On cytochrome c oxidase I. The extinction coefficients of cytochrome a and cytochrome a3

BIOCHIMICA ET BIOPHYSICA ACTA BBA

65360

ON CYTOCHROME c OXIDASE 1. THE EXTINCTION COEFFICIENTS OF CYTOCHROME a AND

CYTOCHROME as

B. F. VAN GELDER Laboratory of Biochemistry', University of Amsterdam, Amsterdam (The Netherlands) (Received August 20th, 1965)

SUMMARY

1. In agreement with GIBSON, a shoulder at 420-424 mfl is present in the absorption spectra of preparations of cytochrome c oxidase (ferrocytochromec :oxygen oxidoreductase, EC 1.9.3.1) reduced with Na 2S 20 4 • An examination of the spectra in the presence and absence of cyanide showed that this is due to the presence of equal amounts of non-reducible cytochrome a and non-reducible cytochrome aa' 2. Cytochrome c oxidase was titrated with NADH and phenazine methosulphate. It was found that each mole ofNADH reduced I mole of haem a and I atom of copper. 3. Functionally inactive copper present in the enzyme preparation, added Cu(II) and Fe(III) salts and mitochrome, a modification product of cytochrome c oxidase, are not reduced by phenazine methosulphate, under the conditions of the titration. 4. The values of LlSmM (reduced minus oxidized) at 605 mfh and 445 tiu», respectively, were: cytochrome a, 19.4 and 82; cytochrome aa, 4.6 and 82; cytochrome aaa' 24.0 and 164. 5. Concentrations of cytochrome a, calculated on the basis of the widely used value of 16.5 for LfsmM (A ao5 m,u-Aa30 m,u), are 150% too high.

INTRODUCTION From the experiments of KElLIN AND HARTREE1 it was clear that cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) contains two differently bound haem a groups. They named the components with the different haem a groups: cytochrome a and cytochrome a3 • Although cytochrome a and cytochrome aa have not been chemically separated • Formerly Laboratory of Physiological Chemistry. Postal Address: Amsterdam (The Netherlands).

Biochim, Biophys, Acta, !IS (1966) 36-46

J. D.

Meyerplein 3,

EXTINCTION COEFFICIENTS OF CYTOCHROMES a AND a 3

37

from one another, the relative proportions of the two components in cytochrome c oxidase-r" and their contributions to the absorbance of the difference spectrum are known 9 - 1S. Thus, it is possible to calculate the difference between the extinction coefficients of the reduced and oxidized forms of both cytochrome a and cytochrome a3 , We have examined this problem by a method introduced by MASSEy14 to determine the extinction coefficient of cytochrome c. MASSEY showed that phenazine mcthosulphate, reduced by succinate in the presence of succinate dehydrogenase (succinate: (acceptor) oxidoreductase, EC I.3.99. I), quantitatively reduces ferricytochrome c. By using an excess of ferricytochrome c and plotting the increase in absorbance at 550 mf-l against the number of reducing equivalents of succinate added, MASSEY was able to determine the molar extinction coefficient (reduced minus oxidized) for cytochrome c. These measurements were confirmed by VAN GELDER AND SLATER15, who showed further that succinate and succinate dehydrogenase could be replaced by NADH, which reacts non-enzymically with phenazine methosulphate. It was found also that reduced phenazine methosulphate could reduce cytochrome c oxidase. It is the purpose of this paper to consider the extinction coefficients of cytochrome a and cytochrome as separately and in the complex, while the following paper? will deal with the ratio of cytochrome a and cytochrome as present in cytochrome c oxidase. Some of the results have been reported in a preliminary form 2- 4 , l O. METHODS

Reagents NADH (g8% pure) and phenazine methosulphate were obtained from Sigma Chemical Co. EDTA, cyanide and most of the other chemicals used were Analar reagents supplied by British Drug Houses Ltd. "Pro analysi" ammonium sulphate was purchased fromE, Merck A.G. Emasol 4130 (polyoxyethylenesorbitan monooleate) was a preparation of the Kao Soap Co., Tokyo, Japan. Asolectin, a crude mixture of soybean phosphatides, was obtained from Associated Concentrates, Woodside, Long Island, New York, U.S.A. Neocuproine (2,g-dimethyl-I,Io-phenanthroline) was purchased from Fluka A.G., Buchs S.G. o-Phenanthroline hydrochloride "pro analysi" was obtained from E. Merck A.G. Cytochrome c and cytochrome c oxidase Cytochrome c was purified from horse heart according to MARGOLIASH16. The A 550 tau (reduced) :A 280 uui (oxidized) was 1.22-1.30. The concentration was determined from the absorbance of the reduced cytochrome, using a millimolar extinction coefficient of 29.5 (ref. IS). Cytochrome c oxidase was prepared by the method described by YONETAN19,17, slightly modified. 3.5-4 kg of fresh horse-heart mince were washed 4-6 times with tap water of 0°. From the washed mince a Keilin and Hartree heart-muscle preparation was prepared-", The heart-muscle preparation was suspended in 0.1 M phosphate buffer (pH 7.8) using a Waring Blender, the pH was adjusted to pH 7.8 with 3 M ammonia and the final volume was brought up to 2.16 1. Biochim, Biophys, Acta, lIS (1966) 36-46

B. F. VAN

GELDER

240 ml 20% (w/v) of cholate (pH 7.8) and 360 g of solid ammonium sulphate were added. The solution was left standing for z h at 4-5° and then brought to 35 % saturation with 144 g solid ammonium sulphate. After standing 20 min at 4-5° it was centrifuged 4S min at 10 000 X gat 0°. The precipitate was discarded and pulverised ammonium sulphate (ro g/Ioo ml) was gradually added to the supernatant to bring it to So% saturation. After standing 10 min at 4-S ° the solution was centrifuged 10 min at 10 000 X g. The brown viscous precipitate was dissolved in 300 ml of a solution (pH 7.4) containing 0.02 Mphosphate and 1% cholate, 27% saturated with ammonium sulphate. After standing 2 h at 4-S ° the preparation was centrifuged at 2S 000 X g for IS min. The precipitate was discarded and saturated ammonium sulphate (pH 7.8) was added to the supernatant to 40% saturation. After centrifuging immediately at 10 000 X g for S min the viscous precipitate was dissolved in the phosphate-cholateammonium sulphate solution mentioned above. The centrifugation, precipitation of the supernatant with saturated ammonium sulphate and solution of the precipitate was repeated. At this stage the cytochrome c oxidase was usually free of other cytochromes as determined by visual spectroscopy. If this was not the case the precipitation with saturated ammonium sulphate was repeated. Finally the cytochrome c oxidase was dissolved in 0.02 M phosphate-a % cholate (pH 7.4) and kept at the temperature of liquid nitrogen. The concentration of cytochrome c oxidase is expressed in terms of its haem-a content, using LlemM (reduced minas oxidized) at 60S mp of 12.0. Measurement of cytochrome c oxidase activity Cytochrome c oxidase activity was assayed manometrically with differential manometers at 2So essentially as described by SLATER19 . The manometer constants, calculated according to VAN DORP AND SLATER2 0 , were 0.5-0.7,ul 02/mm manometer fluid. The reaction mixture contained, in a volume of 1.1 ml, 30 mM ascorbate, 45 mM phosphate, 0.1 mM EDTA, I mg asolectin, 0.9% Emasol, 0.01-0.1 mM cytochrome c and about 5/-lg cytochrome c oxidase. The pH was 7.3. The reaction time was 45 min. The concentrated cytochrome c oxidase was diluted with a solution containing 0.25 M sucrose, 1% Emasol and 0.1% asolectin. The asolectin sol was prepared according to GRIFFITHS AND WHARTON 21 • Meas~trement

of spectra The spectra of the preparations were measured with a Cary Model-I4 recording spectrophotometer, recording the absorption spectrum from 700 to 400 mp in I min, or with a Zeiss PMQ II spectrophotometer. The preparation was diluted-in 0.1 M Na 2HP04 and 1% Emasol. The reduced spectrum of cytochrome c oxidase was measured aerobically in the presence of a few grains of dithionite. After the absorbance at 444 mp became constant, the spectra were measured. Titration lvith NADH and phenasine methos~tlphate The titration was carried out under anaerobic conditions. A r-em Thunberg cuvette contained 0.1 M phosphate buffer, 1% cholate and 10-100 pM cytochrome c Bioohim. Biophys. Acta, lIS (I966) 36-46

EXTINCTION COEFFICIENTS OF CYTOCHROMES a AND a3

39

oxidase in a volume of 2.25 ml. The final pH was 7.3-7+ A reference cuvette contained the same solution. One compartment of a two-compartment stopper of the Thunberg cuvette contained various amounts of NADH in a volume of 0.2 ml and the other 0.05 ml O.OI % phenazine methosulphate. The contents of the reference cell were diluted with 0.25 ml water. The Thunberg tube was flushed six times with purified nitrogen, which had been washed through a IO% pyrogallol-tro'z, KOH solution, and evacuated. The reaction was started by tipping in NADH and phenazine methosulphate. The increment of absorbance was followed on a Cary Model-ra spectrophotometer until the slope of the absorbance against time was constant. By extrapolating to zero time the immediate increment in absorbance was found. The NADH concentration was determined from the total absorbance at 340 mil, using an 8mM of 6.22 (see ref. IS). A nalytical methods The protein concentration was determined by the biuret method in the presence of hydrogen peroxide, according to YONETANI 17 . Iron was determined by the 0phenanthroline method, as described by YONETANI 17. Copper was determined as the Cu(I)-neocuproine complex after wet ashing with cone. H 2S0 4 and H 20 2 by the method of SMITH AND MCCURDy 22. Haem a was determined by adding pyridine and 3 M NaOH to dithionite-reduced preparations to a final concentration of IS % and 0.3.5 M, respectively. The absorbance at 587 mfl- was determined and the haem a calculated assuming a 8m M of 30 (ref. 23). The same value was obtained whether or not oxygen was removed from the solution before adding the Na 2S20 4 •

RESULTS Absorption spectra of cytochrome c oxidase The absorption spectra of our cytochrome c oxidase preparations were similar to the reported spectraO,12,21,24-27. The maxima for the reduced enzyme were at 604-605 mfl-, 518-520 mp and 443-444 mzz ; for the oxidized enzyme the maxima were at 598-600 mfl- and 42I-424 mfl-. The shoulde« in the reduced y-band All our preparations have i~ the reduced spectrum an asymmetric y-band with a small shoulder at 420-424 m!". Since protohaem could not be detected (as pyridine protohaemochrome), it is probable that the shoulder is due to a non-reducible modification product of cytochrome c oxidase, as suggested by GIBSON et ai». In contrast, however, to the conclusion of GIBSON, PALMER AND WHARTON 5 that mainly cytochrome aa is modified, the data assembled in Table I show that the same amounts of cytochrome a and a3 are modified. This follows from the constancy of the ratios ,1A 445 mp, (reduced minus oxidized) :LlA s 05 mp, (reduced mima oxidized) and LlA445 mp, (reduced minus oxidized), measured in the presence of cyanide rzl .11 446 lilt (reduced minus oxidized), measured in the absence of cyanide, despite considerable variation from preparation to preparation of the ratio A 444 mil (reduced) :A 424 mil (reduced). If more cytochrome aa than cytochrome a were modified, the ratio L1A 445 mil (reduced mimts oxidized) :L1A eo5 mil (reduced mimts oxidized) would vary in the same direction as the ratio LlA 444 mp, (reduced) :LlA424 mp, (reduced), while the ratio L1A 445 mit (reduced Biochim. Biophys. Ada, lIB (1966) 36-46

B. F. VAN GELDER

40

minu« oxidized), in presence or absence of cyanide, would vary in the opposite direction. It should be noted that the ratios are calculated after complete reduction of cytochrome c oxidase, i.e. after the absorbance at 444 mf-l has reached its maximum and did not change over a further 10 min. The maximum in absorbance is mostly reached after 20-30 min. As has been shown by LEMBERG et af.l 2 the reduction of a 33 +CN to a 3 z+CN is very slow. In the presence of cyanide the absorbance was TABLE I SOME SPECTRAL CHARACTERISTICS OF DIFFERENT CYTOCHROME C OXIDASE PREPARATIONS

Except where indicated, the cytochrome c oxidase was reduced by Na.S.0 4 •

Prep. A 444 trI# (reduced) : .'1,12'1 "'#

(reduced)

86 87 85

z3 84 71 89

1.60 1.7 2 1.79 1,85 1. 87 1,96 2.15

LlA H 5 mil {reduced minus oxidieed}: LlA oo5 mit (reduced minus oxidized)

LlA 445 trI# (reduced minus oxidized) a,: LlA 4,15 "'1' (reduced minus oxidieed )»

6·9 6·7 6.9 7·3

0·5° 0.5 1 0.5 0 0.4 8' 0·5° 0.5°" 0.5 0

6·7 7. 0

, Reduced anaerobically with IS ~!M cytochrome c and 50 mM ascorbate. •• 9.S/tM cytochrome c oxidase was reduced anaerobically with 12 ~M NADH and 1.3 ~!M phenazine methosulphate, a In presence of 10 mM KeN. b In absence of cyanide.

measured within 5 min of addition of difhionite. The same values were obtained when reduced phenazine methosulphate or ferro cytochrome c was used as reducing agent (see Table I). The activity of cytochrome c oxidase The activity of cytochrome c oxidase was determined manometrically with ascorbate as reducing agent. Qo. (,ul Oz/mg protein/h) values, at infinite cytochrome c concentration-", of 21 000-24 000 at 25 0 were usually found, with an occasional value up to 29 000. The reduction oj cytochrome c oxidase with NADH and phenazine methosulphate Fig. I shows the effects on the absorbance at 445 mf-l of the addition anaerobically of phenazine methosulphate, in the presence and absence of NADH. As can be seen phenazine methosulphate alone caused a small increase in the absorption. In this particular experiment the reduction rate was constant after 3 min. This rate is strongly pH dependent, e.g. at pH 7.6 LlA 445 m.u/min was 0.025 while at pH 7.3 it was only 0.001. In the absence ofphenazine methosulphate or NADH there was no reduction at all of cytochrome c oxidase within I h. The rapid increment in absorbance due to phenazine methosulphate was determined by extrapolating to zero time, as shown. Biochim. Biopbys. Acta,

r r S (I966) 36-46

EXTINCTION COEFFICIENTS OF CYTOCHROMES

a AND a3

41

0.6,-----...,-----,-----.,------,

_ _ 1I

4

8 Time (min)

12

16

Fig. 1. The reduction of cytochrome c oxidase with phenazine methosulphate, under anaerobic conditions, (I) in the presence and (II) absence of NADH, measured at 445 mft.

NADH and the same amount of phenazine methosulphate showed a large rapid increase in absorbance, but after 3 min the rate of reduction had become the same as with phenazine mcthosulphate alone. The absorbance increment due to NADH was found by subtracting the extrapolated absorbances in absence of NADH from that obtained in its presence. The absorbance increment found in this way is plotted against the NADH concentration in Fig. 2. Titration of cytochrome c oxidase

Fig. 2 shows the results of the titration at 445, 605 and 830 mf'. From the slope of the straight lines extinction coefficients (reduced minus oxidized) of 82, 12.0 and 0.g8, respectively, per millimolar NADH could be calculated. 0.3

0.04

0.2

0.02

a
~ a

E 0.2

111

"'

SJ

""j'

a,

~

10

20

30

°0

"

""

'
4 8 12 NADH (PM)

0.1

2

3

Fig. 2. Titration of cytochrome c oxidase, under anaerobic conditions, with NADH and phenaztne mcthosulphate at 830 mp, 605 mp and 445 tsui. The final concentrations of cytochrome c oxidase were, based on the haem a content: roo pM, 58 pM and r6 ~tM at 830 m/-", 605 m/-" and 445 m/-", respectively. The titration was carried out as described in the text. The absorbance increment was corrected for the absorbance increment in a similar experiment without any NADI-1 added. At 830 m!t the Cary Model-r a spectrophotometer was supplied with a slidewire of 0-0. r.

In order to calculate the true extinction coefficients for cytochrome c oxidase, it was necessary to know whether one mole NADH reduces one mole of the chrornophores responsible for absorption at 445 m,u, 605 mp, and 830 m,u, or two moles. In the case of cytochrome c, one mole NADH reduces two moles of ferricytochrome c (refs. 14,15). Biochim. Biophys, Acta, II8 (l966) 36-46

B. F. VAN

GELDER

Calculated on the iron and haem a (determined by pyridine haemochrome) contents of the cytochrome c oxidase preparation, LlemM (reduced minus oxidized) values of ro.g and II.8, respectively, were obtained at 605 m{l. These may be compared with the values, based on total iron contents, of 10-4 reported by GRIFFITHS AND WHARTON 21 and 11.0 by YONETANI 17. Since these values are only slightly less than the extinction coefficients per millimolar NADH, and only one reducing equivalent is required to reduce ferrihaem a to ferro haem a, it appeared most probable that a second reducing equivalent is used to reduce another electron acceptor in cytochrome c oxidase. The side-chains of the porphyrin molecule (e.g. the formyl group) could be excluded as the acceptor of the second reducing equivalent, since cytochrome c oxidase reduced by NADH and phenazine methosulphate had the same absorption spectrum as that given with dithionite and yielded the same pyridine haemochrome, Using the procedure of GRIFFITHS AND VVHARTON 21 , 28, it was found that the second acceptor of electrons from phenazine methosulphate in the cytochrome c oxidase is Cu(II), shown to be present in cytochrome c oxidase by BEINERT and coworkers 2 0 - 31 and by TAKEMORI 3 2 . 'When an excess of NADH in the presence of phenazine methosulphate was used, 0.gO-LI5 atoms of Cu(I) per haem a molecule" were formed. With limiting amounts of N ADH 0.g8 atom Cu(I) was formed per molecule of haem a reduced" (see ref. 7 for the procedure). Fig. 3 shows a complete titration at 605 tup: In this case the "blank" measurement with phenazine methosulphate alone was not subtracted. The titration is quite

0.5

t 0.4 '" ~ 0.3 -sa

0.2

o.lL o -10

0!:---1:':O-~20::-----='30:::----'4'="O---:5:':0:------' NADH (,uM)

Fig. 3. Titration of cytochrome c oxidase, under anaerobic conditions, with NADH and phenazine methosulphate at 605 m!l. The final concentrations were: 0,09 M phosphate buffer (pH 7.3), 0.9% cholate and 46.6 !IM cytochrome c oxidase, based on haem a content. The titration was carried out as described in METHODS . • • reduced with an excess of Na 2S 20 •.

sharp, the oxidase being completely reduced when an amount of NADH (including the amount obtained by extrapolating to zero absorbance increment) equal to Lor times the haem a content of the enzyme was added. I t is of particular interest that, although the preparation contains considerable amounts of copper in excess of haem a (CuJhaem*a = 1.47), some of which might be reducible by dithionite, the reduced phenazine methosulphate under the conditions of the titration reduces only one atom of copper per haem a molecule. Indeed as can • These haem a concentrations were calculated from the absorbance at 605 m!1 (see METHODS.

Biooh.im, Biophys. Acta, IIS (1966) 36-46

EXTINCTION COEFFICIENTS OF CYTOCHROMES

a AND as

43

TABLE II THE EFFECT OF ADDITION OF

Addition

Fe(III), Cu(II)

OR MITOCHROME ON THE TITRATION AT

605 mp.

LJA/mM NADH

None 12.0 Fe(III) - 0,13 roM 12.0 Cu(II) - 0.13 mM I1.8 Mitochrome* 12.1 * Prepared by ageing cytochrome c oxidase at room temperature for 3 weeks, and removing the sediment by centrifuging for 10 min at 20 000 X g. The a band had almost disappeared while the y band was split into two, at 442 m/l and 418 m/l, respectively (cj. ref. 25).

be seen in Table II the addition ofCu(II) had no effect on the titration. Nor had the addition of Fe(III) or mitochrome, which also might be present in some preparations (see Table II). Extinction coefficients of cytochrome a, cytochrome as and cytochrome c oxidase Since the ratio of cytochrome a and cytochrome as in cytochrome c oxidase is I (see refs. 2-8) and the contribution to the difference spectrum (reduced minus oxidized) of a and as is known O- 13 , the extinction coefficients of the twocyto chromes can be determined. It was found that cytochrome a contributes 50% to the difference spectrum at 445 m/-l (see Table I) and 8I % at 605 m/-l (see refs. 9-I3). In Table III the various extinction coefficients are listed. Since it is known that TABLE III THE EXTINCTION COEFFICIENTS OF CYTOCHROMES

a

AND

as

AND OF CYTOCHROME

aa s

L1 BmM (reduced-oxidieed}

Cytochrome a Cytochrome as Cytochrome aa s (a

+ as) *

605 mfl

445

19.4 +6

82 82 164

24,0

* Based on a molecule containing

2

mu.

haem a molecules.

cytochrome a and cytochrome as are present in equal amounts in cytochrome c oxidase, its extinction coefficient should be based on two haem a molecules. Thus the Llsm M (reduced minus oxidized) at 605 mfl is 24.0 instead of 12.0, which is based on only one haem a molecule. DISCUSSION

The enzyme activity ofthe preparations used in this study (Q02 = 21000-29000 at 25°) is comparable with those of GRIFFITHS AND WHARTON 21 (Q02 = 50 000-60 000 at 38°, which becomes 24°00-:28500 at 25°, when the QI0 OfLlS found by MINNAERTs3 Biocliin». Bioplws. Acta, rr8 (1966) 36-46

44

B. F.

VAN GELDER

is applied). The straight lines obtained in the titrations (Figs. 2 and 3) suggest rather strongly that both types of ferrihaem a molecules and the copper atoms (it will be shown in the following paper" that two types of copper are present) are reduced at about the same speed suggesting that the acceptors are not acting independently of one another. GIBSON AND GREENWOOD 34 have also shown that in the titration of reduced cytochrome c oxidase with molecular oxygen, the enzyme reacts as a single unit and that 1 molecule of oxygen oxidizes 1.8 molecules of reduced cytochrome c oxidase. In this respect, reduction by dithionite would appear to differ from that by reduced phenazine methosulphate, since ELLIOTT 3 5 , LEMBERG AND MANSLEy36 and GIBSON AND GREENWOOD37 have shown that dithionite reduces cytochrome a faster than cytochrome a3' However, the experimental conditions are quite different. In the experiments with dithionite, amounts of reducing agent stoicheiometric with the enzyme are required. In our titration procedure, reduced phenazine methosulphate is present only in catalytic amounts (the total concentration of phenazine methosulphate is about 1/20 of that of the enzyme). It is now well-established that copper undergoes oxidation and reduction in the enzyme. This has been demonstrated by electron-spin resonance spectroscopy (refs. 29-31,30-40), chemical copper analysis 28 ,32 and kinetic studies". Our titration shows that when one molecule of haem a is reduced, simultaneously one atom of copper is reduced. This is in complete agreement with the chemical copper determinations of TAKEMORI 3 2 , GRIFl~ITHS AND WHARTON 28 and our own 3,7. The reaction equation of the reduction of cytochrome c oxidase by NADH and phenazine methosulphate may be written as: (PMS)

NADH

+ cytochrome c oxidase [Fe(III), Cu(II)] ~ NAD+ + H+ + cytochrome c oxidase [Fe(II), Cu(I)].

It is interesting to note that, although the preparation contained copper in excess of haem a, under the conditions of the titration only one atom of copper was reduced per haem a molecule reduced. When Cu(II) was added to the enzyme no change in the slope of the straight line was found, suggesting that only the active copper could be reduced. This has also been demonstrated by unpublished measurements of the electron-spin resonance carried out by H. BEINERT AND B. F. VAN GELDER. When cytochrome c oxidase was completely reduced by reduced phenazine methosulphate, the signal of the enzymatically active copper disappeared, while that of the enzymically inactive copper is still present even IS min after the reduction was completed. This would strongly suggest that, under the conditions of the titration, the inactive copper could not be reduced or at least that the reduction is much slower than the reduction of the active copper. BEINERT AND PALMER31 showed that cytochrome c and ascorbate do not reduce the inactive copper, while dithionite reduces both the enzymatically active as well as the inactive copper. Similarly, added Fe(III) and mitochrome, a modification product of cytochrome c oxidase'", do not affect the slope ofthe line, again suggesting that if they are reduced the reduction is much slower than the reduction of the haem a and the copper of cytochrome c oxidase. Since the preparations used have been shown to contain modified cytochrome c oxidase (cf. ref. S) that is not reduced by Na 2S20 4 or by reduced phenazine methoBiochim. Biophys, Acta. r r S (1966) 36-46

EXTINCTION COEFFICIENTS OF CYTOCHROMES a AND a 3

45

sulphate, it is not surprising to find that the extinction coefficient based on the titration with NADH is about 16% higher than that based on the iron determination. The titration procedure has the great advantage that it is not disturbed by the presence of enzymatically inactive iron or copper compounds. It is, however, surprising that the extinction coefficient based on the haem a content should agree so closely (within r%) with that based on the titration. It is possible that the value for the SmM of pyridine haemochrome used, viz. 30 (ef. ref. 23) is not correct under our conditions. The values given in the literature 2a , 41 , 4 2 vary between 26 and 33. If the lowest value was used, the extinction coefficient, LlAo05 mit (reduced minus oxidized) would become 10.2, the same as the value based on the iron content. Alternatively, it is possible that modified cytochrome aa 3 does not form a pyridine haemochrome a under the conditions employed. This does not, however, seem very likely, since mitochrome is converted to pyridine haemochrome a (see refs. 25043). A knowledge of the extinction coefficients of cytochrome a and a 3 , and of cytochrome oxidase (which contains equal amounts of the two cytochromes and may be designated cytochrome aa 3) , is important for the calculation ofthe relative concentrations of the cytochromes in mitochondria and particulate preparations derived from mitochondria. We should like to emphasize that the extinction coefficient of cytochrome e oxidase, i.e. cytochrome a phts cytochrome a 3 , should be based on two haem a molecules, and not on one haem a molecule, as is the invariable practice in the literature. The most commonly used extinction coefficient is the value of r6.5 for the LlSmM (A005 m/t-A o30 mil) of the reduced enzyme, based on one haem a (ref. 44). According to our data this should be 20.5 per haem or 41 per molecule of cytochrome aaa' The discrepancy could be accounted for by the presence of non-reducible haem a in YONETANI'S preparations (ref. 44). Thus, concentrations of cytochrome a determined on the basis of YONETANI'S extinction coefficient should be multiplied by the factor 16.5/41, i.e. 0+ NOTE ADDED IN PROOF (Received January r Sth, r966) Since submitting the above paper, a report by VANNESTE AND VANNESTE 45 has come to our attention. Based on the assumption that cytochrome a constitutes onehalf of the total haem a-containing proteins in the cytochrome e oxidase preparation, they calculated the following values for LfsmM (reduced minus oxidized): cytochrome a, 20 at 605 mil, 57.2 at 445 mp; cytochrome as, 4.8 at 603 m/J, IIZ at 444 mp. Although the values for the a-band are in reasonable agreement with ours, they differ very markedly in the Soret region. The reason for this discrepancy is that, as discussed above, equal amounts of non-reducible cytochrome a ancl cytochrome aa contribute to the absorption spectrum. ACKNOWLEDGEMENTS The author wishes to thank Professor E. C. SLATER for his advice and encouragement during the course of this work, Mr. A. O. MUIJSERS for his collaboration in some of the experiments and Mr. R. LE CLERQ for his technical assistance. The Cary spectrophotometer was purchased from funds made available by the Rockefeller Foundation. Biochim, Biopbys. Acta, II8 (I966) 36-46

B. F. VAN GELDER REFERENCES AND E. F. HARTREE, Proc: Roy. Soc. Landon Ser, B, 127 (1939) 167. GELDER AWn A. O. MurJSERS, Biochim, Biophys. Acta, 81 (1964) 405. 3 E. C. SLATER, B. F. VAN GELDER AND K. MINNAERT, in T. S. KING, H. S. MASON AND M. MORRISON, Osidases and Related Redox Systems, Wiley, New York, Vol. 2, 1965, p. 667· 4 B. F. VAN GELDER, Proc. 6th Intern. Congr . Biochem., New Y01'k, 1964, Abstracts, p. 791. 5 Q. H. GIBSON, G. PALMER AND D. C. WHARTON, j. Bioi. Chem., 240 (1965) 915. 6 W. H. VAN NESTE, Biochem . Bio pliys. Res. Commun., 18 (1965) 563. 7 B. F. VAN GELDER AND A. O. MUIJSERS, Biochim . Biophys, Acta, lI8 (1966) 47. 8 G. E. MANSLEY, J. T. STANBURY AND R. LEMBERG, Biochim, Biopliys. Acta, lI3 (1966) 33· 9 T. YONETANl, J. Bioi. 235 (1960) 845. 10 B. F. VAN GELDER AND E. C. SLATER, B'iochim. Biopliys. Acta, 73 (1963) 663. 11 S. HORm AND M. MORRISON, j. Bioi. Chem., 238 (1963) 2859. 12 R. LEMBERG, T. B. G. PILGER, N. NEWTON AND L. CLARKE, Proc. Roy. Soc. London Ser. B, I D. KElLIN 2 B. F. VAN

cu-«,

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Biochim, Biophys, Acta, lIS (1966) 36-46