration Study of Guaiacol Oxidation by Horseradish Peroxidahe. Ctul. 9. Bioclzent. 53, 649-657 Titration of guaiacol by hydrogen peroxide in the presence of a catalytic amount of horseradish peroxidase shows that the reduction of hydrogen peroxide proceeds by the abstraction of two electrons from a guaiacol molecule. In the same way, it can be demonstrated that 8.5 mol of guaiacol can reduce, at low temperature, 1 mol of peroxidase compound I to compound 11. Moreover, the reaction between equal amounts of compound I and guaiacol at low temperature produces the native enzyme. A reaction scheme is proposed which postulates that two electrons are transferred from guaiacol to compound I giving ferriperoxidase and oxidized guaiacol with the intermediary formation of compound 11. The direct two-electron transfer from guaiacol to compound I witl~duta dismutation of product free radicals must be considered as an exception to the general mechanism involving a single-electron transfer. Santimone, hl. (1975) Titration Study of Guaiacol Oxidation by Horseradish Peroxidase. Cart. 9. Biochern. 53, 649-657
Le titrage du guaiacol par le peroxyde d'hydrogkne en prksence d'une quantitC catalytique de peroxydase du raifort montre que-la rCduction du peroxyde d'hydrogkne se fait par abstraction de deux Clectrons de la molkule de gua'iacol. De la mCme f a ~ o nil, peut Ctre dCmontrC qu'une demi-molCcule de guai'acol peut rkduire, a basse tempkrature, une molCcule du compost2 peroxydasique I en composd II. De plus, la reaction entre des quantitks Cgales de csmposC I et de guaiacol B basse tempdrature produit l'enzyme native. Le schCma rdactionnel proposC postule que deux Clectrons sont transfkres du guaiacol au composC I donnant la ferriperoxydase et le guaiacol oxydC avec formation intermkdiaire du compose 11. Le transfert direct de deux electrons du guaiacolau composC I sans une dismutation des radicaux libres du produit doit Ctre considdrk comme une exception au mdcanisme gknCral impliquant le transfert d'un seul electron. [Traduit par le journal]
Introdhetion It is currently believed that most of the oxidation processes involving the electron exchange between an electron donor and the peroxideperoxidase system occur through the classical mechanism of Chance (1-6) and George (7-10). The first step in this mechanism involves the transfer of the two oxidizing equivalents of H 2 0 2to the ferric enzyme. The compound I thus obtained then exchanges one oxidizing equivalent with 1 mol of the electron donor. This oxidoreduction reaction leads to compound I1 and to
the oxidized donor. Compound 11 then reacts with a second mole of donor and is reduced to ferric peroxidase. Thus in this classical reaction scheme, two molecules of an electron donor are needed to reduce one molecule of compound H to ferriperoxidase i.e., only one of the two electrons of a molecule of the donor is exchanged with peroxidase. Various experimental results have shown that peroxidases can catalyze the oxidation of two electron donors such as sulfite (Il), iodide (12), and ethanol (13). The oxidation of these donor
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650
CAN. J.
BIOCHEM.
n~oleculesoccurs with a two-electron transfer in one step and does not involve compound 11. Moreover, the stoichiometry observed for the reaction of H z 0 2 or con~pound H and these donors is 1:l. 'The direct two-electron transfer fr~m these substrates to compound I, thus forming peroxidase without the intermediate formation of compound 11, is generally considered as an exception to the mechanism of ChanceG e ~ r g e(1-10). It is of interest to determine whether other two-electron donors could be oxidized by peroxidase in a two-electron process, either with the intermediate formation of compound HH or not. Since guaiacol, as we shall demonstrate, is a two-electron donor, this substrate has been chosen in the present study. Materials and Methods The horseradish peroxidase isoenzyme used in the present work, designated P2 (14), is very similar to isoenzyme C of horseradish peroxidase previously described by Shannon et al. (15). It is isolated and purified from the commercial enzyme preparation obtained from Fluka. The purification method (14) was derived from that of Maaaa st al. (16). The hemoprotein thus obtained was homogeneous on analytical centrifugation and electrophoresis. Its Reinheitsaahl (R.2.)was close to 3. Some sf the physico-chemical characteristics of this enzyme preparation have already been reported 4 14). The kinetics of compound I reduction was recorded with a Spectralux 1800 spectrophotometer equipped with a Sefram recorder. This apparatus was modified so that it was possible to inject rapidly a reagent into the medium and record the absorbance change at a fixed wavelength. Mixing was effected by a mechanical stirrer. The stirring and injection apparatus was positioned outside the path of the light beam. The equipment for low-temperature spectrophotometry was derived from that designed by Douzou (17). Its characteristics have already been described (18). The antifreeze solvent was methanol - aqueous phosphate buffer, rased in a 58-50 volume ratio. Denaturation of the protein during the preparation of the solutions at low temperature was avoided by an appropriate balance between the temperature and the volume ratio of the solvent. The theoretical titration curves of an electron donor by hydrogen peroxide were obtained by computer (Siris 10070) sin~ulation.Depending on the number of transferred electrons and the degree of polymerization of the product, one has to solve equations of the second, third, fourth, sixth, and eighth degrees. This has been done with the so-called Polrt (19) Fortran program.
Results It is well known, that during the oxidation of guaiacol, the enzymatic reaction is followed by a
VOL. 53. 1975
non-enzymatic polymerization process leading to a brown product, P. The structure of this product is still unknown. The polymerization process is certainly rapid with regard to the enzymatic reaction, for the overall rate is proportional to the peroxidase concentration, at least for a Barge range of substrate concentratior~s. The titration curve of guaiacol oxidation by hydrogen peroxide with a catalytic concentration of peroxidase (Fig. 1) shows a change of slope for a 1:1 ratio of peroxide to guaiacol. This result could be interpreted by a 1:1 stoichiometry in the overall process. However, deviation from the strict titration curve is observed, which could be attributed to an eEect of reversibility, to the nature of the reaction product, or to secondary reactions competing for hydrogen peroxide. This observed deviation casts doubt on the conclusion that the stoichiometry of the reaction is 1:l. Therefore, theoretical titration curves have been simulated assuming that either a one-electron donor (Fig. 2) or a two-electron donor (Fig. 3) is oxidized by hydrogen peroxide. Moreover, calculations were effected with differeizt equilibrium constant values supposing that the reaction product is a monomer (scheme A of Figs. 2 and 3), a dimer (scheme B of Figs. 2 and 3), or a tetrainer (scheme C of Figs. 2 and 3). The curves obtained clearly indicate that, for an equilibrium constant not lower than 100, a change of slope is observed that indicates the number of transferred electrons. When the change of slope is observed for a 1:2 ratio of peroxide to donor. this implies a one-electron
FIG. 1. Titration of guaiacol by hydrogen peroxide in the presence of horseradish peroxidase P2. Absorbance changes are plotted against the ratio [I-1202]j[guaiacoBl. The concentrations of peroxidase and of guaiacol are 0.94 X M and 1.56 X 10-8 M, respectively. The pH is 7.0 (0.2 M phosphate buffer).
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SANTIMONE : GUAIACOL OXIDATION
65 1
FIG.2. Theoretical titration curves of a one-electron donor, AH2, by hydrogen peroxide. The oxidation product is supposed to be either a monomer (A), a dimer (B), or a tetramer (C).The overall equations corresponding to the various situations are presented in the same figure. The initial concentrations of the donor are supposed to be either 1 (A), 2 (B), or 4 (C) in arbitrary units. Curves are computed for different values of the equilibrium constant K. Concentrations of the product are given in arbitrary units.
oxidation of the donor molecule. When it is obtained for a 1 :1 ratio, then two electrons are abstracted from the same donor molecule. It is noteworthy that this conclusion can be drawn whatever the reason for the deviation observed on the theoretical titration curves. Then, the titration curve of guaiacol oxidation by hydrogen peroxide (Fig. 1) is only compatible with the
view that an exchange of two electrons occurs between one molecule of guaiacol and PI2O2. Indeed, a change of slope can be observed for a 1 :1 ratio of peroxide to guaiacol. Though this conclusion seems unquestionable, it was interesting to obtain information about the molecular weight of the reaction product. The brown product, B, of guaiacol oxidation was
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652
CAN. 9 . BIOCHEM. VBPL. 53, 1975
FIG.3. Theoretical titration curves of a two-electron donor, AMs, by hydrogen peroxide. The oxidation product is again supposed to be either a monomer (A), a darner (B), or a tetramer (C). The overall equations corresponding to the various situations are presented in the same figure. The initial concentrations of the donor are supposed to be either 1 (A), 2 dB), or 4 ( C ) in arbitrary units. Curves are computed for different values of the equilibrium constant K . Concentrations of the product are given in arbitrary units.
thought at first to be tetraguaiacol(20), then dihydroxydimethoxydipheny1(2 I). As was pointed out by Saunders (21), the structure of tetraguaiacol cannot account for the visible spectrum of P. Recently, the n~olecular weight of this product has been determined by mass speetrometry (22) and the value obtained was 458, thus obviously incompatible with the view that guaiacol has been oxidized into dihydroxydi-
~netboxydiphenyl.However, in the light, P is slowly reduced to a new product (23) with a molecular weight of 246 and that seenas very likely to be dihydroxydimethoxydiphenyl (2 1). Since the mcsleeular weight of guaiacol and of the main reaction product are 124 and 458, respectively, it appears that the non-enzymatic polymerization process leads to the formation of a tetramer and that. during this reaction, part of
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SANTIMONE: GUAIACOL OXIDATION
I
Compound H /
I
I
FIG.4. Spectra of peroxidase, compound 1, and compound II in the Soret and in the visible regions. Compound 1 is obtained by the addition of 26.6 X 10-3 pmol of H202to the same amount of horseradish peroxidase P2. Compound E l is obtained by the addition of 13.3 X ctmolof guaiacol. The final volume of the reaction medium is 3 ml. The pH is 7 (8.08 IM phosphate buffer). The antifreeze solvent is methanol - aqueous buffer (volume ratio 58-50). The temperature is -58 "C.
the oxidized guaiacol molecule has been lost. That guaiacol is a two-electron donor is verified in Fig. 4, which represents the spectrum in the Soret and in the visible regions of compound I1 obtained by the addition of 0.5 mol of guaiacol to B mol of compound I. It is also possible to confirm the fact that guaiacol is a two-electron donor molecule. by a titration of compound I at low temperature. At -50 "C the rate of spontaneous reduction of compound I to compound IT is relatively slow and can be recorded by an increase in absorbance at 410 nm, which is the isosbestic point between peroxidase and compound I1 (curve b of Fig. 5A). However, when small amounts of guaiacol are introduced, into the medium, the transformation t o coimpound 11 is much more rapid (curve a of Fig. 5A). The absorbance changes measured upon successive additions of guaiacol allow one to determine the percentage of compound I reduced. The exact titration curve (curve b of Fig. 5B) is calculated subtracting spontaneous reduction of this compound to the total reduction (curve a of Fig. 5B). It is very important t o note that the slope of the straight line obtained shows that I mol of compound I is fully reduced t o compound I1 by only 0.5 mol of guaiacol. Since the transition from
compound 1 to compound Il needs one reducing equivalent, this confirms the idea that guaiacol is a two-electron donor nlolecule. The titration of compound I by an equimolar amount of guaiacol, giving rise to the appearance of ferriperoxidase, can be followed (curve I of Fig. 6) at -58 "C and 394 nm. which is the isosbestic point of compound I and compound II. Under the same experimental conditions, mixing of compound 1 and a one-electron donor, such as ferrocyanide, in equimolar concentrations, must give rise to the compound TI and not to the ferriperoxidase, so that no absorbance change is observed (curve 2 of Fig. 6). A similar result has been obtained by Bouzou using luminol as an electron donor (14). Since conlpound I is a twoelectron acceptor, while guaiacol is a twoelectron donor, it seems reasonable t o assume that the transfer of the two oxidizing equivalents of compound I is effected on a single guaiacol inole without a free radical dismutation (24, 25). As will be seen in the discussion, this point is very important. Discussion The classical Chance-George (1-10) mechanism corresponds to the following reactions:
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CAN. J. BIOCHEM. VOL. 53, 1975
Time [min]
FIG.5. Titration of compound I of horseradish peroxidase P2 by guaiacol. A. Curve a shows the kinetics of the ompound I formation followed by its reduction by guaiacoI. Absorbance changes are recorded at 410 nm (isoskstic paint fferriperoxidase- compound 11). Compound I is formed by injection of 27.8 X 10-3 rmol sf H202 (arrow 1) into t k same amount of petoxidax. The positive absorbance increments ob:ained upon successive additions of 2.32 x 10-3 pmol of guaiacol (mows 2) correspond to the reduction of compound I by both the guaiacol and the endogenous &nor, Curve b r e p m s the spontaneous reduction of compound 1 by the endogenous donor. B. Percentage of corrmpou~.ctI reduced by both the guaiacsl and the endogenous donor (curve a) and by only the guaiacol (curve la). The final volume of the reaction d u r n is 3 ml. The pH is 7 (0.01 M phosphate bufler). The antifreeze solvent is methand - aqueous buffer (volume ratis 543-50). The temperature is - 50 "C.
PI
E+HzOz+CpdI accounts for the stoichiornetry of the oxidation of single-electron donors by hydrogen peroxide Cpd I +-AH2 4 Cpd I1 f AH [2]! in the presence of perpxidase, in which only one PI. CpdI'I A)-IZ-+E + A M electron of the doppr is exchanged with the where E, Cpd 1, and Cpd I1 are ferriperoxidase, peroxidase. The overall reaction cpmpound I, and cornpound IT, respectively, andi.FM2is a one-electron donor. This scheme [4] H 2 0 2 2AH2-+ 2H20 2AH
+
+
,
is +
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SANTIMONE: GUAIACOL. OXIDATION
0.03 pmol ferrecyanide
FIG.6. Reaction of peroxidase compound I with guaiacol at low terflgerature. Equimolar amounts (0.03 #md)of peroxidase and H z 0 2are mixed in the antifreeze solvent at - 50 "C. The absorbance changes are recorded at 397 i m (isoskstic point compound I - compound 11). The maximum a A value corresponds to the transformlion of fmigeroxidase into compound I. After several minutes (arrows), 0.03 pmol of either guaiacol (curve I ) or ferrscyzuri& (curve 2) are introduced into the reaction medium. For the one-electron donor (ferrocyanide), no spectral change is detected at 397 nm. This is due to the accumulation of compound 11. When guaiacol is added, the return to fmiperoxidase is observed (curve 2). The final volumes of the reaction media are 3 mI each. The pH is 7 (0.81 M phosphate buffer). The antifreeze solvent is methanol - aqueous buffer (volume ratio 50-58).
George (8), and Yamazaki and Souzu (25) have suggested that free radicals (AH') would be formed from substrates as intermediates in peroxidatic oxidation, and one of the possibilities of their disappearance is a dismutation reaction : In this case, the overall process of the oxidation of a two-electron donor by hydrogen peroxide would be the sum of the two following reactions: [ti] H202+2AH2-i2H2O+2AHS
+
171 2AH' -+ AH2 A Thus, titration results obtained with a catalytic amount of peroxidase (Figs. 1-3) could be interpreted by the one-electron exchange between compound I and compound 11, and this electron donor through the Chance-George mechanism (Eqs. 1-3), followed by the radical dismutation (Eq. 5). The two-electron transfer between one molecule of compound I and one molecule of guaiacol giving ferriperoxidase (Fig. 6) could be explained by the same reactions.
On the other hand, the radical dismutation cannot account for the fact that compound I is fully reduced to compound I1 by half a mole of guaiacol (Figs. 4 and 5). As previously reported (1 1, 12), when a two-electron donor is oxidized by compound I. it is more likely that the two oxidizing equivalents of compound I are transferred to a single mole of the two-electron danm rather than separately transferred on two distiw moles of donor. This may not apply to the guaiacol reaction, however. According to Yarnszaki and Piette (261, the free radicals formed in the enzymic oxidation seem to be slow substrata for the enzymes, decaying principally by dismutation or dimerization. However, to take into account the results of the reducing titration of compound I to compound 11 at low temperature (Fig. 51, it is possible that free radicctls formed reduce compound I, at least in the case of guaiacol. The two electrons sf guaiacol are thus transferred on compound 1 by the following proposed mechanism, suggested by the ChanceGeorge mechanism (1- 10).
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656
[$I PI
CAN. 4.
BIOCHEM. VQE. 53,
1945
+ AH2 Cpd 11 + AH' Cpd 1 + AH' >- Cpd 11 + A
in this case. On the other hand, when 2 mol of guaiacol are added, the 2 11101of the compound I1 formed with 1 mol of gaiaiacol react with the Overall, only 0.5 mol of guaiacol is necessary for two reducing equivalents of the second nlole of the reduction of 1 moI of compound I to com- guaiacol, giving 2 mol of ferriperoxidase through pound HI. The same mechanism can be applied the reactions of Eqs. 10 and 1I . The comparison for the reduction of compound II to ferric of Figs. 6 and 5 clearly shows that the overall compound I reduction to ferriperoxidase (Fig. 6) peroxidase. as slower than the transition of compound I to cornpound HI (Fig. 5). The two-phased kinetics of the compound I reduction to ferriperoxidase, Cpd I1 AH' + E A 11 predicted by the above considerations, cannot be Thus when 0.5 naol of ganaiacol is added to 1 mol seen in Fig. 6 because, at the wavelength used of compound I, compound I1 is formed quanti- (397 nm), the transition of conzpound I to comtatively; when 1 11101 of guaiacol is added, ferri- pound I1 cannot be observed. peroxidase is formed. Though the stoichion~etry Although in the proposed reaction scheme of the proposed mechaizisin is difTere~atfrona that (Eqs. 1, 9, 10, 12) two electrons are transferred of the Chance-George mechanism (1-1 01, these Bi0n.a the gbiaiacol molecule to the enzyme, it two nzschanisms are not inconsistent, since in should be noted that this scheme is probably both cases the reduction of the electron donor over-sinaplified. Indeed, little is known about the AH2 involves the participation of compound J number of internzediate steps and the reversibiiity and compound HI. When peroxidase is obtained of these reactions. For instance, it is possible from compound I through reactions of Eqs. 8 that the radical reactions of Eqs. 9 and/or 11 and 11, the free radical AH' may be released from might be mediated by the reaction of Eq. 13. the compound TI - radical intermediate complex followed by the reaction of Eqs. 84 or 15: which must be formed (27). Then, this radical AH' 0 2 + 0 2 A binds to compound 11, forming again a com- U33 pound I1 - radical intermediate complex before e>2> Cpd B + 0 2 Cpd t l giving ferriperoxidase. These reactions can prob- 1141 0 2 " Cpd II - 3O 2 E ably be written as follows: [I51 Cpd I
+
+
+
+
+ +
el21 Cpd 1
+ AH2
?:-
Cpd 11-AH t .Cpd I %
.k
+ AH'
E + A However, the direct formation of ferriperoxidase in Eq. 1%is incompatible with the observation of quantitative compound II formation when 0.5 mol of guaiacol is added to 1 mol of compound I. This incompatibility can be explained when one remembers that the transition of compouizd B to conzpound HI is much more rapid than the transition of compound HI to ferriperoxidase (28, 29). Therefore, when I rnol of guaiacol is .added to 2 mol of compound 1, one reducing equivalent of guaiacol is necessarily utilized by 1 mol of compound I through the reaction of Eq. 8, which is rapid. Then, the second reducing equivalent, which has the possibility to react through the reaction of Eqs. 9 or B B , proceeds through the reaction of Eq. 9, which is more rapid. Thus 2 rnol of compound 11 are obtained
+
+
+
Therefore, the sum of the reaction of Eqs. 13 and 14 or 15 is the reaction of Eqs. 9 or % 1. It is noteworthy that, in contrast to the mechanisms previously proposed for other two-electron donors (11-19, the oxidation of guaiacol occurs with the intermediate formation of compound II. Probably the most obvious conclusion that can be drawn from the present results is that the reaction mechanism of the oxidation process is not unique, as was thought at first, but largely dependent on the nature of the electron donor. We wish to express our gratitude to Professor J. Rimrd, in whose laboratory this work was carried out. We thank Professor H. B. Dunford for carefully reading the manuscript and for helpful criticism. We are grateful to Dr. P. Penon for stimulating discussions. The skilful technical assistance of Mrs. M. Wsudstra is also gratefully acknowledged. 1. 2. 3. 4.
Chance, B. (1940) Science 92,455-46 1 Chance, B. (1943) J . B i d . Chew. 151, 553-577 Chance, B. ( 1 949) Arcla. Biochern. 22, 224-252 Chance, B. (1949) Arch. Biocbtern. 21, 416-430
SANTIMONE: GUAIACBL BXIDAT ION
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5. Chance, B. (1951) Adv. Enzymol. 12, 153-190 6 . Chance, B. (1949) Arch. BiocBaem. 24, 389-409 7. George, P. (1952) Adv. Gaful. 4, 367-428 8. George, P. (1953) BiocBrem. J. 54, 267-276 9. George, P. (1953) Biochem. J. 55, 228-230 10. George, P. (1952) Biocinem. J. 52, 51 1-517 11. Roman, R. & Dunford, H. B. (1973) Carl. J. Ghem. 51, 588-596 12. Roman, R. & Bunford, H. B. (1972) Bioclremisbq. 11, 2076-2082 13. Thomas, J. A., Morris, D. R. & Hagger, L. P. (1970) J . Biol. Chem. 245, 3 135-3 142 14. Santimone, M. (1973) Thkse, Universitk d'AixMarseille 11, Centre de Luminy, France 15. Shannon, L. M.. Kay, E. & Lew, J. Y. (1966) J . Biol. Chem. 241, 2166-2172 16. Mazza, G., Charles, Cl., Bouchet, M., Rimrd, J . & Reynaud, J. (1968) Biochim. Biopitys. Acta 167, 89-98 17. Bouzou, P., Sireix, W. & Travers, F. (1970) Pro@. ~Vabl.Acad. Sci. U.S. 66, 787-792 18. Ricard, J. & Job, D. (1974) Eur. J. Biochem. 44, 359-374
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19. I.lW.B. Scientijc Subroutine P ~ c A N ~360 P ,A. C M . 03X Version 111. Programmer's Manual, pp. 181-183 20. Bertrand, C, R. (1903) C . R . Acad. Sci. Paris 137, 3 269-1 272 31. Both, H. & Saunders. B. C. (1956) J. CBlem. SOC. (part X), 940-948 22. Santimone, M. & Dou, PI. J. M. (1974) C . R . Acad. Sci. Puris 278, (series C), 1469-1471 23. Santimone, M. (1974) C . R . Acad. Sci. Puris 279, (series D), 951-954 24. Yamazaki, I., Mason, H. S. & Piette, L. M. (1960) J . Biol. C i ~ e m235, . 2444-2452 25. Yamazaki, I. & Souzu, H. (1960) Arch. Biocioent. Biopktys. 86, 294-381 26. Yamazaki, I. & Piette. E. H. (1961) Biochim. Biopizys. Actn 50, 62-69 27. Hosoya, T. (1960) J. Biochem. Tokj'e, 47, 794-803 28. Chance, B. (1952) Arch. Biochem. Biophys. 44. 40441 1 29. Yonetani, T. (1966) J . Biol. Ckem. 241, 2562-257 1
