THE JOURNAL OF BIOLOGICAL CHE~STRY Vol. 241, No. 11, Issue of June 10, PP. 2562-2571, 1966

Printed in U.S.A.

Studies on Cytochrome c Peroxidase

IV. A COMPARISOK OF PEROXIDE-INDUCED COMPLEXES OF HORSERADISH AND CYTOCHROME c

PEROXIDASES*

(Received for puhlicat.ion, December 10, 1965)

TAKASHI YO~ETANI

From the Johnson Reseawh Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104

SUMMARY

The stoichiometry between horseradish peroxidase (HRP) and H202 in the formation of Complexes I and II was spectro- photometrically studied by titrating HRP with HZ02 in the presence and absence of added reducing agents. The molar stoichiometry between HZOz added and Complex II formed was found to be 1: 1 in the presence and absence of added reducing agents. No evidence was obtained that more than 1 mole of Complex II was formed per mole of HzOz added. The titration of Complex II with ferrocyanide confirmed George’s finding that Complex II retained only 1 oxidizing equivalent per enzyme hematin unit. The oxidation of ascorbate and ferrocytochrome c upon additions of stoichio- metric quantities of HzOz in the presence of HRP exhibited two-phased kinetics: a very rapid initial oxidation followed by a slower secondary oxidation. These results were con- sistent with the currently accepted mechanism of the HRP reaction of Equations 2, 5, and 6.

The titration of an enzyme-HzOz complex (Complex ES) of yeast cytochrome c peroxidase with ferrocyanide indicated that Complex ES retained 2 oxidizing equivalents per enzyme hematin unit. No reducing equivalent of added donors was consumed in the process of the formation of Complex ES from cytochrome c peroxidase. The added donors were oxidized only in the process of the conversion of Complex ES to cytochrome c peroxidase. These results were con- sistent with the cytochrome c peroxidase mechanism of Equations 7 and 8.

Complex ES of cytochrome c peroxidase was compared with Complex II of HRP.

It is well established that horseradish peroxidase is rapidly con- verted, by the addition of a stoichiometric quantity of H202, to a green compound called Complex I (4) and that the Complex I, so formed, transforms on standing to a red compound called Complex II, which in turn decomposes gradually to regenerate

* Supported by Research Grant GM-12202-02 from the United Stat,es Public Health Service. The preceding papers of this series are References 1 to 3.

HRP’ (5). The rate of transition from Complex I to Complex II as well as that from Complex II to HRP appears to be dependent on the purity of HRP preparations used. For example, in par- tially purified enzyme preparations HRP was rapidly converted to Complex II upon the addition of H202, and the formed Com- plex II decomposed rapidly (5, B), while in the highly purified enzyme preparations, a mixture of Complexes I and II was ob- tained from HRP on the addition of Hz02, and these complexes decomposed at much slower rates (4, 7-9). Chance (9) showed that the transition of Complex I to Complex II was greatly accelerated in t,he presence of reducing agents. He subsequently showed that the transition of Complex I to Complex II was a l-eq reduction (10). Since Complex II? of HRP was reported by George (11) to carry 1 oxidizing equivalent per enzyme hema- tin unit, the relationship among HRP, HzOz, and Compleses I and II was described as shown in Equation 1 (10, 11)

+HzOz +AH HRP ___f Complex I p

(E-~~0) (E-fLOJ 1-eq reduction Step 1 Step 2

+AH (1)

Complex II f HRP (E-OH) l-eq reduction (E-HzO)

Step 3

where E-H20 = peroxidase, E-Hz02 = a peroxidase derivative carrying 2 oxidizing equivalents, E-OH = a peroxidase derivative carrying 1 oxidizing equivalent, and AH = a hydrogen donor.

Therefore, the varying effects of Hz02 mentioned above added to HRP were interpreted as due to the presence in HRP prepara- tions of varying quantities of endogenous donors which promoted the reactions of Steps 2 and 3 of Equation 1 in the absence of added donors.

Abrams, Altschul, and Hogness (13) reported that, by the addition of a stoichiometric amount of HzOz, cytochrome c peroxidase from bakers’ yeast was converted to a red compound (Complex KY). Subsequently George (11, 12) studied Complex ES of cytochrome c peroxidase in comparison with Complex II

1 The abbreviation used is: HRP, horseradish perosidase. 2 The term “Compound” was used by George (11, 12) instead

of “Complex.” 3 Formerly designated as Complex II since its absorption spec-

trum resembled closely that of Complex II of HRP (cf. References 1 to 3 and 11 to 13).

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of HRP. He found that Complex ES derived from partially purified preparations of cytochrome c peroxidase was titrated to cytochrome c peroxidsse by l-eq reduction with ferrocyanide and thus considered that the reaction mechanism of cytochrome c peroxidase was essentially identical with that of HRP (cf. Equa- tion 1) although the formation of Complex I had not been re- ported in the cytochrome c perosidase system.

Although previous investigators (5, 6, 8, 10, 13) had reported the molar stoichiometry between HzOz added and Complex II or ES formed from HRP or cytochrome c peroxidase to be l:l, George (12) claimed that approximately 2 moles of Complex II or ES per mole of HI02 added were formed from HRP or cyto- chrome c peroxidase. However, our recent re-examination of the stoichiometry of the cytochrome c peroxidase syst)em (2) revealed that only 1 mole of Complex ES was formed per mole of Hz02 added. Furthermore, Complex ES, derived from a highly puri- fied preparation of cytochrome c peroxidase (l), was found to retain 2 oxidizing equivalents of HzOz per enzyme hematin unit (2) instead of 1 oxidizing equivalent as reported by George (11).

In this paper we re-esamined the stoichiometry among HRP, HzOt, and Complexes I and II by titrating HRP with Hz02 in the presence and absence of added reducing agents such as as- corbate, ferrocytochrome c, and ferrocyanide. Complex II of HRP was compared with Complex ES of cytochrome c peroxidase by titrating these complexes with reducing agents such as ferro- cyanide and ferrocytochrome c.

EXPERIMENTAL PROCEDURE

Cytochrome c Peroxidase-A purified preparation of cytochrome c peroxidase was made from bakers’ yeast as described previously (1). Its purity index, which was defined as the ratio of absorb- ance at 408 rnp to that at 280 rnp (AK,~:&~), was found to be 1.2 (I). A maximal turnover rate of this enzyme in the perox- idatic oxidation of horse heart ferrocytochrome c was determined to be 2500 see-i at infinite concentrations of Hz02 and ferrocyto- chrome c and at 23” in potassium phosphate buffer, pH 6.0, with an ionic strength of 0.05 (1, 3).

Horseradish Peroxidase-A purified preparation of HRP (Type V) was purchased from Sigma. The preparation was dialyzed against potassium phosphate buffer, pH 6.0, with an ionic strength of 0.1, at 4” for 24 hours. The purity index of this prep- aration was found to be 2.8. The specific activity of this HRP preparation was not measured under the conventional conditions for purpurogallin number assay (14). The specific activity is given in the text in terms of rate constants, ka and lcq (lo), which were measured under appropriately defined conditions. Some experiments were duplicated with a preparation of HRP from Boehringer. Results obtained with Sigma and Boehringer prep- arations were found to be essentially identical; therefore, only results obtained with the Sigma preparation are reported in this paper.

Donors-Horse heart cytochrome c was purchased from Sigma. This was further purified by chromatography on’ Amberlite CG-50. Ferrocytochrome c was prepared as described elsewhere (15). Sodium ascorbate (Sigma) and potassium ferri- and ferro- cyanides (Baker) were used without further purification.

Concentrations of cytochrome c peroxidase, HRP, and ferro- cytochrome c were determined spectrophotometrically by the use of the following extinction coefficients: eqa8 (cytochrome c perox- idase) = 93 rnrvr-l cm-i (1) ; ~403 (HRP) = 90 rnM-’ cm-l (14) ;

er,50 (ferrocytochrome c) = 27.6 rnM-i cm-r (2) ; and Ae5~o (ferro- minus ferricytochrome c) = 19.6 rnM-i cm-i (2).

HeOz-Superoxol (30% Hz03 produced by Merck) was diluted with 0.01 M potassium phosphate buffer, pII 7.0, containing 1 mM EDTA. The concentration of HsOs was assayed by the cytochrome c peroxidase-ferrocytochrome c system as described elsewhere (2).

Bugler-Potassium phosphate buffer, pH 6.0, with an ionic strength of 0.1, was used as a reaction medium throughout this investigation unless otherwise noted.

The quantity of a reactant was expressed as a final concentra- tion of the reaction mixture. Usually 5- to 10.~1 portions of ap- propriately concentrated solutions of a reactant were added to 2.00 ml of reaction mixtures in order to keep the volume change of reaction mixtures, upon each addition of a reactant, less than 0.5 70.

Spectrophotometric measurements were carried out with a Cary recording spectrophotometer, model 15, which was equipped with a cell compartment thermostatically controlled at 23”. pH measurements were made with a Radiometer pH meter, model 25.

RESULTS

Horseradish Peroxidase System

DiJerence Extinction Coescients of Complex II minus Horse- radish Peroxidase-An accurate determination of the absorption spectrum of Complex II of HRP is difficult to carry out by a simple addition of Hz02 to HRP because under these conditions a mixture of Complexes I and II is obtained and these complexes change their concentrations continuously with time. Chance (6-10) indicated previously that, during the steady state of the perosidatic oxidation of donors, HRP was retained in the form of Complex II. Therefore, it is possible to determine the spectrum of Complex II by a rapid wave length scanning during the steady states. In order to determine the duration of steady states and to ascertain the complete absence of HRP and Complex I during steady states under various conditions, the kinetics of HRP and Complexes I and II were studied by measuring absorbance changes at 395, 410.5, and 426 mp, respectively; these wave lengths are isosbestic points of Complex I-Complex II, HRP- Complex II, and HRP-Complex I pairs, respectively (e-10).

The kinetics was studied with 1.45 pM HRP in the presence of excess Hz02 (5 to 200 PM) and ascorbate (10 to 200 PM). In agreement with Chance’s observation (6-lo), HRP and Complex I were found to be absent during steady states under these condi- tions. The amplitude of the 426 mw absorbance increase during steady states was found t,o be independent of the concentration of HzOz and ascorbate, although the duration of steady states was a function of the concentration of HzOz and ascorbate. The differ- ence spectrum of Complex II minus HRP shown in Fig. 1 was recorded by a rapid wave length scanning (2.5 rnp see-1) during the steady state, which lasted for 60 sec. Difference estinction coefficients of Complex II minus HRP in the wave length range from 380 to 460 rnp are indicated in the right-hand ordinate in Fig. 1.

Kinetic Titration of Horseradish Peroxidase uith H302 in Ab- sence of Added Donors-Experiments were carried out by titrating HRP with varying concentrations of HzOz. The kinetics of formation and decomposition of Complexes I and II was followed by measuring absorbance changes at 410.5 and 426 mp, respec-

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2564 Cytochrome c Peroxidase. IV Vol. 241, No. 11

i E

-2

(426mp) HRP-

O2 Added

HRP- (410.5mp)

1.45pM HRP

IOOpM Ascorbote -

I 380 400 420 440 460

Wove Length (rnp)

FIG. 1. A difference spectrum between Complex II and HRP

1.45pM HRP In PO,, Buffer, pH 6.0,O.l~ I .

/ Slower Scanning

’ Time Course ’ c

FIG. 2. Kinetics of formation and decomposition of Complexes I and II of HRP in the absence of an added donor. Absorbance changes of HRP upon additions of H202 were measured at 426 mr (for Complex II) and 410.5 rnp (for Complex I).

tively, as shown in Fig. 2. Complex I was formed very rapidly. tions cannot be compared accurately without a flow apparatus After its concentration had reached a maximal value within 1 set As seen in Fig. 2, the time period from the initiation of reactiul,

after the initiation of the reaction, Complex I decomposed slowly to the maximal formation of Complex II was found to vary de- with time. Complex II appeared to be formed less rapidly than pending on [HsO2]. Maximal absorbance increments at 426 and

Complex I, although kinetics of the initial phase of their forma- 410.5 rnp after each addition of Hz02 were plotted against [Hz021

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in Fig. 3. As shown in Fig. 3B, absorbance increments at 410.5 mp increased linearly to [H202] with a slope value of -28 mM-1 cm-l and reached a plateau value of -0.041 cm-1 at 1.48 pM H202. A difference extinction coefficient at 410.5 rnp of Com- plex I minus HRP of -28.3 rnM-’ cm-‘, which was arbitrarily calculated from maximal increments in 410.5 rnp absorbance in the presence of excess H202, was found to be in close agreement with the corresponding coefficient of -30 mM-1 cn-1 obtained by Chance (16) from flow experiments. Therefore, it was con- cluded that the molar stoichoimetry among HRP, Hz02, and Complex I in the formation of Complex I from HRP was ap- proximately 1: 1: 1 under the given conditions.

However, a maximal increment in the 426 rnp absorbance at each addition of H202 did not increase proportionally to [Hz02]; less than 1 mole of Complex II was always formed per mole of Hz02 added (cf. Fig. 3A). Thus, under the present conditions, we could not confirm George’s observation that the addition of 1 mole of Hz02 to HRP resulted in the formation of more than 1 mole of Complex II (12). It should be pointed out that an ap- preciable concentration of Complex I always remained at the time when Complex II was maximally formed, after each add i- tion of HzOz, as can be seen in Fig. 2. The concentration of Complex I which remained when Complex II was maximally formed was plotted against [Hz021 in Fig. 4. Each sum of the concentration of Complex I remaining and the concentration of Complex II maximally formed plotted against [HzOz] fits re- markably well the theoretical curve which was drawn on the basis of a molar stoichiometry of 1: 1 between H202 and Com- plexes I and II (cf. Fig. 4).

Kinetic Titration of Horseradish Peroxidase with HzOz in Pres- ence of Excess Bscorbate-Experiments were carried out by titrat- ing HRP with varying concentrations of Hz02 in the presence of 20 PM ascorbate. The ascorbate concentration of 20 PM was

chosen in preliminary experiments in order to record kinetics of formation and decomposition of Complex II in an appropriate time scale. Formation and decomposition of Complex II were followed by measuring absorbance changes at 432 rnp; this wave length was used in order to compare the present results with those of the titration in the presence of ferrocytochrome c which will be described later. Since HRP and Complex I are approximately isosbestic from 425 to 450 rnp (16), there was no gross difference in the Complex II kinetics whet.her measurements were carried out at 426 or 432 unp except that the difference extinction coeffi- cient of Complex II minus HRP was smaller at 432 than at 426 mp: 27 and 45 mM+ cm-‘, respectively (cf. Fig. 1).

Spectrophotometric examinations at 395, 410.5, and 432 rnp revealed that the conversion of HRP and Complex I to Complex II was virtually complete when the stirring in of Hz02 was finished (cf. Fig. 5). When the concentration of HzOz was less than that of HRP, the Complex II which formed rapidly decomposed, as shown in Fig. 5, A and B (cf. traces at 432 mp). When the con- centration of Hz02 exceeded that of HRP, the concentration of Complex II remained constant for a considerable period of time before Complex II began to decompose rapidly, as shown in Fig. 5C (cj. the trace at 432 mp).

A maximal increase in the 432 rnp absorbance after each addi- tion of HzOn was plotted against [HzOz], as shown in Fig. 6. The result clearly indicates that 1 mole of Complex II was formed by the addition of 1 mole of Hz02 to HRP in the presence of ascor- bate as a hydrogen donor.

The kinetics of oxidation of ascorbate under the same condi-

tions was followed by measuring the absorbance decrease at 268 rnp, as shown in Fig. 5. B difference extinction coefficient at 268 rnp of ascorbate minus dehydroascorbate was determined to be 12.4 rnM-’ en-l under these conditions; values of 10 (at pH

t Slope = -28mM-‘crf’

AC 410.5

=-28.3mM-‘cm-’ t

I I I I 1 I I I

0 I 2 3 4 5 6

(PM) [Hz $1

FIG. 3. Titration of HRP with Hz02 in the absence of an added donor. Maximal increments in the 426 and 410.5 xnp absorbances of HRP upon additions of H&z (cf. Fig. 2) were plotted against [H202]. A is a plot of the concentration of Complex II maximally formed with respect to the concentration of HpOr. The broken line is theoretically drawn on the basis of a molar stoichiometry of I:1 between Complex II and Hz02. B is a plot of maximal in- crements in the 410.5 mp absorbance with respect to [HsO~]. The concentration of Complex I is readily calculated by applying the difference extinction coefficient indicated.

I I I I 1 I

1.45+l HRP O= Moximolly Formed II

L!L I / I 6

:#: L

‘H’

H, 02(pM)

FIG. 4. Plots of the concentrations of Complex I, Complex II, and Complexes I plus II with respect to the concentration of H20a. Concentrations of Complexes I and II were measured at the time when Complex II was maximally formed after each addi- tion of H202 to HRP in the absence of an added donor (cf. Fig. 2). The broken lines are drawu on the basis of a molar stoichiometry of 1:l between the concentration of Complexes I plus II and the conceutration of HaOz.

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decrease. When the concentration of Hz02 was less than that of HRP, approximately 507’ of the ascorbate consumed was oxi- dized in the rapid initial phase, and the rest (about 507,) of the ascorbate disappeared in the slower secondary phase as shown in Fig. 5, A and B (cf. traces at 268 mp). Transition points of these two phases are indicated by arrows. When the concentra- tion of Hz02 was more than that of HRP, the amplitude of the initial decrease in the 268 mp absorbance was approximately constant in a [H202] range from 1.76 to 6.60 FM; it ranged from 0.0075 to 0.0085 cn-l ( = 1.21 to 1.29 pN ascorbate). When the concentration of HRP was increased from 1.45 t.o 3.5 PM, the amplitude of the initial decrease in the 268 mp absorbance in- creased to 0.0150 cm-l ( = 2.4 pN ascorbate), as shown in Fig. 50. Therefore, there appears to be a direct relationship between the rapid phase of ascorbate oxidation and the concentration of HRP. By comparing the kinetics of formation and decomposi- tion of Complex II with the kinetics of oxidation of ascorbate, we may interpret these results to indicate that the ascorbat,e con- sumed in the initial phase was used in the I-eq reduction of Con- pies I to Complex II (cj. Step 2 of Equation 1) and the ascorbate consumed in the secondary phase was used in the I-eq reduction of Complex II to HRP (cj. Step 3 of Equation 1). Thus, Chance’s finding (9, 16) that the conversion of Complex I to Complex II in the presence of an added donor was considerably faster than that of Complex II to HRP or k7 >> kd was substanti- ated for the first time by measurement of the kinetics of donor disappearance. The kg values, which were obtained under the present conditions, are summarized in Table I. The present kq value of 3 x lo3 M-I set-1 should be compared with those of 2.8 x lo3 (at pH 7.0) and 1.3 to 3.3 x lo4 (at pH 4.7) M-I se0 reported by Chance (9). The presently employed mixing technique was inadequate to evaluate k7 values from the rapid initial phase of ascorbate oxidat’ion.

Kinetic Titration of Horseradish Peroxidase with H202 in Pres- ence of Excess Ferrocytochrome c-Experiments were carried out by titrating HRP with varying concentrations of Hz02 in the presence of 4.5 to 10 PM ferrocytochrome c (cf. Fig. 7). The

TABLE I

Activity of Complex II of HRP and Hz02 toward ascorbate

In potassium phosphate buffer, pH 6.0, with an ionic strength of 0.1

t.45pM HRP + 2QM Ascorbate In PO, Buffer, pH 6.0,(+

. Time Course

FIG. 5. Kinetics of formation and decomposition of Complex II (at 432 rnp) and of oxidation of ascorbate (at 268 rnp) upon addi- tions of HIOn to HRP in the presence of ascorbate as a hydrogen donor. Xote two-phased kinetics of ascorbate oxidation as ob- served in the 268 rnp absorbance decreases. Arrows indicate transition points between two phases.

AC 432 = 27mM-‘cm-’

1

Concentration

ks ka H,O, t/-M

FIG. G. Titration of HRP with Hz02 in the presence of ascor- bate (cf. Fig. 5). Maximal increases in the 432 rnp absorbance were plotted against [H,O?]. The results indicated that 1 mole of Complex II was formed upon the addition of 1 mole of HzOz to HRP in the presence of ascorbate.

HRP CoYPx A

.-

scorbate

#N(W)

20 (40)

PM PM pN seco N x-1 St?-’ IO’ M-1 see 0.44 0.43 0.048 0.112 2.8 0.88 0.85 0.090 0.106 2.6 1.10 1.07 0.105 0.100 2.5 1.32 1.23 0.150 0.120 3.0 1.76 1.33 0.186 0.140 3.5 2.20 1.37 0.186 0.135 3.4 4.40 1.40 0.186 0.132 3.3 G.60 1.44 0.186 0.132 3.3

20 (40) 6.60

PM

1.45

3.5 3.50 0.470 0.135 3.4

4.6) and 9.35 (the pH value unspecified) 111~~~ Cm-l were previ- ously reported by Chance (9) and Morton (17), respectively.

Kinetics of oxidation of ascorbate exhibited an interesting feature which has not been reported hitherto. Ascorbate disap- peared in two phases, a very rapid initial disappearance which was followed by a slower secondary disappearance. The rapid initial decrease in the 268 rnp absorbance was due to neither an increase in [Hz021 nor the conversion of HRP to Complex II since they caused an increase in absorbance at 268 mp rather than a

- - D Maximally formed. 6 Initial steady state rate of ascorbate oxidation at the time

when Complex II was maximally formed.

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1.45/~M HRP + 4.5$,l Cytc” In PO, Buffer, ptd 6.0,O.l~

+ Time Course

FIG. 7. Kinetics of formation and decomposition of Complexes I and II (at 408 and 432 rnp) and of oxidation of ferrocytochrome c (at 550 rnp) upon additions of H20, to HRP in the presence of ferrocytochrome c as au added electron donor.

enzyme-substrate kinetics was followed by measuring absorbance changes at 408 and 432 rnp; ferro- and ferricytochrome c are isosbestic at these wave lengths. The kinetics of oxidation of ferrocytochrome c was followed by measuring the absorbance decrease at 550 mp. As shown in Fig. 7, Complex II was maxi- mally formed 1.8 to 8.5 set after the initiation of the reaction. A comparison of the absorbance change at 432 rnp revealed that Complex I was virtually absent when Complex II was maxi- mally formed; absorbance decreases at 408 mp, shown in Fig. 7, were contributed mainly by the conversion of HRP to Complex II (cf. Ae = -12.5 rnM-i cm-l from Fig. 1). The plot of the concentration of Complex II with respect to that of Hz02, which was obtained under these conditions, was found to be essentially identical with that of Fig. 6. Therefore, it can again be con- cluded that 1 mole of Complex II was formed by the addition of 1 mole of HzOz to HRP in the presence of 5 to 10 PM ferrocyto- chrome c as an added electron donor.

Kinetic curves measured at 550 rnp during the oxidation of ferrocytochrome c again exhibited two phases, as shown in Fig. 7, although the transition from the initial phase to the slower sec- ondary phase was observed to be less distinct in this case than that observed at 268 1nl.L for the oxidat,ion of ascorbate (cf. Fig. 5). This was probably due to the smaller value of the k7 :lcd ratio for ferrocytochrome c than that for ascorbate. The Jc( value for ferrocytochrome c, which was calculated from initial slopes in the slower secondary phase of the 550 mK kinetics, was equal to 5.0 f 1.5 x 104M-’ set-I. This value was considerably smaller than a kd value of 107 M-I se0 for ferrocytochrome c at pH 4.7 reported by Chance (18).

Kinetic Titration of Horseradish Peroxidase with HzOz in Pres- ence of Excess Ferrocyanide-A titration was carried out with 1.50 pM HRP with varying concentrations of HzOZ in the presence of 5 pM ferrocyanide. The result of this titration was essentially identical with that obtained in the presence of ascorbate: 1 mole of Complex II was formed by the addition of 1 mole of HzOz to HRP in the presence of ferrocyanide as an added electron donor.

Kinetics of oxidation of ferrocyanide could not be studied in these experiments since absorbance changes in a region from 260 to 450 mp, which accompanied the oxidation of ferrocyanide to ferricyanide, were too small to be measured accurately (Ae < 1 IllM-l cm-i).

Titration of Complex II of Norseradish Peroxidase with Ferro- cyanide-Upon an addition of 12 PM Hz02 to 11.8 PM HRP, ap- proximately 12 PM Complex I was immediately formed, as indi- cated in Fig. 8 by a rapid decrease in the 410.5 mp absorbance of 0.330 cm-i: Ae410,5 (Complex I minus HRP) = -0.028 PM-’

cm-1 (14). As indicated by a subsequent increase in the 410.5 mp absorbance, which was accompanied by an increase in the 426 rnp absorbance, the Complex I, so formed, was gradually converted to Complex II on standing: At,,, (Complex II minus HRP) = 0.045 PM-’ cm-l (14). Approximately 600 set after the initia.tion of reaction, the concentration of Complex II reached a maximal steady state level of 5.4 pM while that of Complex I steadily decreased during the period, as can be seen in Fig. 8. The titration of Complex II with ferrocyanide was car- ried out both before and after Complex II was maximally formed (cf. Fig. 8, A and B, respectively). Upon additions of 1.25 PM

ferrocyanide, the Complex I present in the mixture was rapidly

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4 4,:~ b A’A=Cu$-1 1 1

I Time Course --

HRP ’ pN-‘cd

FIG. 8. Titration of Complexes I and II of HRP wit,h ferro- cyanide. Kinetics of formation and decomposition of Com- plexes I and II followed by measuring absorbance changes at 410.5 and 426 xnp, respectively; these wave lengths were isosbestic point,s of HRP-Complex II and HRP-Complex I pairs.

FIG. 9. Effect of ferrocytochrome c (Cyt. c) added before and after the addition of Hz02 to cytochrome c peroxidase (CCP). Note that the concentration of the ferrocytochrome c oxidized was independent of whether the cytochrome c was added before or after the addition of Hz02 to cyt#ochrome c peroxidase. In other words, no cytochrome c was oxidized in the process of forma- tion of Complex ES from cytochrome c peroxidase and Hz02.

and stoichiometrically converted to Complex II, as previously shown by Chance (10). After Complex I had been fully con- verted to Complex II, Complex II was titrated with 2.5 PM

ferrocyanide (cf. Arrows a and c in Fig. 8) followed by 1.0 PM ferrocyanide (cf. Arrows b and d in Fig. 8). The decrease in the 426 mp absorbance of Complex II per aliquot of ferrocyanide added was found to be 0.046 f 0.002 pN-’ cm-l. Similar ex-

periments carried out at 1 PM HRP gave the same value for this coefficient. The comparison of this coefficient value of 0.046 pN+ Cm-’ with a value of the AE~~~ (Complex II minus HRP) of 0.045 PM-I cm-i indicated that Complex II of HRP retained approximately 1 oxidizing equivalent per enzyme hematin unit: 0.045 PM-' cm-i:0.046 ~CIN-’ cm-i = 0.98 N Mb'. These results essentially confirmed George’s finding (11) that Complex II of HRP contained 1 oxidizing equivalent per enzyme hematin unit as well as the assumption proposed by George (11) and proved by Chance (10) that the conversion of Complex I of HRP to Com- ples II was a l-eq reduction.

Cytochrome c Peroxidase System

Effect of Ferrocytochrome c ddded before and after Addition of Hz02 to Cytochrome c Peroxidase-Cytochrome c peroxidase could be stoichiometrically converted to Complex ES upon the addition of H202 at least within a range of enzyme concentration from 0.1 to 5 MM (2). The conversion of cytochrome c peroxidase to Complex ES occurred very rapidly, and no indication of the formation of a compound similar to Complex I of HRP was ob- tained during the conversion (l-3). The effect of ferrocyto- chrome c which was added to cytochrome c peroxidase before and after the addition of HzOz was studied, as shown in Fig. 9. The reaction was followed by measuring changes in the 550 mp ab- sorbance. In the experiment shown in Fig. 9A, 21 pM ferrocyto- chrome c (Arrow b) was added to 4.8 PM cytochrome c peroxidase. Since cytochrome c peroxidase had no cytochrome c oxidase activ- ity, no aerobic oxidation of ferrocytochrome c occurred under these conditions. Upon three successive additions of ~.~O~MHZO~

(Arrows c, d, and e) to the reaction mixture, ferrocytochrome c was partially oxidized. Because of a high turnover rate of cytochrome c peroxidase in the peroxidatic oxidation of ferrocytochrome c (1, 3), the oxidation of ferrocytochrome c upon each addition of Hz02 was completed as soon as HzOz was added, as indicated by the vertical decrease in the 550 nm absorbance upon each addition of H202. The concentration of ferrocytochrome c oxidized was found to be precisely equal to the concentration of oxidizing equivalents of Hz02 added. In the experiment shown in Fig. SB, 1.30 MM H302 (Arrows f, g, and h) was added three times to 4.8 pM cytochrome c peroxidase (Arrow a) to form 3.9 PM Complex ES. Fcrrocytochrome c, 21 PM (Arrow i), was added to this mixture. A portion of ferrocytochrome c was immediately oxidized. The concentration of the ferrocytochrome c oxidized was readily est,imated by a comparison of Fig. 9, A and B. Fer- rocytochrome c, 7.7 PM, was oxidized by 3.9 pM Complex ES. The final level of the 550 rnp absorbance of this reaction mixture (Fig. 9B) was found to be equal to that of the previous mixture (Fig. 9A). These results clearly indicated that 7.7 pN oxidizing equivalents were retained in 3.9 PM Complex ES and that all the oxidizing equivalents of HzOz added were retained in Complex ES. In other words, no oxidizing equivalents of Hz02 were con- sumed in the process of conversion of cytochrome c peroxidase to Complex ES, and Complex ES contained 2 oxdizingequivalents per enzyme hematin unit.

Effect of Ferrocyanide Added before and after Addition of Hz02 to Cytochrome c Peroxidase-The experiments of Fig. 10 were carried out with 1.7 pM cytochrome c peroxidase and 1.5 pM HzOz. The reaction was followed by measuring changes in the 424 rnp absorbance of Complex ES: Aeq24 (Complex ES minus cyto- chrome c perosidase) = 0.042 PM-' cm-l (2). In Fig. lOA, 1.5 FM Complex ES was formed by the addition of 1.5 PM Hz02 to

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Issue of June 10, 1966 T. Yonetani 2569

1.5pM H202 A Time Course B I.OpM K4 Fe EN),

I I.5pM H,O,

I

I.OpM ‘K4 Fe (CN),

FIG. 10. Effect of ferrocyanide added before and after the addition of H,O, to cytochrome c peroxidase (CCP). Note that ferrocya- nide was oxidized only after Complex ES was formed from cytochrome c peroxidase and H20, under these conditions.

1.7 PM cytochrome c peroxidase. Complex ES was then titrated by successive additions of ferrocyanide (1.0 or 0.5 PM). The rate of reduction of Complex ES by ferrocyanide was relatively slow under these conditions; the second order rate constant of the reaction was calculated to be 3.5 x lo3 ~-1 se0 at pH 6.0. An average decrease in the 424 mp absorbance per aliquot of ferro- cyanide added was found to be 0.023 -f 0.002 JJN-' cm-l. Ferro- cyanide, 1 KM, is assumed to have 1 FN reducing equivalent.* Similar experiments carried out at 10 PM cytochrome c peroxidase gave the same value for this coefficient. The comparison of this coefficient value of 0.023 pN-I en-l with a value of the AC,,, (Complex ES minus cytochrome c peroxidase) of 0.042 PM-I

cm-1 led to the conclusion that Complex ES retained approxi- mately 2 oxidizing equivalents per enzyme hematin unit: 0.042 PM-~ cm-‘:0.023 pN-' cm-l = 1.85 N ?0.

Fig. 10B illust,rates an experiment in which 1.50 pM Hz02 was added to 1.7 PM cytochrome c peroxidase in the presence of 1.0 PM ferrocyanide. On the addition of 1.5 PM H202, 1.5 PM

Complex ES was found to be formed immediately, as indicated by a rapid initial increase in the 424 rnp absorbance of 0.062 cm-l ( = 1.5 PM Complex ES) upon the addition of Hz02 in Fig. IOB. The Complex ES so formed was subsequently reduced by the ferrocyanide present in the mixture; the value of the 424 mp absorbance decrease per aliquot of ferrocyanide added was found to be 0.023 ~N-I cm-i. A subsequent addition of ferrocyanide also gave an absorbance decrease of 0.022 p~-l cm-l. It should be pointed out that the rate of formation of Complex ES from cytochrome c peroxidase and HaOz was very much faster than the rate of reduction of Complex ES by ferrocyanide under these conditions; second order rate constants of these reactions were 1.4 x 108 (3) and 3.5 x lo3 (this paper) M-I see-l, respectively. These results clearly indicated that Complex ES could be stoichiometrically formed from cytochrome c peroxidase and

4 No correction was made for the effect of the absorbance change at 424 to 426 rnp due to the oxidation of ferrocyanide to ferri- cyanide, since it was found to be relatively small: Aed26 < 0.001 /AN-~ cm-l.

H202, in the presence and absence of ferrocyanide, and that the ferrocyanide present in the mixture was not oxidized in the process of formation of Complex ES but was consumed in the reduction of Complex ES.

DISCUSSION

Horseradish Peroxidase System-The mechanism of the per- oxidatic oxidation of hydrogen donors catalyzed by HRP was described in 1951 (19), as shown in Equations 2, 3, and 4,

HRP (E-H20) + Hz02 + Complex I (E-HeOz) + Hz0 (2)

Complex I (E-HBOP) --t Complex II (E-H202) (3)

Complex II (E-HtOJ + AH* ---f HRP (E-H20) + A + Hz0 (4)

where B is the oxidized donor and the other abbreviations are as defined for Equation 1. The conversion of Complex I to Com- plex II (Equation 3) was assumed to be an intracomplex isom- erization.

The subsequent refinement of the HRP mechanism from Equations 2, 3, and 4 to Equations 2, 5, and 6, which are cur- rently well accepted, owed a great deal to the contributions of George (11, 12, 20) and Chance (10, 16).

HRP (E-HsO) + Hz02 + Complex I (E-HzOZ) + Hz0 (2)

Complex I (E-HPOB) + AH + (5)

Complex II (E-OH) + A + Hz0

Complex II (E-OH) + AH + HRP (E-H20) + A (6)

In 1952 George (20) assumed that the transition of Complex I to Complex II was a 1-eq reduction rather than an isomerization (cj. Equation 5). This assumption was based upon his finding (11) that Complex II was converted to HRP by a I-eq reduction with ferrocyanide (cf. Equation 4), along with Chance’s previous observation (9) of the accelerated conversion of Complex I to Complex II in the presence of added donors. Chance (10) sub- sequently demonstrated that Complex I was converted to Com-

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2570 Cytochrome c Peroxidase. IV Vol. 241, No. 11

plex II by I-eq reduction with ferrocyanide and thus substanti- ated George’s assumption of Equation 5. A set of Equations 2, 5, and 6 was presented by Chance (10) as a probable mechanism of the peroxidatic reaction catalyzed by HRP. Although he had previously presented Equations 5 and 6 (20), George (11, 12) apparently did not derive the mechanism of Equations 2, 5, and 6, since Equations 2 and 5 did not conform to t,he molar stoichi- ometry of 1:2 between Hz02 added and Complex II formed which he determined in the absence of added donors (12).

plex &S retained 2 oxidizing equivalents per enzyme hematin unit and thus was consistent with our previous conclusion of Equations 7 and 8 (a),

Cyto-c P (E-H,O) + Hz02 ---t Complex ES (E-He02) + H20 (7)

Complex ES (E-HzOZ) + 2,4H +

cyto-c P (E-HQO) + 2A + Hz0 (8)

where cyto-c P is cytochrome c peroxidase. The present result of the titration of Complex II with ferro- It should be pointed out that Complex I has never been de-

cyanide (cf. Fig. 8) confirmed George’s important finding (11) tected in a cytochrome c peroxidase system under the same condi- that Complex II retained 1 oxidizing equivalent per hematin unit tions at which Complex I could be readily detected in an HRP (cl. Equation 6). The two-phased kinetics of the donor oxida- system. tion, as shown in Figs. 5 and 7, is consistent with Equations 5

George (11, 12) attributed this to the presence in cyto-

and 6. It has been shown for the first time that the added hy- chrome c peroxidase preparations of an excess endogenous donor

drogen donors are oxidized at two steps within the over-all reac- which promoted a rapid transition of Complex I to Complex ES. However, such an interpretation fails to give an adequate ex-

tion, namely, the rapid formation of Complex II from Complex I and the slower decomposition of Complex II to HRP.

planation of the fact that Complex ES of cytochrome c peroxidase is more stable than Complex II of HRP (1).

The molar stoichiometry of 1: 1 between H202 added and Com- plex II formed, which was determined in this paper in the pres- ence as well as absence of added donors, confirmed the results of Keilin and Mann (5) and Chance (6, 8, 10) and conformed to the HRP mechanism of Equations 2, 5, and 6.

Cytochrome c Peroxidase System-The present titration of Complex ES with ferrocyanide (cf. Fig. 10) indicated that Com-

The results of Figs. 9 and 10 clearly indicated that the added donors were oxidized in the process of the conversion of Complex ES to cytochrome c peroxidase. In contrast to the formation of Complex II of HRP from Complex I, no reducing equivalent of the donor was consumed in the process of the formation of Complex ES from cytochrome c peroxidase.

Current controversies over stoichiometry, reaction, and chemi-

TABLE II

Comparison of Complex ES of cytochrome c peroxidase with Complex II of HRP

Peroxidase property

Cytochrome c peroxidase Stoichiometry

Complex ES:H20, Nature of Complex ES

Aerea (Complex ES - cytochrome c peroxidase) AAds (Complex ES)/K,Fe(CN), Oxidizing equivalents per Complex ES

Nature of reaction Cytochrome c peroxidase + Complex ES Complex ES 4 Cytochrome c peroxidase

Horseradish peroxidase Stoichiometry

Complex I:H202 Complex II:HzO*

Nature of Complex I A~1o.s (Complex I - HRP) Oxidizing equivalents per Complex I

Nature of Complex II Aeaz6 (Complex II - HRP) AAw, (Complex II)/KaFe(CN)8 Oxidizing equivalents per Complex II

Nature of reaction HRP ---f Complex I Complex I + Complex II Complex II 4 HRP

Unit

mole nlole-1 2 lb

rnrv-l cm-l mN-’ cm-l N M-l

40.5 -40.5

1

42 -23

2

I-eq oxidation (Complexation)c l-eq reduction 2-eq reduction

mole mole-l mole mole-l

(1) 2

1 1J

1 1

rnrv-l cm-l N M-’ (2)

-30 (2)

-28 (2)

rnM-l cm-l mn-* cm-l N M-’

44 -44

1

47 -47

1

45 -46

- I - .-

George (11, 12)

(Complexation)c (Complexation)E (Complexation)c (1-eq reduction) l-eq reduction I-eq reduction I-eq reduction (1-eq reduction) 1-eq reduction

Investigator (Reference)”

Chance (6, 9, 10) onetani (2, this paper)

a Parentheses indicate that it was not shown, but assumed. b Also by Abrams et al. (13). c Or 2-eq oxidation. rl Also by Keilin and Mann (5).

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Issue of June 10, 1966 T. Yonetani 2571

cal nature of H*Oz-induced complexes of cytochrome c peroxidase 5. KEILIN, D., AND MANN, T., Proc. Roy. Sot. (London), Ser. B,

and HRP are summarized in Table II. 122, 119 (1937).

It is very puzzling that, despite their spectral resemblance, G. CHANCE, B., J. Biol. Chem., 151, 553 (1943).

Complex II of HRP and Complex ES of cytochrome c peroxidase 7. CHANCE, B., Arch. Biochem. Biophys., 21, 416 (1949). 8. CHANCE, B., Arch. Biochem. Biophys., 22, 224 (1949).

retain different amounts of oxidizing equivalents, namely, 1 and 9. CHANCE, B., Arch. Biochem. Biophys., 24, 389 (1949).

2 oxidizing equivalents, respectively, since the absorption spectra 10. CHANCE, B., Arch. Biochem. Biophys., 41, 416 (1952).

under consideration are thought to reflect electronic structures of 11. GEORGE, P., Biochem. J., 54,267 (1953).

their hematin prosthetic groups. 12. GEORGE, P., Biochem. J., 55, 220 (1953). 13. ABRAMS, R., ALTSCHUL, A. M., AND HOGNESS, T. R., J. Biol.

Acknowledgments-1 appreciate the valuable criticisms of Dr. Britton Chance and Dr. Ronald W Estabrook given during the course of this study.

REFERENCES

1. YONETANI, R., AND RAY, G. S., J. Biol. Chem., 240, 4503 (1965).

2. YONETANI, R., J. BioZ. Chem., 240, 4509 (1965). 3. YONETANI, T., AND RAY, G. S., J. Biol. Chem., 241, 700 (1966). 4. THEORELL, H., Enzymologia, 10, 250 (1941).

Chem.; 14i, 303 (1942). 14. KEILIN, D., AND HARTREE, E. F., Biochem. J., 49, 88 (1951). 15. YONETANI, T., AND RAY, G. S., J. Biol. Chem., 240,3392 (1965). 16. CHANCE, B., Arch. Biochem. Biophys., 44, 404 (1952). 17. MORTON, R. A., Absorption spectra of vitamins and hormones,

Adam Hilger, London, 1940. 18. CHANCE, B., in J. T. EDSALL (Editor), Enzymes and enzymes

systenhs, Harvard University Press, Cambridge, Massa- chusetts, 1951, p. 95.

19. CHANCE, B., Advan. Enzymol., 12, 153 (1951). 20. GEORGE, P., Advan. Catalysis, 4, 367 (1952).

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Takashi YonetaniCYTOCHROME c PEROXIDASES

PEROXIDE-INDUCED COMPLEXES OF HORSERADISH AND Peroxidase : IV. A COMPARISON OFcStudies on Cytochrome

1966, 241:2562-2571.J. Biol. Chem. 

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