THE JOURNAL OF BIOLOQICAL CAEM~STRY Vol. 237, No. 2, February 1962

Printed in U.S.A.

Studies on Cytochrome Oxidase

IV. THE CYTOCHROME OXIDASE ACTIVITY*

TAKASHI YONETANI

WITH THE TECHNICAL ASSISTANCE OF PATRICIA RAINFORD

From the Johnson Research Foundation, University of Pennsylvania, Philadelphia 4, Pennsylvania

(Received for publication, July 19, 1961)

Cytochrome oxidasel is the enzyme which catalyzes the oxida- tion of reduced cytochrome G by molecular oxygen. A number of methods have been developed to determine the cytochrome oxiclase activity, but two major systems have been widely em- ployed, as reviewed briefly by Slater (5) and Smith and Conrad (6) : System 1,

Reducing agent Reaction 1 Reaction 2 - cytochrome c p

cytochrome oxidase Reaction 3 - oxygen

where the activity is determined by measuring the rate of oxida- tion of reducing agents, or the rate of oxygen consumption; and System 2,

Reduced cytochrome c Reaction 2

cytochrome oxidase Reaction 3 - oxygen

where the activity is determined by measuring the rate of oxida- tion of reduced cytochrome c.

Smith (6, 7), Conrad (8, 9), Wainio (10-13) et ~2. have exten- sively investigated the effect of various factors on the cyto- chrome oxidase activity measured by System 2 with soluble and insoluble preparations of cytochrome oxidase, whereas among those (14-30) who have employed System 1, Slater (5), using an insoluble heart muscle preparation, seems to have studied the experimental conditions for the cytochrome oxidase assay in the most detail.

There has been some disagreement between the results ob- tained by the two systems. For example, with System 2 the optimal pH for the cytochrome oxidase activity has been found to be in the acidic region (3, 10, 11, 30), whereas with System 1 it has been reported to be in the basic region (22, 24).

The present paper describes a study of the effect of various factors, especially the reactant concentrations and pH, on the

* This work was supported in part by Grant ZG-277 from the United States Public Health Service.

1 In previous papers (l-3) of this series, it has been demon- strated that a purified preparation of beef heart muscle cyto- chrome oxidase exhibits the spectral properties ascribed to cyto- chromes a and aa by Keilin and Hartree (4). However, in this paper no attempt has been made to differentiate the enzyme into cytochromes a and US, and cytochrome oxidase is treated as a single enzyme unit, since low concentrations of cytochrome oxi- dase employed in the presence of relatively high concentrations of cytochrome c do not permit us to analyze the spectral be- havior of cytochrome oxidase during reactions.

cytoohrome oxidase activity of a highly purified preparation of cytochrome oxidase from beef heart muscle (1, 3) determined polarographically by System 1 (ascorbate-cytochrome c-cyto- chrome oxidase-oxygen). The different assay methods of the cytochrome oxidase activity are also discussed.

EXPERIMENTAL PROCEDURE

Materials

Preparation of Purijed Cytochrome Oddase-A purified prepa- ration of cytuchrome oxidase, direct dialyzed preparation (3), was made from fresh beef heart muscle according to a modification (2, 3) of the method of Okunuki et al. (31).

Preparation of Cytochrome c-A crystalline preparation of cy- tochrome c made from beef heart muscle according to the method of Hagihara et al. (32) and a purified preparation of cy- tochrome c (Type V from beef heart) purchased from Sigma Chemical Company were used. No difference between these preparations was found in the present study of the cytochrome oxidase activity assay.

Reaction Medium-Potassium phosphate buffer (0.05 M) con- taining 1 mM EDTA and 1% Emasol4130 (24) was used unless otherwise noted. Emasol4130, a nonionic synthetic detergent (polyoxyethylene-sorbitan monooleate) which was kindly sup- plied by Kao Soap Company, Tokyo, was added to stabilize and reactivate the enzymic activity of the purified preparation of cytochrome oxidase (3, 24, 33, 34).

Redwing Agent-Solutions of ascorbic acid (Eastman Organic Chemicals division of Eastman Kodak Company) neutralized with potassium hydroxide and stored in a frozen state were thawed immediately before use.

Methods

Concentrations of Cytochrome c and Cytochrome Oxdase-The concentration of cytochrome c was calculated by use of the millimolar absorbancy indexes, a (m& x cm-l at 550 rnp, re- duced) = 27.7 (35), or Aa (mM-l X cm-l at 550 rnp, reduced minus oxidized) = 19.0 (35). The concentration of cytochrome oxidase was calculated by use of the difference millimolar ab- sorbancy index, Aa (mM-1 x cm-l at 605 rnp, reduced minus oxidized) = 11 (3), based upon the iron content of heme a in the preparation.

Spectrophotometry-Kinetic observations of the oxidation-re- duction changes of cytochrome c during the cytochrome oxidase

550

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February 1962 T. Yonetctni 551

reaction and the determination of the steady state reduction of cytochrome c were carried out with a split-beam recording spec- trophotometer (36-38) as described in a previous paper of this series (2).

Measurement of Cytochrome Oxidase Activity-The cytochrome oxidase activity was measured by the polarographic method developed by Chance (36, 39). The rate of oxygen consump- tion (pM O2 per second) was calculated from the inclination of the polarographic trace recording the time course of oxygen utilization. The activity was expressed as TN,2 which is the number of electron equivalents utilized per mole of cytochrome oxidase per second. This can be calculated from the following equation.

TN = oxygen consumption (PM %/set) X 4 electron equivalent@

cytochrome oxidase concentration (PM)

The measurement was carried out at 25”. At higher pH values, an autoxidation of ascorbate was observed. However, it was significantly depressed by the addition of cytochrome c, in agreement with the results of Mater (5). A combination of ascorbate and cytochrome oxidase shows no appreciable oxygen uptake. The oxygen consumption due to the cytochrome oxi- dase activity was obtained by subtracting the oxygen consump- tion of the ascorbate-cytochrome c system from that of the ascorbate-cytochrome c-cytochrome oxidase system.

RESULTS

E$ect of Reactant Concentrations on Cytochrome O&&se Activity

Effect of Oxygen Concentration-The polarographic trace re- cording the time course of oxygen utilization by the cytochrome oxidase activity of the present purified system shows a perfectly straight line until the oxygen concentration of the reaction medium becomes less than 5 pM. This is in accordance with the results of Chance (40), who used a heart muscle preparation. The oxygen concentration of the air-saturated aqueous medium is -250 pM at 25”. Therefore, as long as the rate of oxygen uptake is determined from the inclination of the polarographic trace obtained in a region of 5 to 250 j&M oxygen, changes in the oxygen concentration of the medium do not affect the determina- tion of cytochrome oxidase activity. Hence, in the following, the effect of the rest of the reactants of System 1, i.e. ascorbate, cytochrome c, and cytochrome oxidase, will be discussed.

Table I shows the specific activity, expressed in terms of TN, of the purified preparation of cytochrome oxidase obtained by changing the concentrations of three reactants involved in this chain reaction. It is evident that, with increased concentra- tions of ascorbate and cytochrome c, higher values of TN are obtained, whereas decreased activity is obtained with increased cytochrome oxidase concentration. The results are further analyzed to clarify the individual effect of the three variables.

Efect of Ascorbate Concentration-At fixed concentrations of cytochrome c and cytochrome oxidase, higher values of TN are obtained with higher ascorbate concentrations. This effect of

2 The abbreviations used are: TN, turnover number; TN,,, (cyt. c) and TN,,,(cyt. oxidase), maximal turnover numbers at an infinite cytochrome c concentration and at an infinitely dilute cytochrome oxidase concentration, respectively; K,,,(cyt. c), the cytochrome c concentration required for the half maximal ac- tivity.

3 One mole of oxygen utilized corresponds to four electron equiv- alents utilized.

TABLE I

Cytochrome oxidase activity obtained at diferent concentrations of reactants

The activity was expressed as the turnover number (electrons per mole of cytochrome oxidase per second). Measurements were carried out in 0.05 M potassium phosphate buffer, pH 7.0, containing 1% Emasol4130 and 1 mM EDTA at 25”.

Line no.

6 7 8 9

10

11 12 13 14 15

16 17 18 19 20

-

P

_-

-

Lscorbate concen- tration

x 10-o M

5 5 5 5 5

10 10 10 10 10

20 20 20 20 20

40 40 40 40 40

-

cyto- :hrome c concen- tration

K 10-e M

10 20 40 60 00

28.0 18.0 10.0 6.0 3.0 36.0 24.0 17.0 9.8 5.5 40.0 32.0 25.0 16.3 10.0 42.0 38.0 30.0 20.0 13.3 w 59 59 59 59

10 40.0 25.0 16.0 10.0 5.3 20 50.0 35.0 25.0 17.5 9.5 40 52.0 41.0 33.0 25.0 16.3 60 56.0 47.0 40.0 30.0 21.5 m 66t 66 66 66 66

10 44.0 33.0 23.6 15.0 8.7 20 60.0 48.0 35.6 23.8 15.0 40 70.0 55.0 47.6 35.0 25.0 60 70.0 58.0 54.0 43.8 30.0 00 W 69 69 69 69

10 52.0 40.0 26.0 20.0 11.2 20 60.0 48.0 37.5 30.0 19.4 40 70.0 55.0 47.5 41.0 30.6 60 70.0 60.0 55.0 47.5 36.3 co f.W 65 65 65 65

- I-

25

x 10-B M

50 1 100 1 200 1 400

-

1’ 00

-

50 50 50 50

59 59 59 59

66 66 66 66

66 66 66 66

-

* TN,,, (cyt . oxidase) . t TN,,, (cyt. c).

ascorbate concentration is significant at lower cytochrome c concentrations and at higher cytochrome oxidase concentrations. For example, at 200 or 400 X 10WD M cytochrome oxidase with 10 PM cytochrome c, the activity increases nearly proportionally to the ascorbate concentration. This indicates that under these conditions the ascorbate concentration is rate-limiting. Although at lower cytochrome oxidase concentrations with higher cytochrome c concentrations, the activity is no longer proportional to the ascorbate concentration, ascorbate is still rate-limiting. However, at higher cytochrome c concentrations (e.g. 40 or 60 PM), with the lowest concentration of cytochrome oxidase tested (25 x 10WD M), the ascorbate concentration is no longer rate-limiting, as indicated by the fact that the activity does not increase with increased ascorbate concentration.

E$ect of Cytochrome c Concentration-At fixed concentrations of cytochrome oxidase and ascorbate, the relationship between the activity and the cytochrome c concentration is shown as a rectangular hyperbolic curve. This is in agreement with other workers (2, 5, 17, 19, 22, 23, 25-27). At higher cytochrome c concentrations, higher values of TN are obtained. Slater’s pro- cedure (5), with a Lineweaver-Burk plot (41), gives the activity at an infinite cytochrome c concentration, which is now referred to as TN,,, (cyt. c). Fig. 1 shows a Lineweaver-Burk plot of

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552 Cytochrome Oxidase. IV Vol. 237, No. 2

I I 1 I

20 mM Ascorbote 400

P

I [ Cytochrome c]

104XM-I

FIG. 1. A Lineweaver-Burk plot of the relationship between the cytochrome oxidase activity and cytochrome c concentration at different cytochrome oxidase concentrations. The values were calculated from Lines 11,12,13, and 14 of Table I. The lines des- ignated 25,50, 100, 200, and 400 were obtained with 25, 50, 100, 200, and 400 X HP M cytochrome oxidase, respectively.4

TABLE II

Relationship between cytochrome oxidase concentration and Km (cyt. c)

The values of K,,, (cyt. c) were calculated from the Lineweaver- Burk plot of the relationship between the cytochrome oxidase activity and the cytochrome c concentration (cf. Fig. 1).

*I%:- concen- tration

x 10-r hr

5 10 20 40

Km (cyt. cl

Cytochrome oxidase concentration

(X 10-9 M)

400 j 200 1 100 1 50 1 25

x 10-6 M

160 75 39 19 * 91 43 23 * * 67 34 17 * * 50 23 12 * *

* Not measurable accurately.

Km (cyt. c)/[cytochrome oxidase]

400 375 390 228 215 230 167 170 170 125 115 126

380

I

__

Mean value

M/M

385 224 170 120

the relationship between the activity and the cytochrome c concentration at five different cytochrome oxidase concentra- tions (25, 50, 100,200, and 400 X 1O-S M) with a fixed eoncen- tration of ascorbate (20 mM). Similar relationships can be ob- tained with other ascorbate concentrations (5, 10, or 40 mM). The lines obtained at different cytochrome oxidase concentra- tions intersect the ordinate (the l/TN axis) at the same point. This result indicates that the value of TN,,, (cyt. c) is inde- pendent of the concentration of cytochrome oxidase used, since the reciprocal of the value at the intercept at the ordinate is identical with TN,,, (cyt. c). The values of TN,,, (cyt. c) at different ascorbate concentrations are given in Lines 5, 10, 15, and 20 of Table I. This relationship also indicates another im-

4 The cytochrome oxidase concentrations employed, with re- spect to heme a concentration, are indicated by the following symbols in Figs. 1, 2, 3, 5, and 7: solid triangle, 25 X 1CP M; open triangle, 50 X 1OP M; open square, 100 X 10-g M; solid circle, 200 X 10-S M; open circle, 400 X 10-S M; open double circle, 1600 X 10-S M.

portant fact, which was previously mentioned briefly by Slater (5) and has since then been neglected by others, namely, that the cytochrome c concentration required for the half maximal activity, the so-called apparent Michaelis constant of cyto- chrome c, K, (cyt. c), is not a universal constant but varies with changes in the ascorbate and cytochrome oxidase concen- trations as shown in the left-hand part of Table II. This may be an explanation of the disagreement about values of K, (cyt. c) obtained by many workers who determined the values without special regard to the cytochrome oxidase and reducing agent concentrations (2, 17, 19, 22, 25-27). Furthermore, it is found that, if the ascorbate concentration is fixed, the molar ratio of K, (cyt. c) to the cytochrome oxidase concentration is roughly a constant, as is shown in the right-hand part of Table II. Of course this ratio is a function of the ascorbate concentration, the specific activity of the enzyme preparation used, and the medium conditions such as pH, temperature, and ionic strength. However, if a definite enzyme preparation and definite medium conditions are employed, the ratio is a function of the ascorbate concentration. As illustrated in Table II, the ratio decreases with increased ascorbate concentration. Further analysis of the relationship between the values of the ratio and the as- corbate concentration reveals that the ratio is roughly propor- tional to [ascorbate]--+, i.e. to the negative square root of the ascorbate concentration. Therefore the relationship of the K, (cyt. c), the ascorbate, and cytochrome oxidase concentrations can be written as follows,

K,(cytochrome c) [cytochrome oxidasel

= [ascorbate]+n

where n is equal to 2 under the present experimental conditions. Eflect of Cytochrome Oxidase Concentration-The relationship

between the activity and the cytochrome oxidase concentration in Table I shows that the activity expressed as TN is higher at lower concentrations of cytochrome oxidase, if concentrations of cytochrome c and ascorbate are fixed. This suggests that the activity may exhibit its maximal value at an infinitely dilute

I 1 I I I 1

I 20 mM Ascorbate ‘OF I

I 0.05 TN/set

I I I I I 0 100 200 300 400

[Cytochrome Oxidase] 10qs M

FIG. 2. The relationship between the cytochrome oxidase con- centration and the reciprocal of the activity. The values were calculated from Lines 11, 12, 13, and 14 of Table I. The lines designated 10, 20, 40, and 60 were obtained with 10,20,40, and 60 pM cytochrome c, respectively. The reciprocal of the intercept value at the ordinate of the plots gives the maximal activity at an infinitely dilute cytochrome oxidase concentration, i.e. TN,., (cyt. oxidase).

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February 1962 T. Yonetani 553

PH PH PH

FIG. 3. The effect of pH on the cytochrome oxidase activity at four different cytochrome oxidase concentrations (25, 50, 100, and 200 X 1OW M). The symbols indicating the cytochrome oxidase concentration are identical with those of Figs. 1 and 2 (See text, footnote 4). The activity was obtained in the presence of 20 PM

cytochrome c in 0.05 M potassium phosphate buffer containing 1% Emasol4130 and 1 mM EDTA. The dotted lines are the pH curves of TNmm (cyt. oxidase) (See “Discussion”).

solution of cytochrome oxidase. Therefore, the reciprocal of the activity is plotted against the cytochrome oxidase concen- tration, as shown in Fig. 2. The points at any one fixed con- centration of cytochrome c are located on a straight line. The lines obtained at different concentrations of cytochrome c inter- sect the ordinate (the l/TN axis) at the same point. The reciprocal of this intercept can be considered as the activity at an infinitely dilute solution of cytochrome oxidase, and is re- ferred to as TN,,, (cyt. oxidase). The values of TN,,, (cyt. oxidase) obtained at different ascorbate concentrations are shown in the right-hand column of Table I.

Effect of pH on Cytochrome Oxidase System

The cytochrome oxidase activity of soluble preparations simi- lar to the present preparation has been reported to be optimal

at pH values of 5.7 to 5.9 by a spectrophotometric method with System 2 (3) and at pH 7.0 by a manometric method with Sys- tem 1 (24). The disagreement between these two values for the optimal pH may be due to the involvement of the nonenzy- mic reduction of oxidized cytochrome c by ascorbate in the latter system (34). To understand this discrepancy, the effect of pH on the present reaction system was further investigated.

E$ect of pH on Cytochrome Otidase Activity-Fig. 3 illustrates the effect of pH on the activity at four different concentrations of cytochrome oxidase (25, 50, 100, and 200 X 10eg M) in the presence of 20 pM cytochrome c with three different concentra- tions of ascorbate (5, 10, and 20 mM). The following three facts should be noted:

1. At any fixed set of ascorbate and cytochrome c concentra- tions, lower values of the optimal pH for the activity are ob- tained when the cytochrome oxidase concentration is decreased. At 200 X lo+ M cytochrome oxidase, the optimal pH is found to be higher than pH 7.2, whereas at 25 X 1O-g M cytochrome oxidase, it is found to be 6.5 to 6.7.

2. If the ascorbate concentration is high enough for the sys- tem, for example, at 20 mM ascorbate as shown in Fig. 3, the activities at pH 7.8 at different concentrations of cytochrome oxidase are nearly identical. In other words, the activity, as oxygen consumption, is proportional to the amount of cyto- chrome oxidase added. This indicates that the electrons sup- plied to cytochrome oxidase are in a large excess, or that the electron donor system for cytochrome oxidase, i.e. ascorbate- cytochrome c, is not rate-limiting at pH 7.8.

3. At pH values below 7.8, higher activities are always ob- tained with lower concentrations of cytochrome oxidase; this is in accord with the result of Table I measured at pH 7.0, and in- dicates that, below pH 7.8, the electron donor system for the oxidase, i.e. the ascorbate-cytochrome c system, is always rate- limiting. In other words, cytochrome oxidase is in excess under these conditions. Therefore, the increase in the cytochrome oxidase concentration simply depresses the activity expressed

- Aerobic State *I’- Aerobic Steady Stated f Anaerobic State

I 20pM Oxidized

2-

Cytochrome c

T i me Course FIG. 4. Kinetic records of oxidation-reduction of cytochrome c during the cytochrome oxidase reaction. The experiments were

carried out with a split-beam recording spectrophotometer under the experimental conditions identical with those of Fig. 3.

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554 Cytochrome Oxidase. IV Vol. 237, No. 2

60

PH

FIQ. 5. The effect of pH on aerobic steady state reduction of cytochrome c at five different concentrations of cytochrome oxi- dase (25,50,100,400, and 1600 X lVg M). Concentrations of 20 pM

cytochrome c and 20 mM ascorbate were used under the same con- ditions described in Fig. 3.

I I I I I

- 0.05M Phosphate I % Emosol 4130

- 20pM Cytochrome c

25O

6.0 6.5 7.0 7.5 8.0

PH

Fro. 6. The effect of pH on the rate of reduction of oxidized cytochrome c by ascorbate. The reciprocal of the time required from the addition of ascorbate to the 50% reduction of cytochrome c was used as the relative rate of reduction.

as TN, although it increases the total oxygen consumption of the system.

Effect of pH on Aerobic Steady State Level of Oxidation-Reduc- tion of Cytochrome c---To confirm the results of Fig. 3, the effect of pH on the aerobic steady state levels of oxidation-reduction of cytochrome c was examined. Fig. 4 illustrates kinetic records of oxidation-reduction of cytochrome c during the cytochrome oxidase reaction. Upon an addition of 20 mM ascorbate (a) to an aerobic solution of 20 pM oxidized cytochrome c, cytochrome c is rapidly reduced. Further addition of 0.05 PM cytochrome oxidase (b) induces a partial oxidation of the reduced cytochrome c because of the cytochrome oxidase reaction. The level of oxi- dation-reduction of cytochrome c during this aerobic steady state depends upon the balance between the nonenzymic reduc- tion of cytochrome c by ascorbate, and the enzymic oxidation of cytochrome c by the cytochrome oxidase-oxygen system. After the oxygen in the reaction medium is exhausted (c), cytochrome c returns to a fully reduced state. If a small amount of oxygen

(d) is added to the anaerobic reaction medium, cytochrome c is again partially oxidized and then returns to the fully reduced state, as studied in detail by Chance (42, 43). During aerobic steady states, cytochrome c is 35% reduced at pH 5.9, whereas it is 68 Y0 reduced at pH 7.0. As shown in Fig. 5, aerobic steady state levels of cytochrome c are varied by changing the pH value of the reaction medium. If concentrations of ascorbate, cyto- chrome c, and cytochrome oxidase are fixed, cytochrome c is considerably less reduced during aerobic steady states at lower pH values than at higher pH values. Since the steady state level of cytochrome c is an indicator of the balance between the electron donor (ascorbate) and the electron acceptor system (cytochrome oxidase-oxygen) of cytochrome c, this result indi- cates that the electron donor is more effective at higher pH values, or that the electron acceptor system is more effective at lower pH values. The former possibility is indicated in Fig. 4. Here cytochrome c seems to be reduced more rapidly by ascor- bate at pH 7.0 than at pH 5.9. To determine more quantita- tively the rate of reduction of cytochrome c by ascorbate, the time required from an addition of ascorbate to the 50% reduc- tion of cytochrome c, tt,,,5 is determined. The rate of cyto- chrome c reduction is inversely proportional to the tion value. Therefore, the reciprocal of the t, on value is taken as the relative rate of reduction of cytochrome c by ascorbate in Fig. 6. The reduction rate is increased by increasing the pH value of the reaction medium. This is in accord with the assumption that the electron donor of cytochrome c is more effective at higher pH values, which was presented to explain the results of Fig. 5.

Effect of Cytochrome Oxidase Concentration on Steady State Level of Cytochrome c-If the pH value of the reaction medium and the ascorbate concentration are fixed, the activity of ascor- bate as an electron donor to cytochrome c is constant. There- fore, the steady state level of cytochrome c depends upon the activity of the electron acceptor system (the cytochrome oxi- dase-oxygen system). If the cytochrome oxidase concentration is increased, the total activity of the electron acceptor system, as indicated by the oxygen consumption, also increases. There- fore, when the concentrations of cytochrome oxidase are in- creased, the steady state level of cytochrome c is moved toward the electron acceptor side. Thus cytochrome c is less reduced at higher cytochrome oxidase concentration, as is clearly shown in Fig. 7.

SpeciJic Activity of Cytochrome Oxidase Preparations

SpeciJic Activity as TN-The specific activity of an enzyme has been conventionally expressed in terms of the activity per unit weight of enzyme protein, so that the increase in purity can be directly indicated as the increase in the specific activity, assuming that the enzyme activity is not damaged by purifica- tion. However, this type of expression of the specific activity does not reveal whether the enzyme has been purified without damage to the activity, or whether the activity per unit en- zyme, such as the activity per mole of enzyme, is constant during purification. Since it has been shown (24, 31, 44) that the activity of highly purified preparations of cytochrome oxidase is considerably inhibited by cholate, which is used during purifi- cation of this enzyme, the specific activity expressed per protein weight basis, such as QoZ (microliters of oxygen per hour per milligram of protein), is not suitable to express the cytochrome oxidase activity of purified preparations. For cytochrome oxi-

6 To distinguish the t+ of the reduction from that of the oxi- dation, the t+ of the former is expressed as t+ W.

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February 1962 T. Yonetani 555

dase, specific activity of purified preparations should be ex- pressed in terms of the activity per mole of heme a. In this way, the degree of enzyme damage is indicated by a decrease of specific activity. Therefore, the present expression of the cyto- chrome oxidase activity, namely TN, is ideal for comparing the activity of the present purified preparation with that of the initial heart muscle preparation and for determining the degree of enzyme damage in the purified preparation.

The cytochrome oxidase activity of several insoluble heart muscle and mitochondrial preparations was determined in 0.05 M phosphate buffer, pH 7.2, in the presence of 1 mM EDTA and 20 mM ascorbate. TN,,, (cyt. c) values are found to be 80 to 100 at 25” for the following preparations: beef and horse heart muscle preparations6 collected either by acid precipitation or by high speed centrifugation at 10,000 x g (See reference (1) for preparation procedure), rat liver mitochondria’ (45), and pigeon heart muscle mitochondria (46) .r Therefore, the TN,,, (cyt. c) value of the present purified cytochrome oxidase prepa- ration corresponds to 70 to 90% of that of the initial insoluble preparations.

Changes in TN During Pur$cation-Changes in TN values of the cytochrome oxidase activity during purification were ex- amined. As is evident from the results presented in this paper, the cytochrome oxidase concentration as well as the ascorbate and cytochrome c concentrations must be kept in mind in the assay of the cytochrome oxidase activity. Fig. 8 illustrates the changes in TN value of cytochrome oxidase and some properties during the course of purification of the enzyme from a beef heart muscle preparation according to the method described previ- ously (1, 3, 31). The assay was carried out at fixed concentra- tions of ascorbate (50 mM), cytochrome c (20 j&M), and cyto- chrome oxidase (0.5 FM) and in 0.05 M phosphate buffer, pH 7.2, containing 1 InM EDTA. Preparations of high purity are usu- ally obtained by repeating ammonium sulfate fractionation more than six times in the presence of 2’% cholate (See refer- ences (1, 31) for preparation procedure). In Fig. 8, prepara- tions are named by the number of the ammonium sulfate frac- tionation repeated to prepare them. Since average recovery of cytochrome oxidase in each ammonium sulfate fractionation is found to be about 70%, the amount of cytochrome oxidase of a preparation designated AS-6 is approximately 120/,, O.76 X 100, of that of the initial heart muscle preparation. If the assay is carried out in the phosphate buffer without Emasol 4130, the specific activity decreases significantly during the process of purification. The lowered specific activity of preparations of higher purity, such as Preparations AS-4, -5, and -6, is signifi- cantly increased upon an addition of 1% Emasol 4130 to the reaction medium, in agreement with the previous report (24).

6 Heart muscle preparations collected by high speed centrifuga- tion show somewhat higher heme a content, 1.1 X leg to 1.4 X 10-s moles per mg of protein, and somewhat higher Qoa values. However, no difference in the specific activity as TN,,,(cyt. c) was observed between preparations collected by high speed cen- trifugation and by acid precipitation.

7 Intact mitochondria often shows a low cytochrome oxidase activity, because added cytochrome c is inaccessible to bound cytochrome oxidase. The maximal cytochrome oxidase activity of mitochondrial preparations can be obtained after causing the mitochondria to swell by using hypotonic solutions or low con- centrations of surface-active agents, as reported by Smith and Conrad (7, 8). Therefore, in the present experiments, mito- chondrial suspensions were pretreated with 2% cholate and then diluted to appropriate concentrations for activity assay; under these conditions, cholate shows no appreciable inhibitory effect.

iz 80

2

g 60

I ‘8-l pH7.6- _

E 20 0) z . . d

.p+l5.9, >

0 I I I F& \

0 100 200 300 400 1630

[Cytochrome Oxidase] IO-’ M

FIG. 7. The effect of the cytochrome oxidase concentration on aerobic steady state reduction of cytochrome c at six different pH values (5.9,6.2,6.5,6.7,7.2, and 7.8). The result of Fig. 5 was replotted to demonstrate the relationship between the percentage reduction and the cytochrome oxidase concentration.

100 c Emosol-dialyzed

0.05M Phosphofe,pH 7.2

a._ I % Emcsol-added

- 2 8

2 20

0.5 JJM Cytochrome cwdase

2 0 pM Cytochmme c 50 ml.4 Ascorbate

f I % Emasol~4130

without Emasol - 0’ ” ” ’

Preparation hMp AS-I AS.2 AS-3 AS-4 AS-5 AS-6

Heme component a.&,~ a,ba 0.s 0.~ O,CI ok3 0

pr&in / Hemeo IO6 5X105 3x10’ 2x105 I-l.3xlo5

Lipid content (%I -40 -30 -30 -20 -20 -10

FIG. 8. Changes of TN values during purification and effect of Emasol 4130. The activity was determined polarographically in 0.05 M phosphate buffer, pH 7.2, containing 1 mM EDTA in the presence of 50 mM ascorbate, 20 PM cytochrome c, and 0.5 PM cyto- chrome oxidase at 25”. HMP, a heart muscle preparation; puri- fied preparations are indicated by the number of ammonium sul- fate fractionation repeated to prepare them, such as AS-l, -2, -3, -4, -5, and -6; O-O, preparations prepared and assayed with- out Emasol4130; O-0, preparations prepared without Emasol and assayed in the presence of 1% Emasol 4130; O-0, prep- arations solubilized with, dialyzed against, and assayed in the phosphate buffer containing 1% Emasol4130. General properties of preparations are also illustrated which were obtained by the methods described previously (1, 3).

I f these preparations are solubilized with, dialyzed against, and assayed in the phosphate buffer containing 1% Emasol 4130, the specific activity of these preparations is further increased; the TN value of the Emasol-reactivated Preparation AS-6 is 25 to 27, which corresponds to about 80 to 90% of the specific activity of the initial heart muscle preparation (HMP in Fig. 8). Since the specific activity of Preparation AS-6 prepared and assayed without Emasol 4130 is -1.0, a 25- to 27-fold higher specific activity is obtained by the Emasol treatment in the

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556 Cytochrome Oxidase. IV Vol. 237, Tu’o. 2

case of Preparation AS-6. It should be noted that the Emasol reactivation is not observed in preparations of early stages of purification, but only in those of highly purified stages, as shown in Fig. 8. Since these preparations are apparently soluble in the absence of Emasol 4130, at least, during the assay, the effect of Emasol 4130 is not simply due to solubilization of cytochrome oxidase. It, may be attributed either to the fact, that Emasol removes cholate which is bound to the enzyme and inhibits the enzyme activity, or to the fact that Emasol takes the place of some cofactor which is assumed to be essential for the enzyme activity (47-51) and has been removed during purification. Since not, only Emasol4130, but also many other nonionic syn- thetic detergents, have shown the reactivation effect without exception so far as tested (24), the latter possibility, assuming a requirement of some naturally occurring phospholipid for the cytochrome oxidase activity, seems to be unlikely.

DISCUSSION

Reducing Agent-A number of agents have been used as re- ducing agents of the cytochrome c oxidase system, such as cysteine, p-phenylenediamine, hydroquinone, and ascorbate. Ascorbate is chosen for the present study for the following rea- sons: (a) it can rapidly reduce cytochrome c (5); (b) its oxida- tion product is relatively colorless, making it especially suitable for spectrophotometry (1, 2, 52); (c) its oxidation product is least inhibitory to the cytochrome oxidase activity (5) ; (d) it can reduce only added cytochrome c, but not endogenous cyto- chrome c of a heart muscle preparation (5), which is important in order to compare the activity of a heart, muscle preparation with that of a purified cytochrome oxidase preparation which does not contain endogenous cytochrome c; and (d) it is one of the few substances which has been shown to be useful as the electron donor for the oxidative phosphorylation of the cyto- chrome c-cytochrome oxidase system (53-57), in view of a con- tinuation of these studies with respect to investigations on oxida- tive phosphorylation.

Cytochrome c Requirement, K, (cyt. c)-Since the relationship between the cytochrome oxidase activity and the cytochrome c concentration exhibits a rectangular hyperbolic curve, and the Lineweaver-Burk plot follows the Michaelis-Menten equation (58), Stotz, Altschul, and Hogness (17) and Borei (19) concluded that cytochrome c forms a dissociable enzyme-substrate com- plex with cytochrome oxidase. This conclusion was criticized by Slater (5) for the following reasons: (a) it is unlikely that a rapid equilibrium is established between the cytochrome oxidase attached to insoluble heart, muscle particles and soluble cyto- chrome c; (b) this view does not explain the fact that the ac- tivity depends not, only upon the cytochrome c concentration, but also upon the reducing agent concentration; (c) in contrast to the Michaelis-Menten theory (66), the value of K, (cyt. c) is dependent upon the cytochrome oxidase concentration. The present work confirms that at least the latter two reasons are correct in a soluble system, and is entirely in favor of Slater’s view that the relationship activity-substrate concentration is a rectangular hyperbola, but that it is not necessary to postulate that a dissociable complex is formed.

As is evident from Table I, if the cytochrome oxidase concen- tration exceeds the critical value of 100 X 10-g M, the activity is limited by either cytochrome c or ascorbate concentrations, or by a combination of both. Reactions 1 and 2 are rate-limiting steps of System 1 under these conditions. Since the effect of

cytochrome c and ascorbate concentrations on the activity are correlated, not only with each other, but also with the cyto- chrome oxidase concentration, it is difficult to characterize the effect of one particular reactant without considering its relation- ship to the other reactants. The equation presented in this paper clearly illustrates the interrelationship of these reactants. At a fixed cytochrome oxidase concentration, the cytochrome c requirement for the activity, as indicated by the K, (cyt. c) value, decreases on an increase in the ascorbate concentration, whereas at a fixed ascorbate concentration, the cytochrome c requirement is proportional to the cytochrome oxidase concen- tration.

Maximal Activities, TN,,, (cyt. c) and TN,,, (cyt. oxidase)- To eliminate the rate-limiting effect of Reactions 1 and 2 of System 1, Slater obtained the cytochrome oxidase activity by extrapolation to an infinite cytochrome c concentration (5). Since cytochrome c and cytochrome oxidase are in the electron donor-acceptor relationship, the maximal activity can be ob- tained by extrapolating the activity to an infinitely dilute con- centration of cytochrome oxidase (the electron acceptor) in- stead of extrapolating to an infinite cytochrome c (electron donor) concentration. As shown in Table I, maximal activities obtained by both methods, i.e. the values of TN,,, (cyt. c) and TN,,, (cyt. oxidase), are in reasonable agreement, if appropriate ascorbate concentrations (10 to 40 mM) are employed.

The relationship of Fig. 1 indicates that cytochrome oxidase molecules are competing with each other to accept electrons from reduced cytochrome c molecules.

Variation of pH Optimum of Cytochrome Oxidase Activity-The observed variation of the pH optimum of the cytochrome oxidase activity (Fig. 3) can be explained if the following are noted; (a) the optimum of Reaction 2 is at, pH 5.7 to 5.9 (3); (b) Reaction 1 is more rapid at, higher pH values (Fig. 6); and (c) the optimal pH values obtained by polarographic and manometric methods are considered to be a sum of Reactions 1 and 2. If the pH curve is measured at lower cytochrome oxidase concentrations, the rate-limiting effect of Reaction 1 is less, and therefore the pH optimum moves toward that of Reaction 2 (pH 5.7 to 5.9). The best way to express the pH effect on the cytochrome oxidase activity obtained by polarographic or manometric methods is to plot the maximal activities such as TN,,, (cyt. c) or TN,,, (cyt. oxidase) against pH values, where the rate-limiting effect of Reaction 1 will be minimized. The dotted lines in Fig. 3 show the pH curves of TN,,, (cyt. oxidase), the pH optimum of which moves more toward pH 5.7 to 5.9.

Assay Methods of Cytochrome Oxidase Activity-The following scheme illustrates possible reactions involved in the cytochrome oxidase assay.

1 Reducing agent -

2 cytochrome c -

(calorimetry) (spectro- photometry)

Reactiir41,2,3, Reactions 2 and 3

3 cytochrome oxidase --+ (spectrophotometry with flow technique)

Reaction 3

oxygen (dissolved) +j-- oxygen (gas phase) (polarography) (manometry)

Reactions 1, 2, Reactions 1, 2, 3, and 3 and 4

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February 1962 T. Yonetuni 557

Reaction 1 is a nonenzymic electron transfer. Reactions 2 and 3 are enzymic electron transfers. Reaction 4 is oxygen diffusion from gas phase to aqueous phase. Cytochrome oxidase activity has been measured in the following ways.

1. Measurement of Utilization Rate of Reducing Agents with Reactions 1, 2, S, and d-Since Reaction 1 was often found to be first order with respect to the concentrations of both reducing agents and oxidized cytochrome c (22, 26), the over-all activity measured by this method is significantly affected by Reaction 1, which is not directly connected with the cytochrome oxidase action. In addition, since multiple reactions are involved in this assay, the experimental results are often too complicated to analyze. Therefore, this method has been almost completely replaced by the next spectrophotometric method.

2. Measurement of Oxidation Rate of Reduced Cytochrome c with Reactions 2 and 3 (Spectrophotometry)-It has been shown that the over-all reaction of this system is tist order with respect, to reduced cytochrome c concentration (6-13); this indicates that Reaction 2 is the rate-limiting step of the system. Kinetic analysis of this system was extensively carried out by Smith and Conrad (6, 7), who found that the activity in terms of the first order velocity constant, varies with changes in the total cyto- chrome c concentration of the reaction medium and is propor- tional to the enzyme concentration. These results have been confirmed by others (3, 9, 26, 27).

3. Measurement of Oxygen Utilization Rate in Reaction Medium with Reactions 1, 2, and S (Polarography)-A large excess of re- ducing agents is usually used so that it can reduce oxidized cyto- chrome c rapidly enough to minimize the rate-limiting effect of Reaction 1, and so that one can consider its concentration to be practically constant during measurements. Cytochrome c is also used in a large excess for similar reasons. As demonstrated in the paper, the cytochrome oxidase activity for a variety of combinations of reactant concentrations can be readily meas- ured by this method. Thus the experimental results can be analyzed in detail, although multiple reactions are involved.

4. Measurement of Oxygen Utilization Rate in Gas Phase with Reactions I, 2, S, and 4 (Manometry)-Since the cytochrome oxidase reaction is carried out, in an aqueous medium, the experi- mental conditions, such as reactant concentrations, medium constituents, pH, and temperature, must be practically identical for both polarographic and manometric methods. In manom- etry, the oxygen concentration in the reaction medium should be kept above a critical value, so that Reaction 4 is not rate-limit- ing. The only differences to be considered are the effect of re- action products of reducing agents and the stability of the enzyme preparations, since in manometry the amounts of the reaction products are theoretically unlimited as long as oxygen is supplied, and a longer period of time is usually required for measurement,. Therefore, if appropriate reducing agents and stable preparations are employed, the results obtained by both methods can be directly compared.

Specific Activity and Emasol Effect-Slater (5) reported that the Qoz value of a Keilin-Hartree heart muscle preparation is 3400 at an infinite cytochrome c concentration and at 38”. Since the heme a content of a Keilin-Hartree preparation was reported to be 1 X 10-g moles per mg of protein (59), the Qo2 value of 3400 corresponds to a TN,,, (cyt. c) value of 150. If the tem- perature difference (25” and 38”) is considered, TN,,, (cyt. c) values of 80 to 100 of insoluble preparations presented here are in reasonable agreement with Slater’s value of 150. Previously

Smith (52) and Griffiths and Wharton (28) reported that the specific activity per heme a of their purified cytochrome oxidase is the same as that of their heart muscle preparations.

In order to increase its spectral purity and to decrease its lipid content, the present purified preparation has been made by a fairly drastic procedure including more than six-times re- peated ammonium sulfate fractionations. Therefore, the final preparation (AS-6 of Fig. 8) is considerably susceptible to the inhibitory effect of cholate and thus shows a very low specific activity. An increase of the activity upon the addition of Ema- sol 4130 is significant in a preparation of a low specific activity such as Preparation AS-6. A comparative study between Ema- sol- and cholate-solubilized preparations, previously reported in detail, shows that the Emasol-solubilized purified cytochrome oxidase is in a more native state than the cholate-solubilized one with respect to specific activity, inhibitor response, and spectral properties (24). Therefore, the Emasol-treated prep- aration and the assay in the presence of 1 To Emasol4130 will be considered to be the established standard preparation and assay procedure of our method. For this reason, the present kinetic analysis of System 1 was carried out in the presence of Emasol 4130. If the preparation is made and assayed without Emasol, various kinetic constants and parameters may be different from those reported here, because of a strong inhibitory effect of cholate. Such a cholate effect was not discussed here, since it should be discussed together with that of other inhibitors.

Cytochrme Oxidase Concentration and Cytochrome Oxidase Study-Since spectroscopically pure and enzymically active purified preparations of cytochrome oxidase have become avail- able, a number of workers have investigated properties of this enzyme from various viewpoints. One of the highest concen- trations of cytochrome oxidase available is about 2 x 10-3 M

with respect to heme a concentration (3), whereas the lowest concentration of the enzyme at which the activity can be ac- curately measured is about, 1 x 10mg M (3). All the studies on enzymically active cytochrome oxidase have been carried out within this range of concentration, as shown in Fig. 9; thus the studies have been performed at the maximal gradient of cyto- chrome oxidase concentration of 2 X lo6 M; 2 X 10-S M/I X

10-g M. As shown in the present paper, even in a polarographic assay of the cytochrome oxidase activity, where the maximal concentration gradient, of the enzyme was only 1.6 X 10 (= 4 X 1O-7 ~/2.5 X 10e8 M), the concentration has a considerable effect on the TN values. Therefore, in the analysis of the experimental results, careful attention must be paid to the cytochrome oxidase concentration used.

SUMMARY

1. The activity of a highly purified preparation of beef heart muscle cytochrome oxidase was polarographically investigated in the system, ascorbate-cytochrome c-cytochrome oxidase- oxygen.

2. The specific activity of the cytochrome oxidase depends upon the cytochrome oxidase concentration as well as upon the ascorbate and cytochrome c concentrations.

3. The K, (cytochrome c) is not a universal constant but is a function of both ascorbate and cytochrome oxidase concentra- tions. This is illustrated by the following equation:

K, (cytochrome c) [cytochrome oxidasel

m [ascorbatel-+

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558 Cytochrome Oxicktse. IV Vol. 237, Xo. 2

Conccntrot ion St ud y Subject Investigator (Reference)

-2 .O 10-3M 5 (mM)

k

5

5 0

g 10-4M

E is

0

C

g 10-5M

iit I

c 10-6M 0 (PM) .-

E t; W 0 & 10-7M 0

z -5: .- x o IO-‘M

electron spin resonance of copper- Sonds-Beinert, ( 60 1

volume magnetic susceptibility _ 8. electron spin resonance

copper volency change -

Ehrenberg-Yonetani,l961(6 I)

Griffiths-Whartan,l961 (29)

- magnetic titration - Yonetani, 1960 (62)

- copper-chelotar P[Takemori,1960 (63) Yanetani.196 I (64)

- spectrophotometric titrotton- Chance-Yonetoni,l959(65)

Chance,l952-(40,42,43,69) - steady state properties Smith,l955(52)

Yonetani,l960(2)

-spectrophatometric kinetics --i

Chance,l952-(65-69) Gibson,,1 960 ( 70 1 Okunukr,l959(30,71,72)

monametric assay

polaragraphic assay

-Sloter,l949 (5) - many others, I9 3a4,14-30) _ Chance, 1952-(39,40,,66) - Yonetani, 1960”(2,this paper)

spectrophotometric assay SmithCanrod,l956- f 6 ,7) Wainio,l951-(IO-13,49,50) manyothers,1943-(3,8,9

,X,31, 47,73-76)

FIG. 9. Cytochrome oxidase concentration and various aspects of cytochrome oxidase study.*

4. The maximal specific activity of cytochrome oxidase can be calculated by extrapolating the activity either to an infinite cytochrome c concentration or to an infinitely dilute cytochrome oxidase concentration.

5. The pH optimum for the cytochrome oxidase activity of the system varies with changes in the cytochrome oxidase con- centration; lower optimal pH values are obtained upon decreas- ing the cytochrome oxidase concentration.

6. Reduction of cytochrome c by ascorbate is more rapid at higher pH values and at higher ascorbate concentrations.

7. The steady state level of cytochrome c is moved toward the more oxidized state either by increasing the cytochrome

* In the early literature, the enzyme concentration was often given in terms of milligrams of protein per milliliter. In these cases, the molar concentration of cytochrome oxidase was cal- culated by assuming that an average heme a content of insoluble heart muscle preparations is 1 X 10-g moles per mg of protein, according to Slater (59).

oxidase concentration or by decreasing the pH value of the medium.

8. Different assay methods of the cytochrome oxidase ac- tivity are compared.

Acknowledgments---The author is grateful to Professor Britton Chance for his encouragement and guidance during the course of this study. He also appreciates the valuable help and criti- cism of Dr. Helen Conrad.

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Takashi Yonetani and With the technical assistance of Patricia RainfordACTIVITY

Studies on Cytochrome Oxidase: IV. THE CYTOCHROME OXIDASE

1962, 237:550-559.J. Biol. Chem. 

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