Complexes of cytochrome c oxidase with cyanide and carbon monoxide

ARCHIVES

OF

BIOCHEMISTRY

ANT)

BIOPHYSICS

Complexes Cyanide

From the Bureau

90,

1<%21 (1960)

of Cytochrome and

Carbon

c Oxidase

with

Monoxide’

of Biological Research and the Department of Physiology and Biochemistry, Rutgers, The State University, New Brunswick, New Jersey Received

April

4, 1960

Inhibition by cyanide of the cytochromc c oxidase contained in a particulate preparation of heart, muscle reveals that there are two binding sites for cyanide. One site has a dissociation constant of 5 X 10e6 moles/l.; the other has a dissociation constant of 3 X 10-S moles/l. The first value is obtained when the oxidase alone is preincubated with cyanide. The second binding site is revealed on1.v after preincuhation of the oxidase with both ferrocytochrome c and cyanide. Inhibition by carbon monoxide reveals only one binding site for carbon monoxide. The dissociation constant for the cytochrome c oxidase-carbon monoxide complex is 4 X 1O-5 moles/l. INTRODUCTIOS

Horecker calculated that one molecule of HCN combines with four molecules of cytochrome c oxidase or with four active sites on the enzyme. Stannard and Horecker were unable to arcount for the differences in t’he two experiments. Recent experiments by Wald and AUlen (1) indicate that cytochrome c oxidasc should contain more than one CO-binding sit,e per molecule. They found that, the curve relating the per cent saturation of the enzyme with CO to the partial pressure of CO was slightly inflected. This indicated an illteraction of CO-binding sites. Our interest in studying the complexes of cytochrome c oxiduse with cyanide and carbon monoxide stems from the hypothesis that cyt,ochrome c oxidase is a single enzyme’ and yet exhibits a chemical reactivity characteristic of its several constituents. We have already shown that the enzyme contains copper (5, 6). In t,his paper we present evidence to show that’ the enzyme has two binding sites for cyanide and presumably only one binding site for carbon monoxide.

There are two reports which are of special interest in connect,ion with a study of t,he equilibrium combination of cyanide with cytochrome c oxidase. One of these is by Stannard and Horecker (2) in which they conclude from studies on the rate of oxidation of ferrocytochrome c that one molecule of HCN combines with one molecule of cytochrome c oxidase and that the complex has a dissociation con&ant’ of 5 X lop7 moles/l. at 25-26”. The second is by Albaum, Tepperman, and Bodansky (3) in which it, is noted t*hat, 50% inhibition of cytochrome c oxidase act,ivity is obt,ained at, 2 X 1OF moles/l. sodium cyanide. From the data cont,ained in the second paper, Stannard and 1 Cytochrome c oxidase is identified as the enzyme or enzymes oxidizing ferrocytochrome c, being oxidized by molecular oxygen, and having absorption spectrum maxima in the reduced state at 442-445 rnp and 603-605 m/*. Supported in part by a grant from the Kew Jersey Heart Association. A preliminary report was presented at the annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, April 1956 (1). 2 Present address: Graduat,e Department of Brandeis University, Waltham, Biochemistry, Mass.

EXPERIMENTAL All of the experiments were carried out with a part,iculate preparation obtained from beef heart (5, 7). The activity of the cytochrome c oxidase contained therein was determined spcctrophoto18

metricall>by following the rate of oxidation of ferrocytochrome c (8) in 0.1 M NaHPO~.KH2POr buffer, pH 6.0. The cytochrome c was a preparation made by Sigma Chemical Co. which was GG.(ici pure. The solutions of NaC?; (RIerck, reagent grade) were renewed for each esperiment I)y making serial dilutions of two freshly prepared st,ock solutions: 1.09 X loo* and 3.3 X 10e3 JI. The dilute solutions were kept in the tightly stoppewd H:lsks until used in the experiment. In the first, experiment, 0.9 ml. cyanide ~ws added lo 0.1 ml. of the insoluble heart, muscle I)reparation (1: 100 dilut,ion, containing 0.025 mg. after having been gent,ly protein). The mixture, sh:rkrn, was allowed to stand for 10 min. before c-buffer mixtrire was ‘2 nil. of the ferrocytochrome addrd ‘l’ht activity values ol)tained are t,hose ninrked with solid dots in Fig. 1. In the nest cxlwrimcnt 0.2 ml. of the ferrocytorhromc cAllffer mistl1r.r and 0.9 ml. c,anide were :~llow~l to st:mtl for 10 min. hefore the remaining substrate amount (1 .S ml.) of frrrocytorhrome c-hufl‘er mixture and the 0.1 ml. of the insoluble prcp:tr:ltion were :ttltlrtl. The v:lllles ol)t:tincd are those m:trked with solid sqwres in Fig. 1, Thus preincribation of the ositlx3e :rlone or of frrroc?-tochrome c alone with cay:lnidc gives :tn uninflected curve of inhihitiorl indic:ttivr of :L single site of cyanide binding having :L dissocintion ronstant of at)orit 3 X lo-” n101es/l. In prclimin:iry experiments where the osidase was :Iddccl last, we noted that the reaction in the l,resruce of N:rCS was not) alwi~s first order. Thcrc appcarcd to t,e a progressive enhancement. of the inhibition during t,he co\Irse of the re:tct ion. \l.‘e interprel.ed this to mean that the r;lt,e of formxtiou of the cyanide coml~lrs of cytochrome c oxidasr was far from beiug instsut:mto,ls. It se~~nrd necessary therefore to mix the oxitlase :rncl the sodium cyanide and to ~110~~t,ime for the compl~s to form. Furthermore. since c~:Lnitle :Ippr:rrs t 0 combine more readily wit,11 the wdllced form of cytochromc c oxidnse th:~n with that osidizcd form (9)) wc also ntlded :I sm:dl amount of frrroc!-tochlone r to the mixture to ~I~SIII’P that thp oxitlasc wo\~ltl be in the reduced statr for at least :I brief time during its rwction with 1hr c!-nnidr. Accordingly, 0.9 ml. S:tCN :md 0.2 ml. of :b mixt1lw of ferrocyt,ochronIr c 10.1 to 0.1 1111. mg. i and phosl~h~tte hr~ffcr lverc ntltl~tl of the tlillltr p:wticul:itr prep:lr:itiotl (().0’2,5 mg. protrin) 10 mill. Iwfore the water :rnd slll)str:~te :im01lut of frrrocytorl~romc c iu bllll’rr (0.9 mg. in 1 .S ml.) wew ad&d. The curve of inhibit ion 01). t :tinetl unclrr t hew rirrumstunrcs at the wvpr:iI concent rations of N:LS is prcscntrd iu Fig. 2. Tllr solitl lint on thr graph is the Iwst estim:lte(l rIIrvr t hrollgh the espcrimrnt:~l poitrts.

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Log M NaCN FIG. 1. Efiect of cyanide on the activity of cytochrome c oxidase following preincubation of the oxidase wit,h cyanide (0) and preincub:~tion of 10% of the substrat,e amount of the ferrocytochrome c lvit’h c,vnnide (m). The inflection in the curve is indiwtive of more than one binding site for hydrocyanic acid. The first HCN goes on at :t rehbtivclg low concentration. It may be calculated from the point of SOc/;,inhibition of this binding site (upljer right ordinate) that the complex has a dissociation constant of qqrosimately 3 X 10mxmolts/l. From the reaction, osidase-HCN = osidase + HCS, it is apparent that K = [oxidase] [HCK]/[oxid:tse--HCN] and that K = [HCN] when [osidase] = [oxid:lse+HCS], i.e., when the oxidnsr is 505; in the form of the complex. The second r>-snide gws on :rt :L higher concentration. The dissociation constant for 1his complrs is :~pprosimately 5 X lo-” moles/l. This must, Iw the same site as that in Fig. 1 which has :L dissociation constant of :ibout 3 X 1W6 molw/l. It is to be noted that the midpoint of the inflection in Fig. 2 is at :rt)ollt 3 X 1OY moles/l. and that this compares favornbl>- with the vallie of 5 X lo-’ molrs/l. obt,uinetl by Stnnnard and Horecker 12) from their uniw flectcd curve of inhibition. Experiments with CO were intcudctl to slIpport the concept of the polynic~ric n:itnrr of cytochromc c 0xid:we. rnfort\in:ittlly, as judged l)y an cxperi merit :~lrc:rdy in thr litrratlue. the olltlook LV:IS

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Log M NaCN FIG. 2. Effect of cyanide on the activity of cytochrome c oxidase following preincubation of the oxidase with 1070 of the substrate amount of the ferrocytochrome c and cyanide. The values on the right-hand ordinate mark 50% inhibition of each binding site.

not too encouraging. In 1951 Ball, Strittmatter, and Cooper (10) studied the reaction of cytochrome c oxidase with carbon monoxide. From these data we calculated that one CO molecule inhibited one active site on the oxidase, and there were no inflections in the curve to indicat.e that the enzyme might have more than one active site. In spite of the prospect, we decided to investigate this problem further. The experiments were carried out under conditions where the oxygen was kept constant at 5%. The CO-Ox-N2 mixtures were made by measuring the appropriate amounts of each of these gases at constant pressure into a 30-l. Douglas bag with the aid of a 100.ml. syringe. The syringe, which was mounted on a board with clamps, was fitted with two 3-way stopcocks in series3 and with a stop to prevent the barrel of the syringe from being ejected when the gases were being metered. The sequence employed in preparing a mixture was as follow-s: The Douglas bag and the connections to the empty syringe (lubricated with glycerol) were evacuated 3 Details

will

be supplied

on request.

GREESLEES

wit,h a vacuum pump; the syringe R-as filled with the appropriate gas under pressure, which forced the barrel back to the top and filled the syringe to the 106ml. mark; the gas in the syringe was brought to atmospheric pressure by permitting the excess gas to escape into the atmosphere; the syringe was emptied into the Douglas bag. In this manner it was relatively easy to prepare 5-10 1. of a gas mixture of known composition and to duplicate the mixture exactly for a subsequent experiment. It was necessary to renew- the mixtures each day for it was found that the composition changed, as judged by a decreased inhibition, if the gases were stored in the bags overnight. Each mixture was then bubbled at the rate of about 100 ml./min. for 10 min. through approximately 10 ml. water in a 50.ml. Erlenmeyer flask, and then through both enzyme solutions con tained in a special spectrophotometer cell (11) which was suspended and shaken in a water bath at 25” in the dark. The body of the cell contained 1 mg. ferrocytochrome c in 1.0 ml. water, 1.0 ml. of 0.3 M sodium potassium phosphate buffer (to provide a final pH of 6.0), and 0.9 ml. water. The side arm contained 0.1 ml. of the particulate enzyme preparation (0.025 mg. protein). The flow of the gas was stopped by closing t,he stopcocks, and the cell was t.ransported to the spectrophotometer in a cardboard cylinder whose interior had been blackened. The solutions in the cell were mixed by inversion, and the optical density of the reaction mixture was recorded at predetermined time intervals. The shutter of the spect,rophotometer was kept closed except when the optical density measurements were made. The solution was in the light path of the spectropho-

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3. Effect of carbon monoxide on the activity of cytochrome c oxidase. Addition values from other experiments (0, A) are added at the low concentrations of CO. FIG.

COMPLEXES

OF

CYTOCHHOMP;

tomet,er, exposed to light of 550 mp, for only about 1O70 of the time. A plot of the per cent, activity on the ordinate versus the logarithm of the molar concentration of CO on t,he abscissa (calculated from t)he solubility of CO at 25” and at the various partial pressures) reveals that the experimental point’s fit) a smooth uninflected curve (Fig. 3). Furt~her COIItact, of the oxidasc with t,he CO or the addition of a small amount, of ferrocytochrome c in the prescnce of CO before t,he substrate amount of t.he ferrocytochrome c was added, did not, change the curve. The ronclusion was forced on us that these experimental conditions reveal only one binding site for CO.

Cyanide reveals two binding sites on cytochrome c oxidase for t’his inhibitor, but only after the particulate preparation which contains the enzyme has been preincubated with both ferrocytochrome c and cyanide. It is assumed that the ferrocytochrome c in the presence of the cyanide reduces some part of t’he oxidase and thus makes it, more susceptible to cyanide. Since the air-oxidized oxidase contains copper in the ruprous state (12) and the iron of the heme in the ferric st.ate, it is suggested that the ferrocytochrome c may reduce the iron of the oxidese hemc and make it susceptible to comhiuation wit’h the cyanide. The results presented here must also bc Tiewed with the knowledge that cyt,ochrome c oxidase is probably a polymer. Griddle and Hook (13) have shown that the preparation of Hat& (l-1) is at least a dimer which can be monomerized with sodium dodccvl sulfate. If the oxidase is a dimer, then it contains two molecules of heme and at least two at,orns of copper (6). Whether only one half of t)hc molecule, i.c., one monomer, is susceptible to attack hy t.he cyanide unt,il exposed to fcrrocytochromc c is a clue&ion that caunot be answered unt,il more is know1 about t,he structure of the oxidase. The inflected curve of inhibition with cyanide cannot be interpreted as supporting the cxisteuw of two enzymes, namely, cyt,ochrome a and cytochrome a3 These two enzymes have been propowd as actmingin t,he sequence: cbytochrome c ---) cytochrome a --) cytochroma a3 + oxygen. The iuflwted curve could tlot. be obtained wit,h t,wo enzymes in secluenw bwauae the more susceptible one

‘I

C OXIDASK

would determine the rate of t’he over-all reaction. It is t,o be noted t,hst carbon monoxide is bound to only one site. It is possible then, as has been suggested by us, that the copper of the enzyme is the carbon monoxide-binding site (15) and that the heme does not bind carbon monoxide. The argument that might be raised against this view is that carbon monoxide complexes of copper are not dissociat8ed by light, as is t’he carbon monoxide complex of cytochrome c oxidase. However, it must be considered t#hat the heme may absorb light in the visible region and transfer t,he energy t.o the copper-carbon monoxide complex to dissociate it just as the prot,ein of the molecule is known to absorb light in the ultraviolet region of the spectrum and to cause a dissociation of the caomplex (I(i). 1.

WAINIO,

W.

W.,

Pederation

Proc.

15,

377

(195G). 2. S;TANNARD, J. S., AND HORECKER, B. L., J. Biol. Chew. 172, 599 (1948). 3. AI,BAT:M, H. (i., ‘~‘F:PPF,RMAN, J., AXT) BODANSKY, O., J. Bid. Chem. 163, 641 (19-K). 4. WALD, G., ASD !U,LEX~ 13. W., J. Gen. Physiol. 40, 593 (1957). B., WMSIO, W. W., PERSON. P., 5. EICHEL, AND COOPERSTEIN, S. J., J. Biol. Chew. 183, 89 (1950). W. W., VANDER WBXDE, C., ASI) 6. WAINIO, SHIMIP, N. F., J. l3iol. (‘hem. 234, X%3 (1959) . 7. WAIXIO, W. W., COOPERSTEIN, S. J., KOLLEN. s., AND EICHEL, B., J. ui01. Chew. 173, 1%j (1948). M., 2Jtvzh. Nio8. W-41~10, W. W., ASD rlR0N0~~, them. Biophys. 57, 115 (1955). 9. WAISTO, W. W., Federation Proc. 14, 299 (1955). E. G., HTRITTMATTISR, C. Y., AND 10. &LL, COOPER, O., J. Riol. Chem. 193, 635 (1951). 11. Laza~ow, A., A?~I) COOPERSTEIN, S. J., Sci~rtcc 120, 674 (1954). 12. \'AXDER WESDE, C., AND WAIXIO, W. W., J. Bid. (‘hem. 235, PC11 (1960). 13. CRIDULE, R.. 8., .~NU I~o('K, R. .\I., lliochcm. f2ioph.y.r. Ifeserrrch Commons. 1, 138 (1959) 1-l. HATEPI, Y., Biochim. et Uiophys. dcta 30, 648 (1958) 15. W.41~10, W. W., in “Symposium on Haem:ltin Enzymes, Canberra, 1959” (Morton, R. K., ed.). Elsevicr, 1960. IG. WARBURG, o., “Heavy Metal Prosthetic Cirot~ps and Enzyme Action.” Oxford Lirriv. Press, Oxford. 1949.