The b cytochromes in succinate-cytochrome c reductase from pigeon breast mitochondria

The b Cytochromes

in Succinate-Cytochrome Pigeon

Breast

c Reductase

from

Mitochondria

A quick and eRiuient method for the preparation of succ.inato-cytochromc c reductase from pigeon breast mitochondria using a mixture of ionic and nonionic detergents is described. The spectral c*harartcristicas of t)he cyt,ochrome components of this preparation obtained during the equilibrium potentiometric Gtration in a new scanning spectrophotometer show a close resemblanc~c to those of the intac,t mitochondria. Two b cytochromes are present whose properties can be modified by the detergents and lyotropic anions. The most sensitive cytoc*hrome toward any modific:ttion is cytochrome bT . Bile salts can convert cytochrome bT into a form spectrally indistinguishable from that of by; , :ind the lyotropic anions cause disappearance of

The elegant st’udics of the 1960s of the Wiswnsin &oup (13) have established that the artive unit in clcctron transport at site II cont.ains cytochromtx 0, cl , nonhemc iron, :&my&n A binding site, and a colorless protein atlIed the core protein. Although on the hcmo basis, there was t.kz as much cytochromc Z, as cytochromc> cl , no indication was obtained as to the existence of more than one cytochrome 0 species. On the other hand, steady-st’atc and kin& cxpcriments carrkd out in various laboratories have repcatcdly indicated t’hat in intact mitochondria and submitochondrial pnrticlcs c>-tochromr b behaws nonuniformly as if it. \wrc composed of mow t,han one ccmponw~t (for rwiew SW 5). The definitive cvidcnw for t hc cxistcncct of multiple cytochromcyj h came\, how-ever, only recently with the illtroduction of the equilibrium potrntiometric titration tc&niquct. The studicts of Wilson and Dutton (6) on intac+ mitochondrix rewakd t,hc: prwc~nw of t\\-o cytochromes 0 11ith diffrrcnt halfreduction ~~otonti:~ls: (O& b, (timi., =

+i30 mV) and (b& b, (J2=mi.0= -30 mV). Il’ikstriim (7) and Slatw and his co-workers (S) have fm%hcr argwd that, the 558-nm should(>r of I>-,. c*orrwponds clc facto to a xeparatP b c~?ochromc~. T\\.o distinctI: diffortwt cytochromes 0 with half-roduetlon potcbntinls of - Z:i ml’ and +GO mV \voro further found in the preparation of succin:ttt,~c-tochromc I’ wduct’ase (9, 10) isolat(Jd bv Triton-dcox\-cholak m&hod, dcscribcd in detail in &is papc’r. Thc~ finding \vns confirmed by Davis et al. (11) and Yu et al. (12j using room and low tc~mp(~raturc~ spectral studies. Thch pcwlliar brhnvior of c~ytochromes 6 and particularly b,,. in both intact mit,ochondria and isolated su~cin:ttc-c!-tochromct c wdwtasc suggestc>dthc>ir possibl~~involvcmcn t, in wry vital and delicat(~ mitochondrial function (1%1.5), but thcx mutual wlntionship of thcl t\vo c>.tochromw has not bwn sat’isfactorily rcwlvc~d.

432

EKECIfiSKA

this investigation was that the isolstctd cytochrome b used for t.hc reconstitubion showed a, single symmetric peali with an O(maximum at 562 nm, characteristic of cytochromc bK . Further studies of Berdcn and Slatcr (17), although esscnt.ially confirming the resuks of Ya.mashit,a and Racker (16)) indicated that the cyt~ochrome 2, purified according t,o their prorc>durc \~as over 70 % CO-sensitiw which suggrstcd modification of this hcmoprotcin. In Brlrden and Slatcr’s (17) results again 119 indicat,ion of the presence of b, could bo found by the analysis of their spectral data. In an attempt to resolve this puxzlc and in viw of the post8ulated importance of both b cyt,ochromes in mitochondrial electron transport and oxidat#ive phosphorylation (13-15)) \\-c set out to trace t,he behavior of those cytochromcs in the preparabion of succinatccytochrome c reductaso under a variety of chxperimental conditions. In addition, we present the detailed procrdurc for the isolntion of succinat,c,-c~tochromr c wductase using a mixt’urr: of ionic and Iwnionic detergentas in \\-hich, in contrast to other pwparat,ions, the q%ochromes b (xxhibit prop&ics spectral and tjhermodynamic characteristic: of t,hosc in intact, mitochondria.

Prepamtion oj the n~ilochondria. Pigeon heart, mitochondria, from which succinate -cytochrome c reductase was prepared, were isolated using crystalline bacterial proteillase (Xagarse) essentially as described for pigcon heart mitochondria by Chance and IIagihara (18). The muscle was separated from the connective and fat tiswles, cult into small pieces with sharp scissors, suspended in a medium contairiiug 0.225 31 mannitol-0.075 M sucrosee0.0002 M J$:L)TA (MS15 medium), and homogenized for 10 SW in :I Turmix blender. Proteinase was then uddcd (l-2 mg/g tissue), Thc pII of the suspension adjusted to 8-9 with 1 RI Tris base and digestion carried oul for GO rnin, at, 0°C with constant stirring. -1ftcr li0 min, the sllsprnsion was rehomogenized for 10 SW with a Turmix blender at :t low speed and c,cnt,rifugrd for 10 min at 3OOOq. The residue was discardrd and the supernatant centrifuged for 20 min at 10,OOOy to obtain the mitoqhondrisl fraction. ‘I’ho mit,ochondria WCI’C washed oI,ce in r\ISF: mcxtlillm, twice ill 10 n1~

ET AL. phosphate buffer pH 7.2 anti finally suspended in 50 m&l pTI 7.2 phosphate buffer. Pwpccdion oj sctccir~ale~c~tochronlc! c ,rduciase. To thr washed mitochondria (-20 mg prot:‘ml) 10:; l)O(: and 20’2, Triton S-100 were added to a final concentration of l’,; of each. After 30 min incubation at O”C, the suspension was centrifuged for 20 min at 12,000 rpm in a Servall refrigerated centrifuge. The residue was discarded alltl the supernatanti was brought to 30’y0 saturation with solid ammonium sulfate (pH was adjusted to 7.4 by the addit.ion of 0.55; ammonia in water. The mixture was stirred for 30 min at 0°C and ccntrifugecl for 30 min at 12,000 rpm. The residue rontaining cytochrome oxidase was discarded and the supernut.ant u-as brought t.o 15’;; saturat.ion with solid ammonium sulfate. The mixture was stirred and centrifuged as iu the previous step. Thr pinkcolored sediment (succillate-cytochrome L’ reducatase) was suspended in 50 rnM phosphate buffer 1~11 7.5 containing O.l<;, 1)OC (sodium dcoxycholatc) and O.lc; Triton X-100 (DTP) and dialyzed overnight at 0°C against a mixture COW taining 50 rnh% phosphate buffer 1’11 7.5, 0.05“;, DOC, and 0.05’;1 Triton X-100 (Fig. 1). h small amount of precipitated, colorless matPri31 was separated l)y ccntrifugation for 30 min at 12,000 rpm and the 30 anti 4.5:‘; fractionation steps with solid ammonium sulfate were repeated. The dark-red optically transparent preparation of succinate-cytochrome (: reductase was again dial~~zrd overnight. X;t) precipitate cc~itld be follnd either visrlally or by centrifngation at that st.:tge and the preparation of a protein concentration of 20-30 mg/rnl crluld be stored frozen (liquid nitrogen) for a few wvrteks withol~t snbstantial loss of activity.

Pro&~ This was tletcrmined either by the billret method (mitochondria or submito~hondrial particles) or by the method of Lowry et al. (19) us;ing rystalline bovine strum albumin as a stantlard. (:ytochromc b and (‘1 colltrnts were determilled after collvetsion of their hcme moieties into respective pyritlinca Ilcrriochromogells as t hc described b\, 13asford el trl. (20). (In this procedlire hcme h i;; separated from hemc cl by extrartioil with acitl acetoll?. while heme cl remains in the rcasidllc:.) The millirnol:~r ext,inct,ion coefhcieltts IIW~ for calculatic)n were 34.7 (at 557 nm) and 19.1 (at 552 nm) for c~ytochromes 6 alld cl , respect ivcly. 11dul iron nrd nonhcme iron. These were det,ermined 1)~ the art hophcrlarlttirolilie method according to Hranlby and Masse)- (21), losing frrrolis ammonium sulfate as :I alalrdartl. Fh7uin.s. This was tlct~~rrnillc~cl by the method of

Xppnji rt al. (22). The amount of the peptidebound flavin was obtained by the difference hetween the values with and without trypsin digestion. Lipid. The content was measured as the amount of phoapholipid phosphorus present in the sample. The phospholipids were exkacted by t,he method of Folch et ul. (23) and the inorganic phosphorus determined by the method of Bartlett (24) after digestion of the phospholipid with perchloric arid. Coe?lzynle Q. This was detcrmincd either by the method of Szarkowskx and Klingenberg (25) or by the method of TTatefi (26). Bot,h gave identical results. Sucrznate-cylochrome c reductase. The activity was measured in a medium containing 0.1 -kl phosphate buffer pTI 7.5, 1 mu KCN, 20 m&l succinate, and 0.1 II~M cptochromc c (Sigma type VI) in a fillal volume of 1.0 ml by following the illcrease in absorbance at 550 nm against a reagent blank. 4n extinct,ion coefficient of 19.7 rnM-’ X cm-l was Itsed for the calculation. ru’AD%cytochrome c and durohydroqninone-cytochrome c reductase activities were assayctt in the same medium except or 50 PM tturohydroquino~~e that 1 nm NADII (50:‘;, ethnnolic solution containing 2 IBM EDTA) were llsed as sltbstratcs. One microliter of the prep:irat,ion (0.020.03 mg prot.) was used for the :\c,sR\’ of the succiilwt,e~cytochromr c reductase, i I~IIS 0.057;, Triton S-100 was diluted by 103. Spcrlrul studies. These n-ere done using the Johnson Foundation dual wavelength spcctrophotometer provided with a digital wavelength drive on one monochrolnator, tho other being set :rt the reference wavelength. Operation of the dual u-avclength system is substantially as ttescribcd llc~forr (27) with a 60-11~ vibrating mirror timesharing t.he two wavelengths upon :I single photornllltiplier. In the earlier system (2i) t,he baseline e~tablishetl for 011~condition of the sample was set into R series of potentiometers so that :I pldaed voltage was applied to the drnodes for correcting t hc, baseline. ln the present instrument,, the func1ion performed by the potentiometers is performed by digiktl memory. The sample in the initial condit ion is plared iii the cuvette and the dynode voltage applied to the photomultiplier chukg the nl(l:tsIIre interval is automatically adjusted as to obt nin :i flat bnselinc. At the same t,ime, the (‘ont rol volt,nge applied to supply the dynodes is memorized by a 1% bit A-to-11 converter and a 12 bil by 1024 word shift register memory thus renrembering the cl?;node volt,agc appropriate to 1024 steps over :i 100&200 nm iutervat of the wavelength drive. After the baseline memorization pr<~cedurc is completed the feedback loop for the tl>.lrotlc vr)tt:igc is c,pen ant1 cl\-node voltages :\I)-

propriate to the measllre interval arp read out from the mrmc,ry in a T)-to-.4 cirrllit for all SIICCPHsive runs. The system has thcl high sensitivity of ttr~l wavelength recording since the step interv:lt of 0.1-0.2 nm is greater than the rrs:olut,ion of the monochromator (0.3 lun) and the stepmotor wavt:length drive affords accurate wavelength registr:ltion. The scanning speed on recording the baseline is limited by the time ronst:nlt of the illtegratol and the slow rate of the high voltage r~gulatot. 10 approximately ii see for 100-200 nm. The bran speed on recortting of sltbsequellt. spectra can l)r greatly increased sinrr the high volt age is being cont,rollrd by the memory illstead of thus high voltage regulator. The ~~)nd motor runs :kt :I maxirn~m~ speed of 4-t set for the 100-200 nm sr:~I(,.

Potentiometric titrations were carried out in an anaerobic cuvette designed by Dutton (28) rising simultaueous measurement, of absorbance (dual wavelength spectrol)hl)tc)mctrr.) and oxidat ioll reduction klotcntial (platinum electrode ill ~OIIjunction with :t c>:rlomel reference clectrod~~) :IS described by Wilson :iu(l I)lltton (6). The orittation-reduction potenti:tl was m:itl~ more clectropositive with l)otassilull-ferri~~~llide and more electroucgative with :I freshly prepared, dilute solution of sodium dithiollitc. The titrations were performed in an atmosphere of Idtrapure argon (02 < 1 ppm) using rcdox indicators specified in the figllre legeud. The data are presented graphtally as i.he logarithm of t,hc ratio of oxidized to reduced forms of the cytochrome:: (abscissa) as :I, function of potential (ordinate). Scparatioll of the components W:LS tit)rntiori cluvc into 1hc individlud performed as described by Dlltt,oll ~1 ctl. (29). Materials Sodilun deoxycholale and horse hrart cy1 (Ichrome (’ type VI wcrc obtained from the Sigma Chemical (:ompany (Ht. Louis, MOj and Tritoll S-100 from the l’ack:trtt Instrrmient Comp:~~l?;. Inc. (I>owners (+rov(x, ILj. The redox mediators were prodllcts of the Sigma Chemical Comp:~tly ; (phcnazinr: rnrt hosulfatej, K & K l,:rbor:ttoric~s, ethosulfate; pyoPlainview, NY ; (phenazine cganine pcrchloratc :tntt 2-OH-I.~-naphtoquiI,ollc 1, and (d~iroquinoiie) Aldrich Chemical Coml~~~y, Milwaukee, WI. I: 11:81JLTS

434

I:IWCIr;JSKA TABLE CohrrOsrTIori

E’/’ A.!,. I

OF SU~~~N.~TI-:-~YTOCH~~~M~; c 1
.-__Heme (cyt orhrome 6) (cyto(~hrome (.I) Iron (total) (nonhcnw) Flavin Lipid

Coenayrnc Q

(pmolesig protein)

2 .5 1 .?I 11.1 7.0 0 .23 -L.(i rng I)hoxpholil)id protein 0.36

&moles/g protein, 0 .6 0.28

2.x P/g

4.5

0 The sssay methods arc giveu in the experimental se&on. b Representative data obt,aincd for pigeon breast rnit,oc:lloncI~i:1..

tion of mitrwhondrial succi]LatC-C~toChrornc, c rcductaw (for the> other preparations described in the litwaturc, SW Ref. 2%). The preparation obt,airwd using a mixture of 11OC and Triton X-100 contains hemrs 6 and Q in thcl amounts of %.,5 ,umolcs/g protein and 1.13 pm&s/g protAn, rcspcctivcly (in approx ratio of 2: 1) and nonhcmci iron (7 pmol(w/g protein), small amounts of flavin: and rocnzymc: Q (Table I). Recowrics as based on hcmochromogcn c cont,cnt) arc betw-ccn 4OG33%. So acid-cxtractablc flavin could be d&c&cd. Spectral propc’rtic>s of cytochromcs 6 and cl in the prcyarat,ion a,w shown in Fig. 2. ‘I%(> addition of ascorbatt~ to t,hc aerobic suspwsion of the>wzymc sclwt~ivcly rcduccs cytochromc cl as dcmonst,ratcd by t,hc appearance of a symmetric O( prnk n-it111A,,,:,, at 554 nm (U). When the spectrum of the reduced q~tochromc cl is taken as a flat hasaline (C) addit’ion of sodium dit,hionit’c causes reduct,ion of cytochromc~s 6 (II). Thr: spw t,rum is slight’ly asymmetric and has A,,,:,, at, Xi4 nm. Addition of csarbon monoxide (E) resu1t.s in disappcaranw of :L pa.rt of the absorbance due t,o modified cgtoehrtrme b and the asymmrttry of the> pr:nl; bwomc>s more’ pronounwd. It should brt mc~ntionrd that, in som(’ proparat,ions no CO-wnsitiw cytochromcl 6 JYRSd(xtc&cld. In our wnrch to identify thcl individual cytochromcs 6 of the succin:ttc!-e3toc,l~rome c rcduct,asc we combined the potcntiomctric titrations with simultancons measurc~mr~nts of the: entire spectrum of R wmprmc:llt at ~1

given oxidatioll-wduction potential. Although basically thr: same information can brh obtained by careful point-by-point titrations carricld rout, at different measuring wavtrlcngt,h thr: prcscnt method is much moro accurate since it, uses the same preparation and the same potential value to record that spcxct,rum of a broad range (100 nm). Thr preparaAion of succinatc-cytochromc c rcductasr: is made anaerobic wit’h sodium dit8hionitc in the prescncc of the rcdox indic::ttors l&d in the legcand of Fig. 3 and the oxidatioll-rcduct,ioll potential adjusted to - 200 mV. ilt this potential cytochromes 6 and cl arc’ c~omplctcly wduwd and no addit.ional absctrha.wn (*hango is observed when the potential is mado more rllectronegativc. Thr spwt,rum of a full), reduced preparation at the Eh of - 200 n-iv is taken as a flat basclinrh. Stcpwisc: addit)ion of small aliquots of more fcsrricyanido makw t,hc potential c~lwtropositivc and at, a certain value, results in oxidation of th(, c~ompownts of succinatt:cvtocliromr~ c rcductast. A set of spwtral diffcrencc curws is t,hus obtained from which the hdf-wduction potc~ntials of individua.1 compownts can b(t wlcul:tt,rd (Fig. 4). In l>ig. 3, thrl tit.rxtion rwricd out in t,ho region b(bt\vwn - 120 mV and +300 mV is for the: salw of clarity divided into three parts, c*orrr~sponding to thrcx: carriers present in tlw snmplc (thr: c~hoic~c:of “dividing potc~ntials” pro& to bn correct as cvidenccd from the, straight liw \vitjll t? = 1 Gtrat,ions

show1 in Fig. 4). ;2t rbach dividing point, \\-hich corrwponds to t)hc rind of the titration

Pigeon

breast

muscle mitochondria

lCh Triton 1c; 1xE

/ Sujrernatnnt I 30’;; Saturation nium sulfate

Residue

/

:11mIo-

LSr&ern:ttani I

Residue

w

;,

haturat1011

monium

am-

sulfate

Supernatant

Iles’idue

Suspend in a mixture containing 0.1“; IWC, 0.15; Triton, and 50 I,~M phosphate buffer pll 7.5 ( IjTP)

1)ixlvac against a mixture 0.05’; l)OC, 0.05’; Triton, phosphate, pll 7.5 overnight I 30:; Saturation ammonium I

Jji.'

(, Saturation suffate

(Succinnte-cyl

c reductnse)

suspend

containing and 50 mu

sulfate

anunonium

in I>TP

1. Flow sheet for the isolation and purifica1iotl of succinate~cytochrome c reductase from pigeon breast. mitochondria. Fro.

436

510

530

550

570

590

A hml

FIG. 2. Difference spectra of the sllccinate-cytochrome c reductase. The succinatecytochrome c reductase was suspended in 0.1 M phosphate buffer pH 7.5 at a protein concentration of 2.1 mg/ml. (540 nm was taken as reference wavelength). A; oxidized baseline. B; ascorbate (5 mM) reduced minus oxidized. C; ascorbnte-reduced spectrum taken as a flat baseline. D; sodium dithionite-reduced sample minus ascorbat,e-reduced. E; sodium dithionite reduced + 30 PCZICO minus asc0rbat.e reduced.

L 500

I

530

560

/

590

A (nm) spectra of the cytochromes of su~cinate~~ytochrome c obtained during anaerobic potentiometric titration of the preparation. The succinatc-caytochrome c reductase (2 mg prot/ml) was stirred under an argon atmosphere in 0.1 M phosphate buffer pH 7.0 (0.00S70 Triton X-100, 0.0087, IIOC). The redox mediators used were: 20 PM diaminodurol, 40 PM eachof phenazine methosulfate, phenazine ethosulfate, and duroquinone, 5 PM pyocyanine and 15 pm 2.hydroxyl-1, 4 nnphtoquinone. The figure shows an oxidative t,itration with potassium-ferricyanide. The reference wavelength was 595 nm and the scanning speed was 1.3 min/lOO nm. Upper traces represent the absorbance rhanges taken in the potential range: -100 mV to -10 mV; middle: -10 to +145 mB; bottom traces: +145 to FIG. 3. Difference

+275

mV.

~rlc,cinate-cgtochromc c rcductase at a rate of ten t,imes greater than that for succinstf. Hoth succinatc and durohydroquinone oxidaCon is nntimycin A sensitive (Fig. 5h). The sigmoid t.itr&on curves for t,he t*\vo sct.ivitics :II‘V superimposed and the titer is one antimj-tin A molecule per one cl (or 1 per two Iwmc3 b) (see also l-4). \\:c have also studied the: antimycin A induced shift in reduced cytochrome b, by nwasuring t’he increase in absorbance at 5(iti nnl

(with

r(fspM

l50-

to

Xi7

nm)

in

n

E m70= + 55mv , 4

I 4

IOO-

J”

50’ /

Eh /

O?

/?

-501 -1001

FIG. 4. Plot of the absorbance changes for the two 6 cytochromes recorded in Fig. 3 as a funct,ion (tii I he c~sidatiorl~red~lction potential (EL). The met hod of separation of the individual componenis is discussed in Ilef. 29. The tot,al reduced value fo! caytochrome 6,~ (566 nm and 558 nm) was taken :I> that at the El, of -200 mV and total oxidized al t.he value of +lO m\:. For cytochrome 6~ total reduced is the value at -10 mV, t,otal oxidized at, +195 rrl\-. Log of the ratio ox/red absorbance is plotted against the E,, (0) 566 nm absorbance in the i?F;i, rauge (-100 m\‘)-(-10 m\‘); (0) 558 urn :\I,srrrbanre in the Kh range (-100 m\:)-(- 10 my); (A I 560 nm absorbance in the E’,, range (+lO mVj+145 mV). Solid lines represent theoretical 1~= 1.

ICnzyme

Activity Alitochondrix fumoles cyt t red/min/mn pmt.) (reductase)

FIG. 5. Ant.imycin A titrat.ion curve of succinate-cytochrome c reductase and durohydroquinone-cytochrome c reductssc activities (A) and antimycin A-induced spectral change (566558 nm) (B) in the reduced succinate-cytochrome c reductase preparation. Succinate-cytochrome c reductase and durohydroquinone-reductsse activities were assayed as described in the method section. The sample used corresponded to 0.4 nmoles hemo c1 of succinate+cytochrome G reductase xct,ivity assay and 0.04 nmoles in the durohydroquinone-cytochrome c reductase act,ivity. The dithionite-redlrced sample corresponding to T.8 nmoles of heme cl was used when the spectral shift (Bj was titrated wit-.h antim?-rin A.

dithionite rcdwcd pwparation of succinatc ~cytochromc f’ rcduct.asc (Fig. 5B). The t,itration

absorbnncc

curve’

givc3

lilicar

increusc

in

\vith respect to antimycin 11 coliceirt~ratiori \Vitli :I sharp cut’ off at 8 ratio of 011~’molwul~ of ant’imycin A per on? (‘1 home. No changes in t,he oxidized cytochromcs cait’hcr in the 01 or in thcb Sorrt region ww observed after the addition of antimycin .I. The e$‘ectoj’ Tritotl. S-100 md clectzyciwiate 011 the spectral properties oj cyiockiwnzc (a re&utase. Bile pigments, cholate, and drosycholate arc the most widely employed tools it) the isolation of the respiratory chain c*omponents. In a few chasesTriton X-100 h:rs been used, c.g., by Jacobs et al. (32) for t.hcbpurification of cytochrome oxidasct and rcccnt,ly by IJsugh and King (31) for KADH dch>,drogcnase. In view of t)he known effect of t,he detergents in gencrnl on the enzymatic propcrt,ics of the isolntcd systems it, seemed dwirnbl(~ to c~st,ablish the degree 31, which they affc>ct the spectral, :mtl thcrmodpamic propertic of thcl isolntcxd auct:inatc~~~?.tocahromc c wdwt:w. It should be strrsscd that in thcx prc~l):lrntifm :id isolatPc1, the ran-

438

2 pHRO 3 OH87

3

#J 4oc

4;o

.T

440

60

550

530

5io

h hm)

FIG. 6. cytochrome pended in value with 2-reduced dithionite

IM’ects of Triton X-100 and pH on the spectral c,haracteristics of succinate c reductase. Succinate-cytochrome c reductasc (1.5 mg prot/ml) was 311s. 30 rnM K-phosphate buff’cr -0.57; Triton N-100, pH was adjusted to the desired 1 N KOTI. O-oxidized spectrum at pH 7.2; 1-redllced with dithionite at pH 7.2; with dithitrnite at pH X.0; 3-reduced with dithionite at. pH 8.i; I-reduced witlL at pH 9.5; 5-reduced with dithionite at. pIT 10.0. 25min

-3m1n IOmln

X (nm)

7. Difference spectrum of the Triton 5-100 and pH-induced spectral change in succ:inate-rytochrorne c reductase. Dithionite-reduced sample in O.ln/;l Triton, 0.03 M potassium phosphate buffer pII 7.0 was t,aken as a flat, baseline in .\. rlftcr addition of Triton (final concn of l’:;), difference spectra were recorded at the time irltervals described in this figure. Difference spectra induced by the addition of Tris-HCl buffer (to the final pH x.0) to the sample of (15 min-g) were subsequent,ly recorded in I3. FIG.

of cnch dctergerlt is 0.05 7; ant1 since the protein concentration is high, thcx ratio of the detergent to protein is very low (0.02 mg Triton X-100 or less, per 1 mg of protein). Exposure of succinatc-cytochrome c wduct,asr t.o 0.15% Triton X-100 at. room temperature causes ncgligiblc changes in t)hc spectral propwt,ies of t’hc complex. Howcvcr ccllt,ratiorl

upon an incrww in Triton conwntration or raising the pH toward mow alkalil~c~ valuw a gradual disappearance of absorbanw of the dithionitc reduced sample in t’lw region of SO-T,fiB nm (Fig. 6) is obsorvcd. In addition, spectral changes in th(x @ and y region arcs noted : t hex .30 and 5% nm absorbance diminishes: the 430 nm p~alc decline and the 418 nm peak of the wdured cytochromo Q

b CYTOCHL:OILII~S

IN SU<:CIPU’ATE-CYTOCHI:OMTi;

appears. .CVhtn the incubation in Triton is cwricd out in the oxidized sample appearancr of the 554 nm peak of the reduced cytocshromcl cl is observed, indicating that, t,he modification of cytochromw b is accompanicxd by the accumulation of thcl wducing cquivalwts in cytochromcl cl.

510

530

c llJ3i)UCTASE

Figure 7 presents similar data but in the form of the diffcrcnce spectrum between the> absorbance change induced by Triton + pH and the fully reduced sample. In this type of experiment the time-course of events can be followd. At an initial stagct the change in absorbance is due to disappc~aranw of :L

550

570

590

A (nml

FIG. X. Differcnc.e spectra of the INN-induced spectral change in succinatc~c~~toc~hrolrlc: c redlwtase. 8u~c‘irlatr-~!-t(,c~hrome c reductase (2.1 mg prot/ml) was suspended in 0.1 Y K-phosphate buffer, 0.00X( ; Triton X-100 pH 7.5. The fully oxidized spectrum (with 20 FM ferricysnide) was taken BY a flat baseline and the dithionite-reduced minus oxidized difference spectra were reccjrdcd. 1)OC wns then added to a final wncentration of l<;h and the time-caourse of the changes was followed hy spanning t.he spwtrn with time intervals of 1.3 min. The inset shows l)OC-itldllcrd difference spectra of the dithionite-redllccd *:mlr,lP. -0,

1

500

5fO

439

560

540

A (nml

l?rc;. 9. 1 )iffcrcncc spec.t IX of suc,c,irr:~tr,~c,ytt)~hroInc c reduct.ase obt:~ined during anaerobic* potcrrtillmetric titrntion of the prcpnration in the prrsencne of 0.571 1)OC. The swcinntec~ytochrome c reductase (2 mg prot./ml) was stirred under an argon atmosphere in 0.1 M phosphate h~rffer pH 7.0 in the presence of 0.50 INK (Triton S-100, 0.00X”,). The redox mediators are specified in the legend of Fig. 3. The referenw wavelength is 510 nm and the scanning speed 1.3 min/lOO nm.

440

ET BL.

EKECIfiSKA

component with double (Y a.nd p peaks, characteristic of cytochromc b, ; at a late1 stage (not shown) it, gradually loses it’s double peak and shifts thcl maximum t,o

3ii:! nm, characteristic of the disappearance of both cytochromes b; DK and b, . From the time-course of the events described above, it can bc concluded that although both cytochromes b are affected by Triton treatment,, cytochrome b, is more sensitive t.han is 0,. Dr~ox~.cholatc~, as Triton X-100, modifies the Ypectral properties of succinatc---cytochrome c reductase. Addition of 0.5 % dcoxycholat,e (Fig. 8) causes the disappearance of absorbance on the long wavelength side of cytochrome b spectrum. The net disappearancc of the 568-nm peak of cytochromr b, is accompanied by a small net increase in the height of the 561 nm peak. Titrations and simultaneous spectral wcordings similar to those of Fig. 3 show that in the presence of 0.5 % DOC the absorbance change induced by the addition of ftrricyanide appears at a much lower oxidntionrrduct,ion pot,ential value than in the abscncc of the detergent (Fig. 9). Two componcnts are still present with half-reduction pot,entials of - 150 mV and -15 mV (IGg. 10). Addition of CO after 60 min incubation in t#heprrsencc of DOC and redox indicators causes disappearance of 70% of t,hc absorb-

O3 -5olz -lOOI

-150

ii

lb

Id0

Ox/Red

FIG. 10. Plot of the course of oxidat,ion-reducof succinate-cytochrome tion of the b cytochromes C reductase against the oxidation-reduction pot.ential (Eh) in the presence of 0.59; 1)OC. Titrations were carried out as described in the method section in the presence of redox mediators specified in the legend of Fig. 3. (Ci-oxidative and reduct,ive titrations of the absorbance changes at 560-575 nm, (A) and (A)-correspond to the two components representing 60 and 407, of the absorbance change at, this wavelength pair. (0) titration in the presence of 1.0 mM CO. Solid lines represent theoretical n = 1.

A

O.d5A

1

h hn)

FIG. 11. The time rourse of thiocyanate-induced

spectral change of cytochrome 6 in the succinat,c-cytochrome c reductase. The sucG~ate-cytochrome c reductase was suspended in 0.1 M phosphate buffer pH 7.5 (2.1 mg prot/ml) and the ascorbate-reduced sample was recorded as a flat baseline. The sample was reduced with dithionite (a) and 0.2 M KSCS was added; at (B) 40 PM CO was int.rod\lced and the (C) spec*t.rurn was obtained. The spectr.1 were recorded contin~~ously with the scaannin, u speed of 1.3 min/lOO nm. The reference wavelengt,h was 540 nm.

h CYTOCHROMI:S

IX SUCCISATR-CYTOCIIROME

550

530

510

441

c REDUCT.4SE

570

590

A (nm)

spectral change in the succinat,e-cytochrome c reductase. CondiFIG. 12. KSCK-induced tions are those of Fig. 11. The dithionite reduced preparation (A) is taken as a flat. baseline. Then 0.2 hf KSCN is added and the spectra are continuously recorded m-it h the scanning speed of 1.3 min/100 nm. The refcrcnw wavelength is 595 nm.

510

530

550

5jo

590

A lnm)

Flu. 13. The effect of CO on KSCN-indrtced spectral change in snccill:Lte-cytoc~t~r[ln~~ wductase. Conditions are those of Fig. 12 except 40 PM CO is present.

:lrw and the rcwaining part titrates as a single component. wit,h a half-reduction potential of 0 mV. Since DOC modifies both the spcct’ral and thermodynamic properties of cytochromes t) it is difficult to state definit& which part of thf> absorbawe can be assigned t,o cytochrome b, and tvhich to bT . The effect oj salts ox spectral properties oj succinate-cytochPorlle c reductase preparation.

ItI has been shown by Imai and Sato (33) that csposure of liver microsomcs to high wnccntrations of neutral salts results in the conversion of the mirrosomal cytochromc

c

P-450 to P-120. Thf~ c#icieiicy of’ various salts in causing the conversion obq-s the order known as Hofmeistcr’s Iyotropic scricls of ions. Addition of 0.2 M IZSCN (potassium thiocyanatc) to thr preparation of reduced succinate-cgtochronw c reducbaw wuws time-dcpcndcnt disappa~rn.nce of absorbance. The differcncc~ spectrum rworded against a baseline in which cytochrome c1 is rcduccd by the previous addition of ascorbate (Fig. 11) she\\-s t’hat’ the total height of the peak of reduced cytochromes h declines and that X,,,Bxgradually shifts from 34 nm (A) to 562 nm (K). The init.ially asymmetric

442

ERECIfiSKA

peak assumes a more symmetric shape characteristic of cytochrome 6, , which slowly declines with time during furthe] incubation with X%X. In order to decide which cg-tochrome 6 is afkctcd by IWCX, treatment, the difference spectrum is r(:corded against the baseline in which both cytochromcs 6 and cl arc rctduced by dithionito. -4s shown in Fig. 12, IG3CS causes disappearance of the spectrum of a component. with a double QI pwl;, at 566 and MS nm. Both peaks disappear simultaneously and thus can bc identified as belonging to cytochrome bT . L21t’hough t,his indicatcbn that, cytochrome b, is mow sensibivc to IISCN trcat.ment, tbnn is OK , two lines of widenw suggest that IZSCN induces simultaneous modification of cytochrome 6, . (1) addition of CO at the point \vhen the decomposition of bT is incomplete (Fig. 1lB) results in an abrupt disappearance of absorbanw and concomit.ant shift of the overall A,,,,, from 562 to 3% nm. (Th(a residual absorbance is due t’o the mixture of unmndificd b, and 6,.) (2) I’otentionwtric titration of the compotwnts absorbing after partial GCiY treat,ment (RSCN is removed by passing the incubation mixture through a Scphadrx G-25 column at. t.hc point, when, as judged 005 02M

KSCN

KI K Br KCI Salt

Seconds FIG. 14. The effect of the anions of Hofmeister’s series on the speckum of succinateecyt,ochrorne c reductase. Succinate-cytochrome c reductase (2.2 mg/ml) was suspended in 0.1 M K-phosphate buffer pH 7.5 in the presence of 0.17; DOC and 0.5yo Triton X-100. At time 0 the anions were added and the set of spectra as those of Fig. 12 were obt.nined. Decrease in absorbance plot,tcd on the abscissa were measured at 586-575 nm.

ET 11r..

from the spectrum, cytochromc bT is warl~ complctrly dccomposcd) rcvwls the prcwncc of only one spcciw with a half-reduction pot,cntlal of -30 mV, \vhich wadily cornbinw wit,h CO to yictld a CO-compound of a wry low absorption cocfficitwt (not sho\sn). When t,hcbincubat,ion with J\SCN is carried Out, in the: pntscncc: of CO (l:ig. 13), similar absorbancct changw duo to disappearanw of the rctduwd qtochromc~s 6 aw obwrwd. Thcrcl is, howwbr, 110 obwrvabk shift in the X,,,;,, during th(l time courw of I of that wcordt’d, when CO is addrd afkr prior cornpletc~ decomposition of cytochromr: 6,. (not sholvn). This suggcwts that if ligand is available during I ligand do(Bs not i~~volw its binding to thcl modified hctmc 6, . Evidwtly various dcgrccs of modification of the>6 (bytochromw arc possible. An intcwsting fact, is that IMCY treatmcnt CRUSCS disappwrancc of 6, dpwtrum, but. total pyridiw hc~mochrc~mogen content, remains constant. Thus, in this case, dccomposition of c\-tochromr ttT and thcl cxtcnsivc spectral changw arc’ uot accompankd by destruction of tho hemct. Th(L qwstion arises whcthw the I protck or \\-hc%hw t.hc> c~xt,ensiw modification of the protrin occurs \vithout heme rcleasc:. httempts to isolate free hcmc b failed, although it is still possible (and probably quite likely) that any hclmc: released binds unspecifically but wry tightly to the mitochondrial protck. The rapid dwomposition of cytochrome 6, by RSCN is not, a characteristic feature only of this reagent but is common to ot’her salts of t.he Hofmcistcr series. The time courses of t,hc sprckal changes induwd by 0.2 11 ICI, T
obrys

the

ions,

SCN-

>

ix.,

Hofmeistcr’s I-

>

seriw

of

Br- > Cl-. Ciumli-

yy

A

30

20

IO

0

mln

(Incubation Time)

cB

IO

100

I-t

IOOOmM

KSCN Concentration VI<:. 1.5. The effect of KSCS on dnrohydroc]llilrolle-c?-tochrome c reductase of nctivitv srlcciIl:lte~cvtochrome c reductase preparation. A. lirlccin:rtr-~?tc)chrome (’ reductase (2 mg prot/ml) was incrlbatetl in 0.1 ~1 phosphate buffer, O.l”;, tl~~r~xyc~holal~. :rlltl 0.2 hl KSCN. At intervals intli~:rtctl 01) the :rlr)~cis~:t the aliqllots were withdrawn :rtltl as,~yrtl for tlr~roh~droq~ri~lone~~~to~l~ror~l~ (’ rrtltlct asc :xtivit,y as described in thr method sec. tion. 13. Pllccinnte-c~torhrome c redllctase (2 nl.5 pro1 ,‘ml) was incnhnted in 0.1 31 phosphate buffer, O.ll’; ‘I’riton S-100, 0.1”; deosycholate, and tliffrlcnt colrcclltr:ltioIls of KSCIG (abscissa) for 5 mill. I)rlroll~tlroc~~~illolle~-c~toc~~t~~)rne c redact ase activity was then assayxl :LS described in the met hod sclction.

The> prcynration of siicoiiiat~-cvtocllromcl r rcductaw by thcx Triton X-lOO-IIOC mc~thod offrrs a numbw of advantqys ovw preparations described in thus li&ratuw. Tht> method is quick and simple>, thcb scp:uw tion of the wductaw from tlicb otliclr mitoc~hondrial complcxw is good and the ~ic+l of thcl prcyaration is high. l~urthwmort~, th(> qwctral arid tlicwnod~nnmi~ propc’rtiw of thr components of thcl succkatc~ cytwhromc> r rc>ductaw closc~ly rcwcmbk thcw of intact mitochondria \vhich is thcl most vital (Sritt,rioii in cwiluating tlio “intadnws” of thcl systrm and its uwfulnws for futuw studies. In addition, thcl functional prupcrtiw of the> componc~nts of the, rcdrwtaw :tro prcwrvcd as manifwtcd by thcl kin& c~orwlations b(+\\ wn cytocl1ron-w c1and OTin the, pr(wnw of antim\.cGi ~1\vhirh :w wscwti~dly idwt k:d to thck c~xtcwsiwly discuswl b(ahnvior of tlicwa wrriclrs in tlrcs iiit:ict rnitoc~llc,iltlria, (10, 34).

Cytoc~hronw /I,,. (Aibits a double O( pc& with two maxima, S nm apart at .i(i(i and 53 nm. Jn potc~lltiornc,t,ri(, titrations and simultnwow qwc+r:d mwsurCmc3~ts the

444

EBECINSKA

points obtained from plotting the 566 and 555 nm absorbance fell OII the same line. Furthermore, trest’ment with KSCN or Triton causes simultancaous disappearance of bot,h peaks. Thus, WC obtain no evidence that the two peaks represent two different components, on the contrary, we feel the\ belong to the same cytochrome (set 7, S). In addition Do the double a peak, cytocabromc>b, has a double p peak, with a maximum at 530 nm and a shoulder at 53X nm. The double LYband of cytochrome b, is not a common fcaturc of the spectral characterist,ics of the cytochromes and suggests a rather unusual “strained” configuration of t,his hemoprotein (cf. ;3Tt). In line with this is bhe great sc>nsitivity of cytochrome b, t’on-ard any changes in environment; deoxycholatc and cholatc modify the spe&ral properties of t,his carrier to a form indistinguishable from t,hat, of cytochrome 6, and lyotropic anions cause disappearance of cyt’ochrome b, spect,rum. Although cytochrome 0, is more sensitive to treatment, wit,h detergents or lyotropic anions than is 0, , thcl modification or decomposition of the former is accompanied b) a modification of the latter. The modification of cytochrome h, , however, does not necessa.rily lead to any changes in its spectral properties but, can be detected by two criteria : change in the half-reduction potential and the CO-sensitivit’y. Change in the halfreduction potential usually precedes the a.cquisit.ion of CO-selAt.ivity. In this cont.cxt it should be stressed tha,t bhc modified c,:tochrome b, can also react’ with CO provldmg that t.he ligand is present during the modification procedure. Nthough neither the detergents nor t,he lyotropic anions can be considcrcd as specific ligands of either of the b cytochromes, it is int’cresting that their modifications tend to occur simultaneously. This may bc a coincidcncc, but may also be and an expression of a close structural functional relationship between the t,wo hemoproteins. In view of the fact that the bile salts, cholate, and deoxycholate modify both cytochromes b and all t,he preparations of succinate-cytochromc c reductasc described in the lit,eraturc arc made with bile salts (l-4, 16) w-e purified succinate-cytochrome

ET /IL.

1: rcductasc from beef heart mitochondria according to the method of Yamashita and Racker (16) and compared it,s spectral and t’hcrmodynamic characteristics with those of our preparation (not shown). W’c found t.hat, in spit{: of high succinate--cytoehromc c roductasc act’ivity (0.95 pmole cyt c red/ min/mgprotein ascompared to 0.3-0.4~moIc in our preparation) t,he two cytochromf>s 6 exhibit’ed indistinguishable spectral properties and t,htbir half-redu&ion potrntials at pH 7.0 wcr(: -100 mV and -10 mV (in addition a third component, was found \vith a half-reduction poterAia1 of +150 my, probably an analog of the b cytochromc rrportctd by DuMon et al. (23) in beef hclart mitochondria). It sc’cms therefore>, that the b cytochromcs prcparc>d with bile salts, in contrast to those prc>scnt in the Trit#on preparation arc already partly modific>d and pcrha,ps :L word of caution should bc issued in intcrpret.ing t,h(l “rr~onstitubion” cxpcrimerits in \\hich thcsc preparations have been used. A further conclusion \\-hich arises from t’his study is that although tb(~ two b cytochromc~s rcprclscnt, t \vo separatcx hcmoproteins (36) \I-hen purified, unless spcGa1 precautions arc> taken, they may clxhibit indistinguishable spectral properties, 11, being convcrtcd to a form resembling h, . In conclusion, it, is worthrvhilr pointing out t#hat the Tritoll-deoxycholatcl prcparntion retains all the spect’ral and thermodynamic characteristics of 11 cytochromes in intact mit,ochondria and thus it is :I useful model systcbm for the studies on c+ctron transport. at sitcl II.

This work

WRS supported

by GM 12202-08.

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