THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 246, No. 24, Issue of December 25, pp. 6669-6674, 1970

Printed in U.S.A.

Absorption Spectra and Enzymatic Properties of Monomer and Dimer States of Cytochrome c Oxidase*

(Received for publication, June 15, 1970)

SAMUEL H. P. CHAN,$ BRUCE LOVE, AND ELMER STOTZ

From the Department of Biochemistry, School of Medicine and Dentistry, The Univewity of Rochester, Rochester, New York 14620

SUMMARY

The spectroscopic and enzymatic properties of a monomeric form of cytochrome oxidase are described and compared with those of the enzyme dimer. The dimer form of the enzyme, isolated by the Yonetani method, is dissociated into a monomer at alkaline pH (pH 9.5 to 11.0) in a medium con- taining a nonionic detergent such as Emasol. The data show that with several preparations of the enzyme the monomeric form retains 83 to 97% of the catalytic activity of the dimer. Ultrafiltration experiments with the use of a membrane that is able to discriminate between monomer and dimer demon- strate that reassociation to the dimeric form, under condi- tions closely approaching those used in the assay, is too slow to account for the observed activity. Therefore, in buffers containing nonionic detergents, exposure of cytochrome oxidase to alkaline pH in the range of 9.5 to 11.0 does not lead to denaturation.

The absorption spectra of the monomer form show that the y-Fez+ maximum is at 441 nm and the a-Fe2f maximum at 604 to 605 mn. The maxima for the y-Fe2f-CO and a-Fez+- CO bands are at 430 to 431 mn and 604 to 605 nm, respectively. The a-band of the Fez+-CO monomer is com- pletely symmetrical, with no shoulder at 593 nm. The y- Fe2+-CO band of the monomer is only slightly asymmetric and lacks the 444 nm shoulder present in the typical cyto- chrome aa3 (i.e. dimer) spectrum. A solution of the monomer was found to react with approximately twice as much carbon monoxide as a solution of the monomer of equal heme A content. Thus, in present terminology, the monomer ap- pears to have many of the properties attributed to cytochrome a3 in the typical cytochrome (~a~ preparation.

It was reported in the previous paper (1) that exposure of cytochrome oxidase in a nonionic detergent to an alkaline pH, in the range of 9.5 to 11.0, led to dissociation of the enzyme. No- leculnr weight studies showed that the enzyme as isolated was a

* This work was supported in part by Grant HE 01322 from the National Heart Institute, National Institutes of Health, United States Public Health Service, Bethesda, Maryland.

i Present address. Section of Biochemistrv and Molecular Biol- ogy, Cornell University, Ithaca, New York i4850.

dimer, with a molecular weight of 200,000, and was converted to a 100,000 molecular weight monomer at the elevated pH. The present paper will report the spectral and catalytic properties of the monomeric form, and the bearing which these have upon the cytochrome au3 concept of cytochromc oxidnse.

The cytochrome aa hypothesis rests primarily on the observa- tions of a component in cytochrome oxidase which does not react with carbon monoxide (cytochrome a), and upon kinetic analysis of the rates of oxidation-reduction followed at both the a and y absorption maxima. (For a review of this subject see Lemberg (2).) Thus, as one example, Gibson and Greenwood (3) con- cluded that their kinetic data could only be interpreted as the sequential reaction of two components Khose spectroscopic properties were consistent with those of cytochromes a and a3. It is recognized, however, that this is an operational distinction and that the two components have not been physically separated.

An alternative concept of the oxidase has been proposed by Okunuki et al. (4). These investigators define cytochrome oxidase as a single hemoprotcin, cytochrome a, complexed with cytochrome c. They have established that their cytochrome a preparation is polymeric and report a molecular weight of 530,000 (5). This species of enzyme has been interpreted as a tetramer based upon a minimum molecular weight of 128,000 (6), although in earlier work it had been thought to be a pentamcr based upon a value of 100,000 for the minimum molecular weight (5). Adopting the hypothesis that the oxidase is a polymer of cyto- chrome a requires the assumption that incomplete reactivity with carbon monoxide is a property resulting from the physical state of the enzyme, rather than to the presence of two hemo- proteins with different reactivities. Several authors (7-10) have discussed the properties of the oxidase in terms of a poly- meric enzyme.

It will be shown in this paper that the monomeric oxidase produced at an alkaline pH retains practically full catalytic activity in the oxidation of reduced cytochrome c. These re- sults are not the same as those obtained by Lemberg and Pilger (11) in their extensive studies of alkali dennturation. However, we note, as did Lemberg and Pilger, that the course of the al- kaline reaction is different in the nonionic detergent used here than it is in the cholate medium which they employed. Lemberg and Pilgcr found that when the reduced enzyme m-as exposed to a pH above 9.5 dcnaturation occurred, and above pII 11.0 this ras followed by format’ion of a Schiff base and loss of cnzgmat,ic activity. They also fom~d that storage in the oxidized state at

6669

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

6670 Properties of Monomeric Cytochrome Ozidase Vol, 245. No. 24

alkaline p1-I produced no apparent change in spectrum, but. that upon reduction the spectral change was immediately evident and to the same extent that would have resulted from storage in the reducrtl state for an equivalent length of time.

XITERIALS riND METHODS

Qtochrome oxidase was isolated from bovine heart by the method of Yonctani (12, 13), with minor modification as de- scribed in the previous paper (I).

I’rrparation of Reduced Cytochrome c--X 1.0 mM solution of reduced cytochrome c was prepared according to the method described by Yonetnni (12). In the Sephadex chromatography step, the small front rumling band of polymeric cytochrome c n-as discarded, and the middle fraction was collected and stored under nitrogen. Lyophilized cytochrome c (horse heart type III, Sigma) was the source material.

Determirzntion of Cytochrome Oxidase Activity--The oxidation of reduced cytochrome c was followed spectrophotometricall~- at 550 nm, at 20” and 1~13 7.0. With the except’ion of the t,em- pernture and ~1-1 giren, the assay was performed exactly as described by Yonetnni (la), after the method of Smith (14). ;\Iolecular activity was calculated from Equation 1, and the maximal molcculnr activity was obtained from the intercept of a Lineweaver-Burk plot of l/molecular activity versus l/[cyto- chrome c] at infinite substrate concentration.

Sedimentation velocity and Archibald “approach to equi- librium” studies were carried out with a Spine0 model E analyti- cal ultracentrifuge, as described in the preceding paper (1). AIlxorption spcctrn were measured with a Unicam model SPSOO, double beam recording spectrophotometer wit.11 fused silica cells of l-cm path length. Ultrafiltration experiments were carried out with an hmicon ultrafiltration cell no. 10 (Xmicon Corpora- tion, Cambridge, Massachusetts) with an XN-100 ultrafilter.

This filter retains solutes with molecular weight,s ~~bovc 100,000. The cell was operated at 15 p.s.i., which gave flop rates of 0.1 to 0.2 ml per min with protein concentrations in the r:u~g;c of O.lyQ.

Carbon Monoxide Titration-A solution of osygxx-flee (‘0 was prepared in the following way. Kater was dcgassed bv heating under reduced pressure and was then saturated with nitrogen, after which it was covered with :I t,hirk l:~yer of ~xwdlh

oil and bubbled with C‘O. Duplicate sumplc~ (3 1111) of monomer and dimer preparations of equal lrcme A1 concentrations were titrated in parallel with this CO solution. The dirner solution was dialyzed against 0.1 RI glycine-NaOH buffer at p1-I 10.9 to produce monomer. The enzyme solutions were dcgassed prior to the start of the titration by placing them in a nitrogen atmos- phere at 0” for 2 hours, with gentle stirring action provided by a Buchler rocking shaker (Buchler Instrument,s, Inc., Fort Lee, New Jersey) operated at very low speed. This resulted in samples of lower t,urbidity than oft’en obtained when the dcgas- sing is carried out by bubbling the samples with nitrogen. Be- fore beginning the titration all samples were brought to 20”, and the cell holder on the Unicam spectrophotomcter was set to 20” (+O.l’) with a Tnmpson bath. Enzyme saml)les wcrc trans- ferred anaerobically to optical cuvettes of l-cm light path and overlaid with paraffin oil. An excess of sodium dithionite (10 mg) was added, and reduction was allowed to proceed for 40 min before the titration was begun. The CO solution was introduced below the paraffin oil with a gas-tight Hamilton 50.~1 precision syringe (Hamilton Company, Whittier, California). The change in optical density at 443 am was used to follow the formation of the CO complex, as described by Horie :mtl 1Iorrison (15). Sdditions of titrant were continued until no further change in optical density was recorded (0.02 optical density units is the limit of photometric accuracy of the Unicam spectrolhotomcter). Good isosbestic points were obtained in all experiments.

Molecular activity = (change in optical density at 550nm) (total reaction volume) (~50 reduced cytochrome c) (reaction time) (enzyme concentration) (volume enzyme added)

TABLE I RESULTS

(1)

Effect of incubation at elevated pH on maximal molecular activity of cytochrome oxidase

The activities were measured at two enzyme concentrations and averaged to give the values in the table. The individual values agreed to within 4y0. The enzyme is in 0.01 M potassium phosphate buffer, pH 7.4, containing O.l$& Emasol. Samples A, B, and C! were brought to pI1 11.0 with 1 x EaOH and allowed to stand for B hours at, 4”.

Enzyme preparation

Maximal molecular activity

Before treatment After treatment

A B C & Ab AC

set 7.5 x 102 7.3 x 102 7.9 x 102 6.G x 102 8.0 x 102 G.8 x 102 7.5 x 102 0 3.6 X lo2

6.8 x 102

a Raised to pH 11.5 for 5 min at room temperature. b Preparation at pH 7.4, stored at 4” for 1 week. c Preparation at pH 11.0, stored at 4” for 1 week. 1 Product of the Kao Soap Company, Tokyo, Japan.

Sbsorption Spectra of Slonomer and Dimer-I?&. 1 shows the spectra for the Fe3+, Fe*+, and Fez+-CO forms of the dimcr. The

Activity of Jrlononzer and Dimer-The m:Gnal activities of typical oxidase preparations before and aft,er conversion to the monomeric form are presented in Table I (I’rcl):rrations -1, B, and C). In these experiments a freshly yrcpnred enzyme sample (pH 7.4) was assayed for activity, after which the p1-I of the sample was raised to 11.0 by the addition of n small volu~ne of 1.0 N NaOH. Samples were kept in the cold room at 4” for 3 hours, which results in complete conversion to the monomer (1). The results show that from 83 to 97% of the activity remain? after treatment’ at pH 11.0 in the ~rl~Rsoll-l)llosp~iate buffer system. At pI1 11.5, however, the enzymc~ is ra])idly inactivated,

It has been observed that the enzyme (tlinlcr) loses activity on st,anding at 4” (13), and that t,his 108-j is xssociatcd with an increase in aggregation (1). Table I illustrates that the enzyme activit’y drops to 487, of the ralue for freshly lxepnred enzyme after 1 week of storage at 4” at pII 7.4, but at pII 11.0 (monomer state) the oxidase retains nearly full activity under these condi- tions of storage.

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of December 25, 1970 X. H. P. Chan, B. Love, and E. Stotz 6671

400 455 500 555 667

WAVELENGTH, NM

FIG. 1. Absorption spectra of the cytochrome oxidase dimer. Curve 1, Fe3+; Curve 2, Fez+; and Curve 3, Fez+-CO complex (3). The medium is 0.01 nr potassium phosphate buffer, pH 7.4, con- taining 0.1% Emasol. The oxidase concentration is 0.019 m&f.

TABLE II

Summary of absolplion spectral data of nlonomer, dimer, and alkali- modi$ed cytochrome oxidase

a (Fez+) ............... 600 a (Fez+) ................ 605406 a (Fez+-CO) ........... 604-605

y (Fe3+) ................ 421-423 y (Fez+) ................ 441 y (Fez+-CO). ........... 430-431

y (Fe”+)/ol (Fez+) ........ y (Fe”+)/y (Fe3+) ........ > (Fc”+)/~ (Fe=CO) ....

3.48 1.09 1.05

-.

-

Absorption maxima (nm) and peak height ratios

MOllOIlICX Dimer

600 606 605 5932

424426 444

429430 444,5

4.84 1.25 1.06

-

I

--

Le$;,‘s

nodified (11)

595 601

437 434

a The subscript s refers to the position of a shoulder in the ab- sorption spectrum.

shoulder at 444 11~ in the Fez+-CO spectrum is clearly evident,

and the other features of the spectra are quite representative of a

typical cytochrome uc13. Some of the spectral data arc tabulated

in Tablc II. 111 contrast to the dimer, the spectra of the mono-

mer, shown in Fig. 2, shoTI- distinct differences from the aa type of sljectrrtm. Generally these are loss of the 444-nm shoulder in

the Fez+-CO spectrum, loss of the 593.nrn shoulder in the Fez+-

CO spectrum, and changes in the ratios of peak heights. In

l)nrticular (see Table II), r(Fe2f)/r(Fe3f) changes from 1.25 in

:he dinner to 1.09 in the monomer, aiitl y(FeZ+)/a(Fe2+) changes from 4.84 in the dimer to 3.48 in the monomer. ‘There is no

cha.ngc in extinction coefficient of y(F?+) when the dimer is converted to monomer, but thcrc is a small shift in position of

-y(Fesf) frolrl 424 to 426 nm in the dimer to 421 to 423 nm in the

mononLcr. The extinction coefficient:: of the I+*+ and Fe2+-CO forms ill the Soret region are lower for monomer than dirner.

The ilrcrcased reactivity of the monomer ITith carbon mon-

ositlc is shown son~emhat more clearly in Fig. 3, in which the cr~b:md sl)cctra of :L more collccntrated solution of the osidase is

prcsentcd, along \\-ith the y-band spectra. 111 the y-band region,

WAVELENGTH, NM

FIG. 2. Absorption spectra of the cytochromc oxidase monomer. Curve 1, Fe3+; Curve 2, Fez+; and Curve 3, Fez+-CO complex. The medium is 0.01 M potassium phosphate buffer containing 0.170 Emasol, adjusted to pH 11.0 with 1 M NaOII. The oxidnse con- centration is 0.019 m&l.

the typical Fe 2f-C0 spectrum with a shoultlcr at 444 nm for the

dimer is replaced by a considerably more so-nm~etricnl peak in the monomer spectrum. The a-band of W+-(Y) with the

pronounced shoulder at about 593 nm in the dimcr is replaced

completely by a symmetrical peak in the n1o11on1cr. It will be noted that the position of the peak labeled ~~OIIOI~I~~L’ I++(‘0 is

at 604 to 605 nm,2 while it might be espectcd to bt at, al)l)rosi-

mately 593 nm (see “Discussion”). Table II aiso gives the data of Lemberg :UI~ l’ilg;cr (11) for

their alkali-modified osidase. In nddit,ion to the :~b~ncr of

enzymatic activity of the alkali-modified oxitla$c (a), there are

several spectroscopic differences between the nlollolncr and the alkali-modified osidase. The most striking diffcrt>nc>e is the

position of the a-band in the alkali~modified cnzymct, \vhich is shifted approximately 5 nm to 595 nm, with ,somc smaller dif-

ferences in the Soret region.

Curbon Monoxide Titratioll-Dill,licate snnil)lcs of n1oilonier

and dimcr (3 ml) at equal heme ,\ concentration (0.008% mar) were titrated Tvit,h CO as described under “RI:rtcrials and

Xet,hods”. The results are shown in Fig. 4. It is clcnr from the data presented that it requires approsimntcly tn-ice w much

CO Want to reach the monomer end point as it does to reach

the same end point in t,he dimer tit,ration. Thcl~e is some curva- ture in the dimer titration curve, which is nlw ~~11 in the data of

Horie and Morrison (15). The end point.< WC at nl)prosinlatel~ 130 ~1 for the dimer and 230 ~1 for the I~OIIOIIW. We have

found by ultracentrifuge analysis (I) that n small :LII~ variable

amount of monomer (of the order of 2 to 5’;) is 1)rcscnt in all

dimer preparations, and this could contributr to :I sn~:rll increase in the apparent (10 upt,ake of the tlimcr prepat~:rtjiorr. Thus the

dimer end point predicted on the basis that tllcl INO~IO~CI~ m-ill

bind exacbly twice as much CO as the dimw wo111tl lx 115 ~1, while the experimental value TKV- 130 yl. ‘I’llis tliscw~xulcy of

about 1Oo/0 reflcct.s the limits in photometric :\ccw~:Ic~, \olume error in titration, and tllc sniall amoulrt of 111oi10111~r I)rc>ent in

the dimer sample.3 TT-e conclude that this cspcriincl~t supports

2 As a further control the spectrum of the monomer was recorded at pH 7.4, immediately after pH adjustment, and found to be identical with the spectrum recorded at pII 11.0.

3 Since in this experiment the type of buffer and t,he pIl of the monomer and dimer solutions were different, a control experiment

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

6672 Properties of Monomeric Cytochrome Oxidase Vol. 245, No. 24

DIMER MONOMER I I I I I

FEN+

1.2 -t&3 -l- FE2’

l.O- 1. FE%

0.8 - Q8

e = 0.6 - 06 E =:

31-3: z 04- 04

400 420 440 460 400 420 440 460

WAVELENGTR. NM

FIG. 3. Absorption spectra of the cytochrome oxidase monomer and dimer in the Fez+ and Fez+-CO states. The data have been replotted on a conventional wave length scale for comparison pur- poses. The medium is 0.01 M potassium phosphate buffer, pH 7.4,

s I I I f I 1

0 I-

.-. -

0 50 100 150 200 250

VOLUME OF CO SOLUTION INpL

FIG. 4. Carbon monoxide titration of monomer and dimer prep- arations of cytochrome oxidase. Both monomer and dimer were 0.00885 PM in heme A. The sample volume was 3 ml and the tem- perature was 20”. The experimental points on each curve represent the results of two separate titrations. The ordinate, percentage of decrease in absorbance, is for the span of absorbance between the maximum recorded in the absence of CO and the minimum re- corded with excess CO.

an increase in CO-combining capacity of the monomer relative to dimer in the expected ratio and supports the interpretation of the absorption spectra of the CO complexes of monomer and dimer given above.

Effect of Sodium Dodecyt Sulfate on Activity-As reported in the previous paper (l), sodium dodecyl sulfate at concentrations of 0.2 and 0.401, causes further dissociation of the 100,000 molecu- lar weight monomer into 4.7 and 5.8 S components. Samples of oxidase were assayed for activity after incubation with sodium

was run in which the dimer solution was adjusted to pH 10 in gly- tine buffer. No significant differences were found in CO uptake due to the type of buffer or the elevated pH.

DIMER MONOMER

I r---l s:+

WAVELENGTH, NM

containing 0.1% Emasol. For the monomer spectra the pH of the mediumwas adjusted to ll.Owith 1 M NaOH. The concentrations of oxidase for the y- and or-band spectra were 0.019 and 0.084 mM, respectively.

FIG. 5. Sedimentation velocity pattern of cytochrome oxidase. The enzyme was converted to monomer, after which the pH was lowered to 7.0 and the sample dialyzed against 0.01 M potassium phosphate buffer, pH 7.0, for 10 hours. Protein concentration, 0.3’%; speed, 60,000 rpm. The boundary at the meniscus is the Emasol micelle and sedimentation is from left to right.

dodecyl sulfate at either concentration for 3 hours and were found to have no measurable activity. Dialysis (10 hours) to remove the sodium dodecyl sulfate did not restore enzymatic activity. The loss of oxidase activity in these experiments was found not to be due to the low concentration of sodium dodecyl sulfate that would have been present after diluting the enzyme for assay.

E$ect of Cytochrome c on Reassociation of Monomer-Since the assay for enzymatic activity was carried out at neutral pH, it is pertinent to inquire whether or not the activity, attributed to the monomer, could have resulted from reassociated dimer formed under the assay conditions. Therefore, several experi- ments were carried out to obtain information on the rate of reassociation of monomer to dimer in both the presence and absence of reduced cytochrome c.

First, in the absence of cytochrome c, the enzyme monomer was brought to pH 7.0 with 1 M acetic acid and dialyzed for 10 hours against phosphate buffer (pH 7.0, 4’). The sedimentation rate was then measured. From the result shown in Fig. 5, it is ap- parent that two major species are present, and calculations from the data show them to be the 6.0 S monomer and 10.5 S dimer. It is clear from this result that in the absence of cyto-

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of December 25, 1970 S. H. P. Ghan, B. Love, and E. Xtotx 6673

chrome c reassociation is a very slow reaction, particularly when compnrcd to the 100 see required for the measurement of oxidase acbivity.

To determine whether reduced cytochrome c is able to accel- erate the rcaasociation of monomer to dimer, the weight average molecular weight of an equimolar mixture of cytochrome oxidase mononler and reduced cytochrome c was measured. This xas done as a funct,ion of time, at neutral pH, by the Archibald method. The weight average molecular weight was found to be 99,600 at 35 to 40 min after mixing, indicating little dimer for- matiolr during this time interval. The precision of values ob- tained at early times is, however, not as high as would be obtained at later times. At 120 min after mixing the weight had risen to 170,000, indicating considerable dimer formation. These molecular weight determinations would qualitatively in- dicate that there is some acceleration in the rate of association in t,he presence of an equimolar concentration of cytochrome c, although again this rate of assocint,ion could hardly account for bhc activity measured in the first 100 set of the assay procedure.

It would be difficult, however, to duplicate the assay condi- tiolls exactly in an ultracentrifugation experiment. In the assay system used the final oxidase concentrat,ion is about 2.5 X 1O-3 PM, and t,he reduced cytochrome c concentration may vary from 10 to 50 ~11. ;1ccordingly, the molar ratio of reduced cgto- chrome c to osidase may vary from 4 to 20 x 103. A method was sought, therefore, which would allow closer duplication of the assay conditions for study of the reassociation. For this purpose, an ultrafiltration experiment was performed. A Diaflo membrane, X-100, was selected for use with an Amicon model 10 ultrafiltration cell. This membrane should distinguish between 100,000 and 200,000 molecular weight molecules. The mem- brane was tested first with a solution of the dimer at neutral pH, and it was found that less than 10% of the enzyme activity could be recovered in the filtrate. This is in agreement with previous results (1) which indicated that, a small amount of monomer was present in the dimer preparation. A solution of monomer at alkaline pH was then filtered, and it was found that more than 90y0 of the activity could be recovered in the filtrate. It was concluded, therefore, that the membrane was able to discriminate between monomer and dimer. sext, two mixtures of cytochrome oxidase monomer and reduced cytochrome c were prepared in the assay medium. The first, Sample A, was 1.6 X 10-Q PM oxidase and 31.8 PM reduced cytochrome c; the second, Sample B, was 1.6 X lop2 pM oxidase and 9.4 pM reduced cyto- chrome c. Samples ,4 and B were allowed to stand for 20 min before filtration was begun. In each case 1.8 ml of filtrate was collected and assayed for activity by the addition of 0.2 ml of a 318 PM reduced cytochrome c stock solution (the final concentra- tion of reduced cytochrome c in the assay was 31.8 PM). The sample activities were compared to controls which had been identically treated but had not been filtered. It is noted that in this experiment the assays were necessarily carried out in the presence of substantial concentrations of oxidized cytochrome c formed during the 20 min of standing prior to filtration. Thus there is some product inhibition, and this makes it difficult t’o exactly reproduce the assay conditions in the filtration experi- ment. However, in the standard assay used here the lower ratio of reduced cytochrome c to oxidase was 4000:1, and in Sample A it was 2000: 1; in both cases there was a large molar excess of reduced cytochrome c. Furthermore, the long period of standing (20 min) in the filtration experiment compared to the 100 see

required for a normal assay, should impose a stringent test of the extent to which dimerization could have occurred during the assay. The results are that 66% of the control activity was found in the filtrate from Sample A, and 61% in the filtrate from Sample B. These results show that most of the monomer is still present after long standing with a large excess of cyto- chrome c. Furthermore, it is not certain that all of the activity lost during filtration is due to dimerization since some protein is adsorbed to the ultrafilter; hence the loss of about 40% in ac- tivity of the filtrate represents an upper limit for the extent of dimerization which could occur in 20 min. It is concluded that reassociation of monomer to dimer could not be responsible for the activity observed with monomeric oxidase and that the monomer is biologically active.

DISCUSSION

Our present interpretation of the spectroscopic results is that the partial reactivity of cytochrome osidase with carbon mon- oxide may be explained in terms of the state of association of the enzyme. This interpretation derives from n comparison of the Fez+-CO spectra of the monomer and dinler prrscntcd in Fig. 3. As noted, the y-Fez+-CO band of the tlimer has a rn:~xinrunl at 430 nm, with the 444.nm shoulder I\-hich i$ tyl)ical of a cyto- chrome au3 mixture. The y-D 4 2f maximum of the monomer was observed to shift from 441 nm to 430 to 431 am when the sam- ple was exposed to carbon monoxide, indicating that the molecule had undergone reaction. The absorption lxmd of the ('0 mm-

pound is now only slightly asymmetrical on the long wa,ve length side, in contrast to the distinct shoulder in the cwc of the dimer spectrum, which is interpreted to mean that the n~onon~c~’ reacts more completely with CO than does the dimcr.

The position of the cu-Fe2+-CO band of the n1onomcr also de- serves comment. The spectra in Fig. 3 ,&OR- tllat the a-Fez+ band decreases in intensity and shifts slightly from a range of 605 to 606 nm to a range of 604 to 605 nm when the molecule reacts with carbon monoxide. The cr-Fez+-<‘0 band seems symmetrical as contrasted with that of the dimer. This again is taken as evidence of more complete reaction of the monomer with CO. It might seem plausible to expect that the a-Fez+-CO band of the monomer would appear at 593 nm, since in a t’ypical cr-Fe*+-CO oxidase spectrum this is the position of the shoulder characteristic of the cytochrome aB-CO complex. In spite of the apparent plausibility of this argument, it would be unwarranted to presuppose that t,he spectral characteristics of the carbon monoxide compounds of a dimer would be retained in a monomer. The complications in spectra which may arise in monomer-dimer situations are well illustrated by the work of Levinson (16) in studies of pyridocyanine dyes, where two bands of the dimer spec- trum become a single band of different Kave length in the mon- omer spectrum. It is of interest in this connection that the LY- Fez+-CO band of the alkali-modified oxidase of Letnberg and Pilger (II), which apparently reacts completely with CO, lies to the red rather than the blue side of the a-Fe*+ 1~~~1 as does the a-Fe*-CO band of the monomer reported hcrc.

The greater reactivity of the monomer toward CO is supported directly by t,he CO-binding results. These results show that the monomer binds almost exactly twice as much CO as the dimer. The maximum uncertainty in this result was lO’;l& and hence rules out integral ratios other than 2 for the total CO-binding capacity of the monomer relative t’o dimer.

The weight. of evidence seems to favor a great increase in the

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

6674 Properties of iifonomeric Cytochrome Oxidase TTol. 245, No. 24

extent of reaction with carbon monoxide of the monomer relative There are several other problems that remain, namely the to the dimer. Thus, using present terminology, the monomer mechanism of the dissociation at alkaline pH, the reason why appears to have ma.ny of the properties attributed to cytochrome denaturation does not occur in buffers containing nonionic sur- a3 in the typical cytochrome aa preparation. factan&, and the structural properties of the dimer which cause

The modification obtained by Lemberg and Pilger (11) was heme A to have different reactivities toward carbon monoxide. obtained as the result of very mild treatment, simply bringing Further studies are required to determine whether the monomers the enzyme to alkaline pH, in the range of 9.5 to 11.0. It should obt.ained from dissociation of the dimer are identical, and also to be recalled, however, that their enzyme was solubilized with elucidate the nature of even smaller subunits and their associn- cholate, rather than with Emasol which was used here, and pos- tion with heme A and copper, even though evidence from several sibly for this reason exposure to alkaline pH led to an inactive laboratories indicate that these do not possess oxidase activity enzyme (2). Lemberg and Pilger did refer t.o some experiments (1, 4, 8, 17). with t~he enzyme in Emasol-phosphate buffer, in which alkaline modification of the oxidase took place more slowly, and con- REFERENCES cluded that the reaction “follows a different course in Emasol solution.” Thus the following differences may be tabulated.

1. LOVE, B., CHAIX, 8. II. I’., AND STOTZ, E., J. Biol. C&m., 246,

Our monomer retains catalytic activity, while activity is lost in 6664 (1970).

2. LE>IBERG, N. R., Physiol. Rev., 49, 48 (1969). the alkali-modified enzyme. The alkaline modification takes 3. GIBSOX, Q. H., AND GREESXOOD, C., J. BioZ. Chem., 239, 586

place in 3 to 15 min compared with 3 hours for monomerization (1964).

at pH 11.0. The alkaline modification causes a pronounced 4. OKUNUIU, K., SEKUZU, I., 0~11, Y., I\~~) MATSUMURA, Y., in

blue shift of the a-Fe*+ band to 595 nm compared with a small K. OKUXUKI, 31. K~XEN, AND I. SEKUZT: (Editors). Slructzlre I ,

ldw shift from 606 nm to 604 to 605 nm upon monomcrization. and fun&on of cytochrimes, University of Tokyo I’ress, Tokvo. Janan. 19G8. v. 35.

The ~u-Fe2f-(‘O bands for the monomer and alkali-modified en- 5. T.~IQ&o&, 6., SEKUZY, I., AND OI~JN~KI, K., Biochim. Bio-

zyme arc at GO4 to 603 and 601 nm, respectively. phys. Acta, 61, 464 (1961).

It is rel)orted here that the enzymatic activities of the mono- G. ORII, Y., ASD OICCSUKI, K., J. Biochem. (Tokyo), 61, 388

mer a.ud dimcr, measured by the rate of oxidation of ferrous (1967).

7. cytochromc c, were nearly equal. This finding is subject to

OKUNUIC~, K., in RI. FLORKIN AND E. STO~TZ (Editors), Co~r- prehensive biochemistry, American Elscvier Publishing Com-

several iutcrl)retatioiis. One possibility is that in the dimer pany, New York, 1966, p. 232. molecule the transfer of XI elechron from cytochrome c to cyto- 8. ATVIBE, K. S., .&SD VESKATAR.~PIAN, A., Biochem. U~O~~?JS. 1,‘~s.

chrome a or a3 is not subject to stcric restrictions such as apply to Commun., 1, 133 (1959).

the carbon monosidt r~eaction, and thus there is no distinction 9. WAISIO, W., J. Biol. Chem., 212, 723 (1955).

10. between cytochrome cc ant1 cytochrome a3 with respect to electron transfer fr011l c>-toclirome c. ,\ second possible interpretation

WAINIO, W., GREBEER, I)., AND O’FARRELL, II., in K. Ortu- NUKI, M. K~XEN, AND I. SEKUZU (Editors), Struclure and function of cytochromes, University of Tokyo Press, Tokyo,

is that the tlimer is composed of one catalytically active and one Japan, 1968, p. 66. inactive monomer, and therefore I-IO increase in activity is to be 11. LE~IBERG, l<.., APITD PILGER, T., Proc. Izoy. Sot. Ser. B Biol. Sci.,

expected upon dissociation. A third possibility is that approxi- 169, 436 (1964).

mately ollc-half of the molecules are denatured under alkaline 12. YONETANI, T., in A. MAEIILY (Editor), Biochemical prepaya-

conditions. It is not felt that the latter is a strong alternative, lions, Vol. 11, John Wiley and Sons, Inc., New York, 1966,

since it would seem reasonable to expect that a range of activi- p. 14.

13. YONETANI, T., J. BioZ. Chem., 236, 1680 (19Gl). ties would bc fou~~tl, depending in part upon the length of time 14. SETH, L., in D. GLICK (Editor), Methods of biochemical analy-

that the enzyme was exposed to alkaline conditions. It was sis, Vol. ZZ, Interscience Publishers, Inc., New York, 1954, observed however t,hat almost full activity was retained in p. 427.

snmples stotetl at pH 11.0 for lengths of t,ime varying from 3 15. HORIE, S., AND MORRISON, M., J. Biol. Chem., 240, 1361 (19F5).

hours to 2 weeks. \\:llile none of the possibilities can be ruled 16. LEVINSON, G., Doctoral dissertation, Universit,y of Michigan,

1956. out on the basis of the data presented here, the first suggest’ion 17. CRIDDLE, 11. S., AND Bocn, R. N., Biochem. Biophys. Res. seems to be the most plausible. Commun., 1, 138 (1959).

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Samuel H. P. Chan, Bruce Love and Elmer Stotz OxidasecCytochrome

Absorption Spectra and Enzymatic Properties of Monomer and Dimer States of

1970, 245:6669-6674.J. Biol. Chem. 

  http://www.jbc.org/content/245/24/6669Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/245/24/6669.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from