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The mechanism of indoleacetic acid oxidase reaction catalyzed by turnip peroxidase
ARCHIVES
OF
BIOCHEMISTRY
The Mechanism
AND
88, 294-301 (1960)
of Indoleacetic Acid Oxidase by Turnip Peroxidase ISA0
From the Biological
BIOPHYSICS
YAMAZAKI
Institute,
Faculty
AND
HIROSHI
of Science,
Received
University
Reaction
Catalyzed
SOUZU’ of Tohoku,
Sendai,
Japan
June 18, 1959
In the iron-reducing activity of peroxidase in the presence of indoleacetic acid as electron donor, the kinetic and stoichiometric evidence supports a free-radical mechanism in which peroxidase catalyzes the formation of 2 moles indoleacetic acid radicals at the expense of 2 moles indoleacetic acid and 1 mole hydrogen peroxide. Inability of peroxidase to catalyze peroxidation of indoleacetic acid under anaerobic conditions is attributed to inactivating action of free radicals upon the enzyme, which is prevented bv the addition of some oxidants. The effects of carbon monoxide and Mn2+ were also examined. INTRODUCTION
The presence, in the extracts of certain plant tissues or fungi, of systems which oxidize IAA2 has been reported by many workers (l-5), and it is now well established that peroxidase itself catalyzes oxygen-consuming oxidation of IAA (6-8). The aerobic oxidase activity of peroxidase has also been observed in the presence of other substrates, such as dihydroxyfumarate (9, lo), phenylacetaldehyde (11), triose reductone (12), and reduced pyridine nucleotides (13). Recently, we have found that methylene blue, cytochrome c, and inorganic iron serve as hydrogen acceptor instead of molecular oxygen in the oxidase reaction catalyzed by peroxidase, provided that a suitable amount of hydrogen peroxide is added (14,15). These H acceptors become reduced when the peroxidase substrate is acted on by peroxidase anaerobically. The present work was undertaken to investigate the mechanism of the
oxidase activity of peroxidase in the presence of IAA when inorganic iron is added as electron acceptor. EXPERIMENTAL Recrystallized TP, with an extinction ratio of the exEm/Em = 2.7-3.1, was used throughout periments. TP concentration was calculated assuming that the value of the molecular extinction of TP is 90 cm.+ mmole-i at 403 rnp. Cytochrome c was prepared from beef heart muscle and purified with ion-exchange resin XE-64. Spectrophotometric measurements were performed with a Beckman model DU or a Hitachi spectrophotometer. The concentration of ferrous ion was determined from the extinction coefficient of ferrous o-phenanthroline complex of 11.3 cm.-’ mmole-i at 510 m/l (16). IAA concentration was determined using the Salkowski color reaction (23). Anaerobic conditions were obtained by gassing the cuvettes 10 min. with N? which had oxygen removed by repeated passage through alkaline solutions of pyrogallol and further over heated copper. RESULTS
1 Present address: The Institute of Low Temperature, University of Hokkaido, Sapporo, Japan. 2 The following abbreviations will be used: TP, turnip peroxidase; IAA, indoleacetic acid; IAA’, free radical of IAA (one equivalent oxidized form of IAA); IAA”, two equivalents oxidized form of IAA.
In previous work (15) on the reactions catalyzed by peroxidase, we have classified peroxidase substrates into two groups: (a) redogenic substrates, including triose reductone, dihydroxyfumarate, ascorbate, hydroquinone, pyrogallol and IAA; and (b) oxi294
INDOLEACETIC
ACID
dogenic substrates, including p- and m-cresol,
guaiacol, resorcinol, m-phenylenediamine, aniline, phenol,, and uric acid. The redogenic substrates are characterized by their capacity to reduce ferricytochrome c in the presence of hydrogen peroxide and peroxidase, a property not possessedby the oxidegenti group. Figures 1 and 2 illustrate the reduction of cytochrome c and inorganic iron catalyzed by peroxidase in the presence of IAA, which belongs to the redogenic group. These reductions. under anaerobic conditions, were observed immediately after hydrogen peroxide was added. Non-enzymic reduction of iron by the added hydrogen peroxide, which was demonstrated by Kuhn and Wasserman (17), was negligible under these experimental conditions. The maximal rate of the red.uction of iron and cytochrome c was obtained at about pH 4.2-4.6. Cytochrome c reduction occurred only when freshly prepared IAA solution was used; the resulting ferrocytochrome c was reoxidized slowly after a considerable amount of cytochrome c had been reduced. The same subsequent slow reoxidation of cytochrome c was previously observed when hydroquinone was em.ployed instead of IAA under these experimental conditions (15). In order to clarify the mechanism, it is helpful first to examine the stoichiometry of
(O.05 + 0
0L I
1 .I5
E z .I0 G d .05
d 0
0
I
2
3 Min.
4
5
FIG. 2. Reduction of iron under anaerobic conditions. Hz02 was added to the reaction mixture 1 min. after IAA addition; 50 pmoles IAA; 10 pmoles HzOz ; 0.1 mmole ferric chloride; 0.3 mmole o-phenanthroline; 0.02 rmole TP; 0.02 M acetate; pH 4.80; temperature 22”. A: 10 rmoles p-cresol. B: control. C: 10 pmoles Mn2+. D: TP-free.
IAA, pM Wz 2 PM 3. Stoichiometry of iron reduction; 0.1 pmole TP; 0.1 mmole ferric chloride; 0.3 mmole o-phenanthroline; 0.02 M acetate; pH 4.80; temperature 22’; anaerobic conditions. A: Dependence of the amount of reduced iron during the initial rapid reaction on the Hz02 concentration at constant IAA level (10 pmoles). B: Dependence of the amount of reduced iron during the initial rapid reaction on the IAA concentration at constant Hz02 level (5 pmoles).
x E 0 lo
0
.20
FIG.
.10-
d d
205
OXIDASE
2 Min.
3
FIG. 1. Reduction of cytochrome c under anaerobic conditions. Hz02 was added to the reaction mixture 1 min. after IAA addition. 0.1 mmole IAA; 6 pmoles cytochrome c; 40 pmoles H&l2 ; 0.02 M acetate; pH 4.80; temperature 22”. A: 0.1 pmole TP. B: 0.1 amole TP and 0.1 mmole NW+. C: 0.04 amole TP. D: 0.01 pmole TP.
the reaction. As can be seen in Fig. 3, it was found that 2 moles IAA consumed 1 mole hydrogen peroxide to reduce 1.7 moles iron during the initial rapid reaction (Fig. 2). Unlike the case of methylene blue reduction (18)) the molar ratio of reduced iron to added hydrogen peroxide in the presence of a sufficient amount of IAA, did not vary greatly under different experimental conditions.
296
YAMAZAKI AND SOUZU REACTIONS KINETICS
In the presence of excess Fe3+, the rate of iron reduction expected from Eqs. (l)-(4) will be given by d Fe2+ v=dt =
l-120e or
IAA,
PM
FIG. 4. Dependence
of the initial rate of iron reduct,ion on the concentration of H202 and IAA (anaerobic conditions); 0.1 pmole TP; 0.1 mmole ferric chloride; 0.3 mmole o-phenanthroline; 0.02 M acetate; pH 4.80; temperature 22”. Concentration of HgOz (A) and IAA (B) are shown in abscissa at constant level of 10 pmoles IAA (A) and 5 pmoles Hz02 (B), respectively.
A free radical mechanism was proposed previously (15, 18) to explain the reduction of methylene blue, cytochrome c, and other H acceptors during anaerobic oxidation of redogenic substrates by peroxidase and Hz02. In the case of IAA oxidation, the following reactions may be suggested:
2O’P) 1 h(H,Od
+
where (TP) represents total concentration of turnip peroxidase. Figure 5 shows changes in absorption at 424 rnp, due to appearance of peroxidaseHz02 complexes. When Hz02 was added to the peroxidase, formation of complex I occurred nearly instantaneously. This was followed by gradual reduction of complex I to complex II, as shown by the increase in optical density. Addition of IAA to this solution caused immediate reduction of the remaining complex I to complex II, followed by slower conversion of complex II to free peroxidase, as indicated by the gradual decrease in absorption. This shows that kz (in the above equations) has a value considerably larger than ka , as Chance (19) has reported in the case of other hydrogen donors. The very rapid formation of peroxidase complex I suggests further that kl(H20z) i> k,(IAA). If this is so, Eq. (6) can then be rewritten 0 = Bk,(TP)(IAA)
TP + HzOz * Complex
I + IAA Complex IAA’
-%
Complex Complex
II + IAA *
+ Fe3+ 2
IAA”
I
(1)
II + IAA’
(2)
TP + IAA’
(3)
-t Fe*
(4)
It is seen that if the amount of Fe3+ is great enough to oxidize all the free radicals IAA’ as fast as they are formed, the over-all reaction expected would be 2IAA
+ Hz02 + 2Fe3+ --t BIAA”
+ 2Fe2+ + 2Hz0
The observed stoichiometry bly well with this.
(5)
agrees reasona-
(6)
(7)
As shown in Fig. 4, the initial rate of reduction of Fe3+ was found to be closely proportional to IAA concentration, but nearly independent of Hz02 concentration over the range investigated, in agreement with Eq. (7). Using Eq. (7), it is possible to evaluate Ic, from the initial rate of iron reduction (Table I). We can also evaluate k~ by direct observation of the reaction of Eq. (3), observed in Fig. 5. The second-order velocity constant, lc, , is easily computed from the course of reduction of complex II to free TP, and is shown in Table I, along with kg obtained from iron reduction. It is also seen in Fig. 5 and Table I that in the presence of ferricyanide, the rate of complex II reduction
INDOLEACETIC
became about double; this will be discussed below. If the mechanism of iron reduction is represented by the reaction sequence of Eqs. (l)-(4), IAA must become oxidized in the presence of TP and hydrogen peroxide, under anaerobic conditions. However, Ray (8) found that no appreciable peroxidation of IAA occurs under anaerobic conditions. The same negative: result was obtained in the presence of 0.1 pmole TP, which was used for the ordinary oxidase reaction (Table 11). Table II shows that consumption of hydrogen peroxide did occur in the presence of some oxidants, such as methylene blue and p-quinone. It also occurred in the presence of IAA alone if higher concentrations of peroxidase were employed. Inability of the lower concentrations of peroxidase to oxidize IAA in the presence of hydrogen peroxide, under anaerobic conditions, may be due to the inactivation of the enzyme during the peroxidase reaction. Such an inactivation is demonstrated by the experiment shown in Fig. 6, in which the determination of peroxidase activity was performed by the guaiacol test. This remarkable inactivation of the enzyme did not occur in the absence of either IAA or hydrogen peroxide and decreased at higher pH or in the presence of some oxidants. By following in parallel the disappearance of IAA, using the Salkowski reagent, and the changes in absorption at 424 rnp, it was observed that the reaction of IAA destruction showed an induction phase, which ended TABLE
I
ESTIMATION OF k3 BY Two ‘DIFFERENT METHODS /
ACID
297
OXIDASE
I
.
I
I
*
‘Jj’
0123457
.
.
8
9
min.
FIG. 5. Formation of peroxidase complex II, and reduction of complex II to free TP by addition of IAA. IAA was added 7 min. after mixing of TP and HzO*. 2.1 pmoles TP; 2 amoles HzOz ; 1 pmole IAA; 0.02 M acetate; pH 4.63; in anaerobic cell with 2-cm. light path at room temperature (13”). Black circles are data in the presence of 0.1 mmole ferricyanide (absorption of ferricyanide is deducted). TABLE
II OF IAA Conditions: 0.4 mmole IAA, 0.4 mmole hydrogen peroxide, 0.02 M acetate, pH 4.30, anaerobic conditions. PEROXIDATION
ElXyIlle concentration
Addition
Remaining hydrogen peroxide after 1 min.
FM 0.1 0.4 0.8 0.8a 0.1 0.1
2
min.
mmoles mm&s
No addition of IAA 10 rmoles methylene blue 40 rmoles p-quinone
0.40 0.33 0.14 0.41 0.27 0.17
a This test shows that there is no catalase tivity in the peroxidase sample.
0.40 0.32 0.11 0.41 0.25 0.10 ac-
Methods “C.
Reduction of iron (Fig. 4) Reduction of iron Reduction of complex II (Fig. 5) Reduction in the presence of 0.1 mmole ferricyanide (Fig. 5) a For the reason mentioned true value of ka is half this.
22 13 13
3.7 2.9 3.0
13
5.9”
in Discussion,
the
after an initial slight increase of absorption at 424 rnp had occurrred. The change in absorption at 424 rncLtook place in two phases. The initial slight increase, before IAA destruction became rapid, might be due to the formation of a peroxidase-hydrogen peroxide complex. The further increase, which occurred after IAA had disappeared, should be attributed to the conversion of the inter-
298
YAMAZAKI AND SOUZU
It exhibited an activating effect only under aerobic conditions without added hydrogen peroxide. A summary of Mn2+ effects on different peroxidase reaction is given in Table III. The addition of p-cresol, which is an activator for the oxidase activity of peroxidase, stimulated the rate of iron reduction but strongly inhibited the reduction of cytochrome c.~
1000
DISCUSSION
Min. FIG. 6. Inactivation of TP in the presence of IAA (14”); 0.3 ml. incubated solution was added to 9.7 ml. solution mixed with Hz02 and guaiacol at the time indicated in the abscissa to determine peroxidase activity. Incubation: 0.1 pmole TP; 0.2 mmole IAA; 0.2 mmole HzOz ; 0.02 M acetate; pH 4.63; anaerobic conditions. Determination of peroxidase activity: 0.4 mmole guaiacol; 1 mmole HzOz ; 0.02 M acetate; pH 4.63; anaerobic conditions.
mediate(s) of IAA oxidation to the final pink-brownish oxidation product(s) as reported by Ray (20). EFFECTS OF CO, Mn2+, AND PHENOLS
It was reported previously (18) that carbon monoxide does not inhibit the reduction of methylene blue catalyzed by TP in the presence of triose reductone and hydrogen peroxide. CO also did not inhibit the reactions catalyzed by TP in the presence of IAA; neither the reduction of iron, when the solution was gassed with CO instead of nitrogen, nor the aerobic oxidation of IAA, in a solution gassed with 80 % CO/ZO% O2 instead of air. Promotive effects of Mn2+ on the aerobic oxidase activity of peroxidase have been reported by many workers. A strong activating effect has been shown in the case of dihydroxyfumarate, reduced pyridine nucleotide, and IAA, and a slight one in the case of triose reductone. On the contrary, the reduction of iron, cytochrome c, and dyes by the peroxidase systems under anaerobic conditions was not activated, but instead slightly inhibited by Mn2+ (see for example Fig. 2).
We have previously concluded (15, 18) that the oxidase activity of peroxidase on triose reductone involves a free-radical mechanism similar to that proposed in Eqs. (l)-(3). It seems likely that strongly reducing free radicals, such as IAA’, would react rapidly with oxygen, under aerobic conditions, accounting for the oxygen-consuming character of the reaction. Considerable evidence for direct reaction with oxygen, of radicals derived from oxidation of IAA (by Mn3+) has been obtained by Maclachlan and Waygood (22). One obstacle to this interpretation of the IAA oxidase reaction appeared to be the observation that peroxidase does not catalyze IAA peroxidation under anaerobic conditions in the absence of an H acceptor. This can probably be explained by the inactivation of the enzyme, which occurs anaerobically in the presence of IAA and hydrogen peroxide, and can be presumed to be due to reaction of IAA’ with the enzyme. Either oxygen or H acceptors, which react with this radical, will protect the enzyme against inactivation and allow IAA destruction to occur. This is shown diagrammatically in Fig. 8, where B represents an H acceptor. ru’ote that inactivation of the enzyme is irreversible, since activity cannot be restored by adding oxidants subsequently. If the reduced acceptor BH is readily reoxidized by peroxidase, the addition of only a small amount of B (or BH) will protect the enzyme against inactivation by the 3 This is due to the reason that iron is reduced irreversibly combining with o-phenanthroline, but ferrocytochrome c is reoxidized by XH free radical (shown in Fig. 9) as reported previously
(15).
INDOLEACETIC
ACID
TABLE EFFECT OF Mnzf
299
OXIDASE
III
ON VARIOUS REACTIONS CATALYZED BY PEROXIDASE
Reactionssystems MI++effect
Conditions H-acceptor Aerobic,
without
added
Hz02
Anaerobic,
with added HzOl
Ref.
H-donor
Oxygen Oxygen Oxygen Methylene blue Cytochrome c Ferric ion
Triose reductone Dihydroxyfumarate IAA Triose reductone Triose reductone IAA
+” + +
H102 Methylene blue Cytochrome c Cytochrome c Cytochrome c Ferric ion
Triose reductone Triose reductone IAA Triose reductone Hydroquinone IAA
-
1: +b
(12) (10, 12) (7) (14) (15) Unpublished
08) (14) This paper (15) (15) This paper
a + indicates promotion; - indicates no effect or inhibition. b These reactions are shown diagrammatically in Fig. 9.
radicals IAA’, as indicated in Fig. 8, cycle B. However, if BH is not reoxidized by peroxidase, as is the case with Fez+, then, in the absence of oxygen, stoichiometric reduction of B will occur, as was observed. It has been suggested by Chance (21) that reaction of peroxidase complex I with a twoelectron donor does not proceed beyond the complex II stage, and reaction of complex II with a fre;sh donor molecule is necessary to reduce complex II to the free enzyme. That something of this kind occurs during IAA oxidation is suggested by the kinetic results described above, which support Eqs. (l)-(4). Additional evidence that the radical IAA’ does not react with complex II is provided by the observation that addition of ferricyanide doubles the rate of complex II reduction. This is explained by assuming that ferricyanide acts as a compound B in Fig. 8, leading to twice the rate of reaction with peroxid.ase as would occur if IAA’ just accumulated.. It was previously suggested (18) that operation of the aerobic oxidase reaction depends upon the formation of HZOz as a result of reaction of oxidized substrate radicals, such as IAA’, with oxygen. This is illustrated in Fig. 9, where YH2 represents the substrate undergoing aerobic oxidation. The results shown in Fig. 7, which indicate formation
FIG. 7. Relation between IAA destruction and optical changes at 4X rnp, under aerobic conditions; 0.2 amole TP; 0.33 mmole IAA; 0.02 M acetate; pH 4.3; temperature 18”. Optical density was determined in a cell with lo-cm. light path.
TP -/
inactivated
TP
FIG. 8. Tentative scheme of IAA oxidation peroxidase as affected by some oxidants (B).
by
300
YAMAZAKI
YH2
(TP
AND
SOUZU
no inhibition by CO was observed. In the case of IAA oxidation, one problem is that many workers have found the stoichiometry to be 1 mole On/mole IAA, and not $60, as would be required for the oxidation of YH2 to Y in the scheme of Fig. 9. However, it seems possible that reaction of IAA’ with O2 might occur in some other way, and have the over-all formulation IAA’
FIG. 9. Tentative scheme for promoting effect of Mn*+ and an ozidogenic substrate (XHS) on the oxidase reactions catalyzed by peroxidase. (H202) shows a trace amount of Hz02 which initiates the reaction. Explanation for promoting effect of Mn2+ is in the text. XH, promote the oxidase reaction when YH radical is produced more rapidly by the reaction of YH2 with XH radical than the direct peroxidase reaction of YH2 (15).
of a peroxidase-Hz02 complex during the induction phase of IAA oxidation, provide evidence that aerobic IAA oxidation depends upon the presence of Hz02 , and that Hz02 appears as a result of reaction of IAA’ with with O2 . Presumably the initiation of the reaction, during the induction phase, depends upon traces of Hz02 either present in the system or derived from autoxidation. It was not possible to determine the identity of the peroxidase-Hz02 complex detected, because-the concentration of peroxidase was too low for observation of absorption bands in the visible region. As summarized in Table III, in all our experiments with peroxidase, Mn2+ promoted oxidations only under aerobic conditions, without added Hz02. It does not affect the rate of anaerobic reduction of H acceptors by redogenic substrates, and can therefore be assumed not to be involved in reactions (l)-(3) between these substrates and peroxidase. It may instead be promoting the reaction between free radicals and 02 to form HfOz , as is indicated in Fig. 9. The mechanism proposed for the oxidase reactions of peroxidase does not involve ferrous peroxidase, so it is understandable that
+ Oz + IAA”0
+ >$HzOz
(8)
thus satisfying the oxygen stoichiometry and the observation that large amounts of Hz02 do not accumulate during the reaction, as they would according to the scheme in Fig. 9. This is in need of further investigation. ACKNOWLEDGMENT The authors are greatly indebted to Dr. Peter M. Ray for helpful criticism and discussion. REFERENCES 1. TANG, Y. W., AND BONNER, J., Arch. Biochem. 13, 11 (1947). 2. TANG, Y. W., AND BONNER, J., Am. J. Botany 36, 570 (1948). 3. WAGENKNECHT, A. C., AND BURRIS, R. H., Arch. Biochem. 26, 30 (1950). 4. GORTNER, W. A., AND KENT, M., J. Biol. Chem. 204, 593 (1953). 5. SEQUEIFLA, L., AND STEEVES, T. A., Plant Physiol. 29, 11 (1954). 6. GALSTON, A. W., BONNER, J., AND BAKER, R. S., Arch. Biochem. Biophys. 42,456 (1953). 7. KENTEN, R. H., Biochem. J. 69, 110 (1955). 8. RAY, P. M., Plant Physiol. 31, s xxvii (1956). 9. SWEDIN, B., AND THEORELL, H., Nature 146, 71 (1940). 10. CHANCE, B., J. Biol. Chem. 197, 577 (1952). 11. KENTEN, R. H., Biochem. J. 66, 350 (1953). 12. YAMAZAKI, I., FUJINAGA, Ii., TAKEHARA, I., AND TAKAHASHI, H., J. Biochem. (Tokyo) 43, 377 (1956). 13. AKAZAWA, T., AND CONN, E. E., J. Biol. Chem. 232, 403 (1958). 14. YAMAZAKI, I., FUJINAGA, K., AND TAKEHARA, I., Arch. Biochem. Biophys. 72, 42 (1957). 15. YAMAZAKI, I., Proc. Intern. Symposium on Enzyme Chemistry, Tokyo and Kyoto, 1967, p. 224. 16. WEBER, M. M., LENHOFF, H. M., AND KAPLAN, N. O., J. Biol. Chem. 220, 93 (1956). 17. KUHN, R., AND WASSERMAN, A., Ann. 503, 203 (1933). 18. YAMAZAKI, I., J. Biochem. (Tokyo) 44, 425 (1957). ~ ,
INDOLEACETIC
19. CHANCE, B., Arch. Biochem. Biophys. 41, 416 (1952). 20. RAY, P. M., Arch. Biochem. Biophys. 64, 193 (1956). 21. CHANCE, B., in “Enzyme Action” (McElroy,
ACID
OXIDASE
301
W. D., and Glass, B., eds.) p. 389. Johns Hopkins Press, Baltimore, 1954. 22. MACLACHLAN, G. A., AND WAYGOOD, E. R., Physiol. Pluntarum 9, 321 (1956). 23. GORDON, S. A., AND WEBER, R. P., Plant Physiol. 26, 192 (1951).
OF
BIOCHEMISTRY
The Mechanism
AND
88, 294-301 (1960)
of Indoleacetic Acid Oxidase by Turnip Peroxidase ISA0
From the Biological
BIOPHYSICS
YAMAZAKI
Institute,
Faculty
AND
HIROSHI
of Science,
Received
University
Reaction
Catalyzed
SOUZU’ of Tohoku,
Sendai,
Japan
June 18, 1959
In the iron-reducing activity of peroxidase in the presence of indoleacetic acid as electron donor, the kinetic and stoichiometric evidence supports a free-radical mechanism in which peroxidase catalyzes the formation of 2 moles indoleacetic acid radicals at the expense of 2 moles indoleacetic acid and 1 mole hydrogen peroxide. Inability of peroxidase to catalyze peroxidation of indoleacetic acid under anaerobic conditions is attributed to inactivating action of free radicals upon the enzyme, which is prevented bv the addition of some oxidants. The effects of carbon monoxide and Mn2+ were also examined. INTRODUCTION
The presence, in the extracts of certain plant tissues or fungi, of systems which oxidize IAA2 has been reported by many workers (l-5), and it is now well established that peroxidase itself catalyzes oxygen-consuming oxidation of IAA (6-8). The aerobic oxidase activity of peroxidase has also been observed in the presence of other substrates, such as dihydroxyfumarate (9, lo), phenylacetaldehyde (11), triose reductone (12), and reduced pyridine nucleotides (13). Recently, we have found that methylene blue, cytochrome c, and inorganic iron serve as hydrogen acceptor instead of molecular oxygen in the oxidase reaction catalyzed by peroxidase, provided that a suitable amount of hydrogen peroxide is added (14,15). These H acceptors become reduced when the peroxidase substrate is acted on by peroxidase anaerobically. The present work was undertaken to investigate the mechanism of the
oxidase activity of peroxidase in the presence of IAA when inorganic iron is added as electron acceptor. EXPERIMENTAL Recrystallized TP, with an extinction ratio of the exEm/Em = 2.7-3.1, was used throughout periments. TP concentration was calculated assuming that the value of the molecular extinction of TP is 90 cm.+ mmole-i at 403 rnp. Cytochrome c was prepared from beef heart muscle and purified with ion-exchange resin XE-64. Spectrophotometric measurements were performed with a Beckman model DU or a Hitachi spectrophotometer. The concentration of ferrous ion was determined from the extinction coefficient of ferrous o-phenanthroline complex of 11.3 cm.-’ mmole-i at 510 m/l (16). IAA concentration was determined using the Salkowski color reaction (23). Anaerobic conditions were obtained by gassing the cuvettes 10 min. with N? which had oxygen removed by repeated passage through alkaline solutions of pyrogallol and further over heated copper. RESULTS
1 Present address: The Institute of Low Temperature, University of Hokkaido, Sapporo, Japan. 2 The following abbreviations will be used: TP, turnip peroxidase; IAA, indoleacetic acid; IAA’, free radical of IAA (one equivalent oxidized form of IAA); IAA”, two equivalents oxidized form of IAA.
In previous work (15) on the reactions catalyzed by peroxidase, we have classified peroxidase substrates into two groups: (a) redogenic substrates, including triose reductone, dihydroxyfumarate, ascorbate, hydroquinone, pyrogallol and IAA; and (b) oxi294
INDOLEACETIC
ACID
dogenic substrates, including p- and m-cresol,
guaiacol, resorcinol, m-phenylenediamine, aniline, phenol,, and uric acid. The redogenic substrates are characterized by their capacity to reduce ferricytochrome c in the presence of hydrogen peroxide and peroxidase, a property not possessedby the oxidegenti group. Figures 1 and 2 illustrate the reduction of cytochrome c and inorganic iron catalyzed by peroxidase in the presence of IAA, which belongs to the redogenic group. These reductions. under anaerobic conditions, were observed immediately after hydrogen peroxide was added. Non-enzymic reduction of iron by the added hydrogen peroxide, which was demonstrated by Kuhn and Wasserman (17), was negligible under these experimental conditions. The maximal rate of the red.uction of iron and cytochrome c was obtained at about pH 4.2-4.6. Cytochrome c reduction occurred only when freshly prepared IAA solution was used; the resulting ferrocytochrome c was reoxidized slowly after a considerable amount of cytochrome c had been reduced. The same subsequent slow reoxidation of cytochrome c was previously observed when hydroquinone was em.ployed instead of IAA under these experimental conditions (15). In order to clarify the mechanism, it is helpful first to examine the stoichiometry of
(O.05 + 0
0L I
1 .I5
E z .I0 G d .05
d 0
0
I
2
3 Min.
4
5
FIG. 2. Reduction of iron under anaerobic conditions. Hz02 was added to the reaction mixture 1 min. after IAA addition; 50 pmoles IAA; 10 pmoles HzOz ; 0.1 mmole ferric chloride; 0.3 mmole o-phenanthroline; 0.02 rmole TP; 0.02 M acetate; pH 4.80; temperature 22”. A: 10 rmoles p-cresol. B: control. C: 10 pmoles Mn2+. D: TP-free.
IAA, pM Wz 2 PM 3. Stoichiometry of iron reduction; 0.1 pmole TP; 0.1 mmole ferric chloride; 0.3 mmole o-phenanthroline; 0.02 M acetate; pH 4.80; temperature 22’; anaerobic conditions. A: Dependence of the amount of reduced iron during the initial rapid reaction on the Hz02 concentration at constant IAA level (10 pmoles). B: Dependence of the amount of reduced iron during the initial rapid reaction on the IAA concentration at constant Hz02 level (5 pmoles).
x E 0 lo
0
.20
FIG.
.10-
d d
205
OXIDASE
2 Min.
3
FIG. 1. Reduction of cytochrome c under anaerobic conditions. Hz02 was added to the reaction mixture 1 min. after IAA addition. 0.1 mmole IAA; 6 pmoles cytochrome c; 40 pmoles H&l2 ; 0.02 M acetate; pH 4.80; temperature 22”. A: 0.1 pmole TP. B: 0.1 amole TP and 0.1 mmole NW+. C: 0.04 amole TP. D: 0.01 pmole TP.
the reaction. As can be seen in Fig. 3, it was found that 2 moles IAA consumed 1 mole hydrogen peroxide to reduce 1.7 moles iron during the initial rapid reaction (Fig. 2). Unlike the case of methylene blue reduction (18)) the molar ratio of reduced iron to added hydrogen peroxide in the presence of a sufficient amount of IAA, did not vary greatly under different experimental conditions.
296
YAMAZAKI AND SOUZU REACTIONS KINETICS
In the presence of excess Fe3+, the rate of iron reduction expected from Eqs. (l)-(4) will be given by d Fe2+ v=dt =
l-120e or
IAA,
PM
FIG. 4. Dependence
of the initial rate of iron reduct,ion on the concentration of H202 and IAA (anaerobic conditions); 0.1 pmole TP; 0.1 mmole ferric chloride; 0.3 mmole o-phenanthroline; 0.02 M acetate; pH 4.80; temperature 22”. Concentration of HgOz (A) and IAA (B) are shown in abscissa at constant level of 10 pmoles IAA (A) and 5 pmoles Hz02 (B), respectively.
A free radical mechanism was proposed previously (15, 18) to explain the reduction of methylene blue, cytochrome c, and other H acceptors during anaerobic oxidation of redogenic substrates by peroxidase and Hz02. In the case of IAA oxidation, the following reactions may be suggested:
2O’P) 1 h(H,Od
+
where (TP) represents total concentration of turnip peroxidase. Figure 5 shows changes in absorption at 424 rnp, due to appearance of peroxidaseHz02 complexes. When Hz02 was added to the peroxidase, formation of complex I occurred nearly instantaneously. This was followed by gradual reduction of complex I to complex II, as shown by the increase in optical density. Addition of IAA to this solution caused immediate reduction of the remaining complex I to complex II, followed by slower conversion of complex II to free peroxidase, as indicated by the gradual decrease in absorption. This shows that kz (in the above equations) has a value considerably larger than ka , as Chance (19) has reported in the case of other hydrogen donors. The very rapid formation of peroxidase complex I suggests further that kl(H20z) i> k,(IAA). If this is so, Eq. (6) can then be rewritten 0 = Bk,(TP)(IAA)
TP + HzOz * Complex
I + IAA Complex IAA’
-%
Complex Complex
II + IAA *
+ Fe3+ 2
IAA”
I
(1)
II + IAA’
(2)
TP + IAA’
(3)
-t Fe*
(4)
It is seen that if the amount of Fe3+ is great enough to oxidize all the free radicals IAA’ as fast as they are formed, the over-all reaction expected would be 2IAA
+ Hz02 + 2Fe3+ --t BIAA”
+ 2Fe2+ + 2Hz0
The observed stoichiometry bly well with this.
(5)
agrees reasona-
(6)
(7)
As shown in Fig. 4, the initial rate of reduction of Fe3+ was found to be closely proportional to IAA concentration, but nearly independent of Hz02 concentration over the range investigated, in agreement with Eq. (7). Using Eq. (7), it is possible to evaluate Ic, from the initial rate of iron reduction (Table I). We can also evaluate k~ by direct observation of the reaction of Eq. (3), observed in Fig. 5. The second-order velocity constant, lc, , is easily computed from the course of reduction of complex II to free TP, and is shown in Table I, along with kg obtained from iron reduction. It is also seen in Fig. 5 and Table I that in the presence of ferricyanide, the rate of complex II reduction
INDOLEACETIC
became about double; this will be discussed below. If the mechanism of iron reduction is represented by the reaction sequence of Eqs. (l)-(4), IAA must become oxidized in the presence of TP and hydrogen peroxide, under anaerobic conditions. However, Ray (8) found that no appreciable peroxidation of IAA occurs under anaerobic conditions. The same negative: result was obtained in the presence of 0.1 pmole TP, which was used for the ordinary oxidase reaction (Table 11). Table II shows that consumption of hydrogen peroxide did occur in the presence of some oxidants, such as methylene blue and p-quinone. It also occurred in the presence of IAA alone if higher concentrations of peroxidase were employed. Inability of the lower concentrations of peroxidase to oxidize IAA in the presence of hydrogen peroxide, under anaerobic conditions, may be due to the inactivation of the enzyme during the peroxidase reaction. Such an inactivation is demonstrated by the experiment shown in Fig. 6, in which the determination of peroxidase activity was performed by the guaiacol test. This remarkable inactivation of the enzyme did not occur in the absence of either IAA or hydrogen peroxide and decreased at higher pH or in the presence of some oxidants. By following in parallel the disappearance of IAA, using the Salkowski reagent, and the changes in absorption at 424 rnp, it was observed that the reaction of IAA destruction showed an induction phase, which ended TABLE
I
ESTIMATION OF k3 BY Two ‘DIFFERENT METHODS /
ACID
297
OXIDASE
I
.
I
I
*
‘Jj’
0123457
.
.
8
9
min.
FIG. 5. Formation of peroxidase complex II, and reduction of complex II to free TP by addition of IAA. IAA was added 7 min. after mixing of TP and HzO*. 2.1 pmoles TP; 2 amoles HzOz ; 1 pmole IAA; 0.02 M acetate; pH 4.63; in anaerobic cell with 2-cm. light path at room temperature (13”). Black circles are data in the presence of 0.1 mmole ferricyanide (absorption of ferricyanide is deducted). TABLE
II OF IAA Conditions: 0.4 mmole IAA, 0.4 mmole hydrogen peroxide, 0.02 M acetate, pH 4.30, anaerobic conditions. PEROXIDATION
ElXyIlle concentration
Addition
Remaining hydrogen peroxide after 1 min.
FM 0.1 0.4 0.8 0.8a 0.1 0.1
2
min.
mmoles mm&s
No addition of IAA 10 rmoles methylene blue 40 rmoles p-quinone
0.40 0.33 0.14 0.41 0.27 0.17
a This test shows that there is no catalase tivity in the peroxidase sample.
0.40 0.32 0.11 0.41 0.25 0.10 ac-
Methods “C.
Reduction of iron (Fig. 4) Reduction of iron Reduction of complex II (Fig. 5) Reduction in the presence of 0.1 mmole ferricyanide (Fig. 5) a For the reason mentioned true value of ka is half this.
22 13 13
3.7 2.9 3.0
13
5.9”
in Discussion,
the
after an initial slight increase of absorption at 424 rnp had occurrred. The change in absorption at 424 rncLtook place in two phases. The initial slight increase, before IAA destruction became rapid, might be due to the formation of a peroxidase-hydrogen peroxide complex. The further increase, which occurred after IAA had disappeared, should be attributed to the conversion of the inter-
298
YAMAZAKI AND SOUZU
It exhibited an activating effect only under aerobic conditions without added hydrogen peroxide. A summary of Mn2+ effects on different peroxidase reaction is given in Table III. The addition of p-cresol, which is an activator for the oxidase activity of peroxidase, stimulated the rate of iron reduction but strongly inhibited the reduction of cytochrome c.~
1000
DISCUSSION
Min. FIG. 6. Inactivation of TP in the presence of IAA (14”); 0.3 ml. incubated solution was added to 9.7 ml. solution mixed with Hz02 and guaiacol at the time indicated in the abscissa to determine peroxidase activity. Incubation: 0.1 pmole TP; 0.2 mmole IAA; 0.2 mmole HzOz ; 0.02 M acetate; pH 4.63; anaerobic conditions. Determination of peroxidase activity: 0.4 mmole guaiacol; 1 mmole HzOz ; 0.02 M acetate; pH 4.63; anaerobic conditions.
mediate(s) of IAA oxidation to the final pink-brownish oxidation product(s) as reported by Ray (20). EFFECTS OF CO, Mn2+, AND PHENOLS
It was reported previously (18) that carbon monoxide does not inhibit the reduction of methylene blue catalyzed by TP in the presence of triose reductone and hydrogen peroxide. CO also did not inhibit the reactions catalyzed by TP in the presence of IAA; neither the reduction of iron, when the solution was gassed with CO instead of nitrogen, nor the aerobic oxidation of IAA, in a solution gassed with 80 % CO/ZO% O2 instead of air. Promotive effects of Mn2+ on the aerobic oxidase activity of peroxidase have been reported by many workers. A strong activating effect has been shown in the case of dihydroxyfumarate, reduced pyridine nucleotide, and IAA, and a slight one in the case of triose reductone. On the contrary, the reduction of iron, cytochrome c, and dyes by the peroxidase systems under anaerobic conditions was not activated, but instead slightly inhibited by Mn2+ (see for example Fig. 2).
We have previously concluded (15, 18) that the oxidase activity of peroxidase on triose reductone involves a free-radical mechanism similar to that proposed in Eqs. (l)-(3). It seems likely that strongly reducing free radicals, such as IAA’, would react rapidly with oxygen, under aerobic conditions, accounting for the oxygen-consuming character of the reaction. Considerable evidence for direct reaction with oxygen, of radicals derived from oxidation of IAA (by Mn3+) has been obtained by Maclachlan and Waygood (22). One obstacle to this interpretation of the IAA oxidase reaction appeared to be the observation that peroxidase does not catalyze IAA peroxidation under anaerobic conditions in the absence of an H acceptor. This can probably be explained by the inactivation of the enzyme, which occurs anaerobically in the presence of IAA and hydrogen peroxide, and can be presumed to be due to reaction of IAA’ with the enzyme. Either oxygen or H acceptors, which react with this radical, will protect the enzyme against inactivation and allow IAA destruction to occur. This is shown diagrammatically in Fig. 8, where B represents an H acceptor. ru’ote that inactivation of the enzyme is irreversible, since activity cannot be restored by adding oxidants subsequently. If the reduced acceptor BH is readily reoxidized by peroxidase, the addition of only a small amount of B (or BH) will protect the enzyme against inactivation by the 3 This is due to the reason that iron is reduced irreversibly combining with o-phenanthroline, but ferrocytochrome c is reoxidized by XH free radical (shown in Fig. 9) as reported previously
(15).
INDOLEACETIC
ACID
TABLE EFFECT OF Mnzf
299
OXIDASE
III
ON VARIOUS REACTIONS CATALYZED BY PEROXIDASE
Reactionssystems MI++effect
Conditions H-acceptor Aerobic,
without
added
Hz02
Anaerobic,
with added HzOl
Ref.
H-donor
Oxygen Oxygen Oxygen Methylene blue Cytochrome c Ferric ion
Triose reductone Dihydroxyfumarate IAA Triose reductone Triose reductone IAA
+” + +
H102 Methylene blue Cytochrome c Cytochrome c Cytochrome c Ferric ion
Triose reductone Triose reductone IAA Triose reductone Hydroquinone IAA
-
1: +b
(12) (10, 12) (7) (14) (15) Unpublished
08) (14) This paper (15) (15) This paper
a + indicates promotion; - indicates no effect or inhibition. b These reactions are shown diagrammatically in Fig. 9.
radicals IAA’, as indicated in Fig. 8, cycle B. However, if BH is not reoxidized by peroxidase, as is the case with Fez+, then, in the absence of oxygen, stoichiometric reduction of B will occur, as was observed. It has been suggested by Chance (21) that reaction of peroxidase complex I with a twoelectron donor does not proceed beyond the complex II stage, and reaction of complex II with a fre;sh donor molecule is necessary to reduce complex II to the free enzyme. That something of this kind occurs during IAA oxidation is suggested by the kinetic results described above, which support Eqs. (l)-(4). Additional evidence that the radical IAA’ does not react with complex II is provided by the observation that addition of ferricyanide doubles the rate of complex II reduction. This is explained by assuming that ferricyanide acts as a compound B in Fig. 8, leading to twice the rate of reaction with peroxid.ase as would occur if IAA’ just accumulated.. It was previously suggested (18) that operation of the aerobic oxidase reaction depends upon the formation of HZOz as a result of reaction of oxidized substrate radicals, such as IAA’, with oxygen. This is illustrated in Fig. 9, where YH2 represents the substrate undergoing aerobic oxidation. The results shown in Fig. 7, which indicate formation
FIG. 7. Relation between IAA destruction and optical changes at 4X rnp, under aerobic conditions; 0.2 amole TP; 0.33 mmole IAA; 0.02 M acetate; pH 4.3; temperature 18”. Optical density was determined in a cell with lo-cm. light path.
TP -/
inactivated
TP
FIG. 8. Tentative scheme of IAA oxidation peroxidase as affected by some oxidants (B).
by
300
YAMAZAKI
YH2
(TP
AND
SOUZU
no inhibition by CO was observed. In the case of IAA oxidation, one problem is that many workers have found the stoichiometry to be 1 mole On/mole IAA, and not $60, as would be required for the oxidation of YH2 to Y in the scheme of Fig. 9. However, it seems possible that reaction of IAA’ with O2 might occur in some other way, and have the over-all formulation IAA’
FIG. 9. Tentative scheme for promoting effect of Mn*+ and an ozidogenic substrate (XHS) on the oxidase reactions catalyzed by peroxidase. (H202) shows a trace amount of Hz02 which initiates the reaction. Explanation for promoting effect of Mn2+ is in the text. XH, promote the oxidase reaction when YH radical is produced more rapidly by the reaction of YH2 with XH radical than the direct peroxidase reaction of YH2 (15).
of a peroxidase-Hz02 complex during the induction phase of IAA oxidation, provide evidence that aerobic IAA oxidation depends upon the presence of Hz02 , and that Hz02 appears as a result of reaction of IAA’ with with O2 . Presumably the initiation of the reaction, during the induction phase, depends upon traces of Hz02 either present in the system or derived from autoxidation. It was not possible to determine the identity of the peroxidase-Hz02 complex detected, because-the concentration of peroxidase was too low for observation of absorption bands in the visible region. As summarized in Table III, in all our experiments with peroxidase, Mn2+ promoted oxidations only under aerobic conditions, without added Hz02. It does not affect the rate of anaerobic reduction of H acceptors by redogenic substrates, and can therefore be assumed not to be involved in reactions (l)-(3) between these substrates and peroxidase. It may instead be promoting the reaction between free radicals and 02 to form HfOz , as is indicated in Fig. 9. The mechanism proposed for the oxidase reactions of peroxidase does not involve ferrous peroxidase, so it is understandable that
+ Oz + IAA”0
+ >$HzOz
(8)
thus satisfying the oxygen stoichiometry and the observation that large amounts of Hz02 do not accumulate during the reaction, as they would according to the scheme in Fig. 9. This is in need of further investigation. ACKNOWLEDGMENT The authors are greatly indebted to Dr. Peter M. Ray for helpful criticism and discussion. REFERENCES 1. TANG, Y. W., AND BONNER, J., Arch. Biochem. 13, 11 (1947). 2. TANG, Y. W., AND BONNER, J., Am. J. Botany 36, 570 (1948). 3. WAGENKNECHT, A. C., AND BURRIS, R. H., Arch. Biochem. 26, 30 (1950). 4. GORTNER, W. A., AND KENT, M., J. Biol. Chem. 204, 593 (1953). 5. SEQUEIFLA, L., AND STEEVES, T. A., Plant Physiol. 29, 11 (1954). 6. GALSTON, A. W., BONNER, J., AND BAKER, R. S., Arch. Biochem. Biophys. 42,456 (1953). 7. KENTEN, R. H., Biochem. J. 69, 110 (1955). 8. RAY, P. M., Plant Physiol. 31, s xxvii (1956). 9. SWEDIN, B., AND THEORELL, H., Nature 146, 71 (1940). 10. CHANCE, B., J. Biol. Chem. 197, 577 (1952). 11. KENTEN, R. H., Biochem. J. 66, 350 (1953). 12. YAMAZAKI, I., FUJINAGA, Ii., TAKEHARA, I., AND TAKAHASHI, H., J. Biochem. (Tokyo) 43, 377 (1956). 13. AKAZAWA, T., AND CONN, E. E., J. Biol. Chem. 232, 403 (1958). 14. YAMAZAKI, I., FUJINAGA, K., AND TAKEHARA, I., Arch. Biochem. Biophys. 72, 42 (1957). 15. YAMAZAKI, I., Proc. Intern. Symposium on Enzyme Chemistry, Tokyo and Kyoto, 1967, p. 224. 16. WEBER, M. M., LENHOFF, H. M., AND KAPLAN, N. O., J. Biol. Chem. 220, 93 (1956). 17. KUHN, R., AND WASSERMAN, A., Ann. 503, 203 (1933). 18. YAMAZAKI, I., J. Biochem. (Tokyo) 44, 425 (1957). ~ ,
INDOLEACETIC
19. CHANCE, B., Arch. Biochem. Biophys. 41, 416 (1952). 20. RAY, P. M., Arch. Biochem. Biophys. 64, 193 (1956). 21. CHANCE, B., in “Enzyme Action” (McElroy,
ACID
OXIDASE
301
W. D., and Glass, B., eds.) p. 389. Johns Hopkins Press, Baltimore, 1954. 22. MACLACHLAN, G. A., AND WAYGOOD, E. R., Physiol. Pluntarum 9, 321 (1956). 23. GORDON, S. A., AND WEBER, R. P., Plant Physiol. 26, 192 (1951).