Purification and spectral characterization of a b-type cytochrome from the plasma membrane of the archaebacterium Sulfolobus acidocaldarius

October 1991

FEBS 10279 Volume 291, number 2, 331-335 0 1991 Federation of huropean Blochcmlcal Socletles 00145793/91/53 50 ADONIS 00144793910098&V

Marita Becker and Gunter Scht&xhtrtute

of &od~ennzatry. Maid

Recetved

Umw~ttv

24 July 1991, revised

version

of L&beck. Lubeck, Gertnony

received 20 August 1991

For the first tune the purlficatlon of a heme& contammg cytochromc from the plasma membrane of an extremely thermoacldophlhc archaebactermm ISdescribed The detergent solublhzed 30 kDa polypeptlde contains two heme- centers and one copper Ion According to Its low temperature spectra and CO-bmdmg properties, It ISlikely to function ds a cytochrome-o hke terminal oxldase m the membrane The purltied cytochrome does not retam catalytic actlvlty, however Cytochrome-6.

Archaebacterla,

Sulfolohs,

1 INTRODUCTION

Electron transport,

Wesplratlon

2 MATERIALS

AND METHODS

2 1 Growl/t of bncrertn

The extremely thermoacidophlhc archaebactenum Sulfolobus acidocaldmus grows aerobically and has been shown to carry out respiration-lmked proton extrusion [l]. The electrochemical gradient of I-I+’across its membrane is !lkely to be used for chemlosmotlcalIy driven ATP synthesis [1,2] It 1s unknown, however, which components of the respiratory system are acting as effective ploton pumps allowing for a pH gradlent >3 It has also been shown that dlfferentlal cyanide sensltlvlty indicates the action of more than one terminal oxldase The membrane-residing respiratory systcm besldcs NADH-dehydrogenase [3] and succmate dehydrogenase [4] contams only a- and b-type cytochromes while c-type cytochromes cu-e absent [5-71. From these we have purified and characterized a novel cytochrome-au, [8,9], acting as a ‘quinol oxlddse’ and using caldanella qumone as substrate. The lsolatlon of b-type cytochromes revealed to be unusually discouraging dbe to the instability under the harsh condltlons necessary for dlssoclatlon of the firmly associated respiratory complexes from the membrane A partial purification has been prehminarily reported [lo]. Here we describe the isolation and spectral characterlzation of a punfied b-type cytochrome from Sul~olobu~ ctctdocakdarm, likely to function as a cytochrome-o m the intact membrane.

Ctrr,crl,l~rrlk/rtc N(frlrcc, G Schafct , inst~tut fth Blechenue, MC~I~II~IUmvcrslttit 7u LUbcck, Ratzcburgcr Allcc IGO. W-2400 Lttbcck. Gcrnldny Fax (49) (451) SO0 4034

schc

Suljblobus actdoculdarm DSM 639 cells were grown aerobically m Brock’s medium [I I] supplemented with 10 mM K2S04. 0 2% sucrose, 0 1% yeast extract (Chbco), pH 2 5, at 75°C Cells were harvested m the early stationary phase, washed once m a buffer contammg 50 mM lmldazole, 1 mM malonate, pH 7 0. and sedunented for 15 mm at 6500 xg at 4OC The pellet wdb stored at -80°C m the same bufferaddltlonally contaming 50% glycerol

2 2 Mnnbratre pt cpurarrotr Membranes were prepared ds dcscrlbed [9], with the excepuon that a buffer contnmmg 50 mM lmldilzole, I mM mafonate, pH 7 0 was used and the cells wele disrupted m a Manton Gaulm press for 5 mm dt 5 x 1O'Pa 2 3 Put r$canon procedut e Membrdne extractlon dnd protem purification were performed at 4’C First the membranes were Incubated m a buffer contammg 50 mM Imldazolc, 20 mM K&O,, pH 7 5, for I h at a final protein concentrahon of about 10 mg/ml After sedimentdtlon at 200 000 x g for 50 mm. the resulting pellet was suspended m 50 mM muda7ole (find1 protcm concentration of 20 mdml) and extrdcted for 75 mm m the presence of 1% dodccylmaltoslde (DM) After 1 II sedlmentatlon dt I50 000 x g, the supernatant wds concentrated (Amicon, PM10 membrane), gently shdken with Blo-Bedds SM2 for at lcdst 2 h, and subsequently passed through a hydroxydpatlte column (2 6 x 26 cm), equlhbrated with 0 7.5 M KH2P04, 0 05% DM, 0 02% N-dodecyl-N,Ndlmethylammomo-3-propdne-suffondte (SBl2), pH 7 5, (f?ow rate 40 ml/h) Protein dnd hcmc III cluatlng fractions were monitored at 280 and 420 nm, respectively Hemc-contammg pcdk frdchons were dndlyzcd by thclr dlthlonlte-reduced mmus oxldlzcd dlfferencc spectra Cytochrome contamrng fractions were conccntratcd, the buffer exchdngcd to 50 mM KHIPO,, pH 7 5, ,tnd loaded on d second hydloxyapdtltc column (3 x 14cm)equ1hbrdtcd with SOmM KH2POo,0 05% DM, 0 02% SB12, pH 7 5 (flow rate 30 ml/h) When 100 ml of the equlhbratlon buffer had pdsscd through, d hnedr phosphate grddlcnt (SO-500 mM KH,POd) III 005% DM, 0 02% SB12, pH 7 5, wa$ dpphcd Cytochromc-h.cnrtchcd frdctlons wcrc pooled dttd conccntrdtcd In cdsc of .i rcsldudt cytochlome-cr Contallllndt~on ttic mdtcrl,tl W‘IS rechrotndtographcd on hydroxyap‘iutc under analogous Londltlons Subscqucatty the mdterldt W.IS to.ldrd on d HILoad/600-

331

Volume291,number 2

October1991

FEBSLE-M-ERS

Superdex 200 column usmg J buffercontammg 50 mM Imldazole, 150 mM NaCI. 0 2% SBl2, pH 7 5 (flow rate I ml/mm) The findi step of purlficatlon was performed on a MonoQ-Y5 column with a lmear gradlent from 0 to 0 5 M NaCl m 50 mM mudazole, 0 2% SBI 2, pH 75 2 4 Spectroscopy A Hewlett Packard HP8450A diode array spectrophotometer was used for spectrd at room temperature, low temperature spectra were measured with a Sigma ZWSII dual wavelength spectrophotometer cqulpped with a self-deagned low-temperature device Pyrldme hemochrome spectra were recorded accordmg to [If] Copper content was deterrnmed usmg a I-htachl 180-80 Zeeman atomic absorption spectrophotometer set at 324 8 nm 2 5 Other mcrkofis Hydroxyapatltc was prepared accordmg to [ 131 Membrane protem was determmed by the bmret method [ 141, membrane extracted protems accorchng to Lowry [I51 m the presence of SDS PAGE were performed by the Laemmh procedure [ 161on 15% gels. protems were vlsuahsed by sliver stdmrng 1191 2 6 Materrals HIEoad/600-Superdex 200 and Mono-Q-515 columns were obtamed from Phdnnacla All chemlcdls were purchased from Merck. Sigma, Serva, Blomol or Flukd

3 RESULTS Prevmus spectroscopic studies have shown that more than one heme-b center is present III the membranes of Su@‘olobus, as Indicated by an absorption maximum at 565 5 nm and the pronounced shoulder at 558 nm m reduced-oxidized difference spectra taken at room temperature [9]. Partial reduction by ascot bate and spectral subtraction from the fully dlthlomte-reduced state suggested a third compound to be present absorbing at 562 nm. In Fig I, a low-temperature spectrum of freshly prepared Sulfolobus membranes IS shown where indeed three individual maxima m the a-region of b-type cytochromes can be clearly dlstmgulshed At low tempcrature the absorptlons are shifted shghtly to lower wave-

Fig I Reduced mmus oxldlzed difference spectrum of a membrane suspcnslon from Srrljulobus rrcr&al&r IUJ taken at hquld nitrogen temperature, I 5 mm I&t path, the medmm contained 50 mM Imldazolc pH 9 5 30% glycerol and 2 9 mg protemlml find1 concentration The reduced sample W‘IS prepdrcd by addltlon of sochum dlthlomte The bottom trdce shows the 1st derrvdtlve. clearly mdlcatmg three InflectIon points m the a-bdnd region of heme-

lengths located at 553.5, 556 2 and 561 nm, respectively Also the P-bands are nicely developed In addltlon, the spectrum shows the typical band of the MI,oxldase and the prominent peak at 582 nm which could not be functionally attributed so far though being the most significant spectral band While we have tentatlvcly assigned it as a cytochrome-a, [6,7], it was suggested to be a constituent part of a terminal aa?-type oxldase described for another spcc~es of Sul~~lohus [ 181 Durmg preparation of b-type hemoproterns from solublhzed membranes the separation from this latter

-r

SO

tmctlan number

BQ

10

20

4b sb tracttan numbor

eb

l-16 2 Elution llroiilc~ oflhrornato8r,tphlc scpdrdtion Ftcps (A) Supcrdcx-200 column. the frdcllons wcrc tortcd utdlvldually dt 28Onm fol protcm, dt 416 nm for oxd~ml, .md ut 426nm for reduced hcmc dbsorpllon, rcspsctlvcly It W,ITobscrvcd tlldl durmg thccoursc of prcpdratlon the hcmes cdn bc pilrtldlly rcduccd (B) Mono-Q 5/S column. 111th15 USC protcm Randhemc wc’c monltotcd ,~utotn~~t~cally dt 280 nm for protcln dnd 420 IIIII a\ un tntcrmcdl,ltc wdvclcn8th foi the hcnlcs Ethel condltlons a\ dcqcrlbsd III the tcu

332

Volume 291, number 2

October 199 I

FEES LETTERS 53

X30

kDa

e

9

2

1 I I

-

45 kDa

FIN 3 SDS-gel elcctropliorcsrs Prolem bdnds were vlsudlued

I fnmn brat extrat,t (lme I. I O,ugW) and purrficd cytochrome-l> (lane 2, 3 @nl) from S14Qblol7rrr o~hrr/hrm~ by s r er sld~~mig, S = liiolcculdr weight ILII kcrs @-gd~dCIOSldd5~*I30 L.D,I,hovmc serum ,Ilburnm 67 kD.1. OvdlbUInln 45 kDd. chymotrypsm 25 kDd, md cytochromc-c 12 3 kD,l)

was the most crtttcal challenge No detergent was found whtch preferenttJly solubtltzcd h-type cytochromes. In contrast. most detetgcnts f‘ttled to ptescrve the nattve state of hetnopt oteins ds concluded from the dtsappearancc of specttal resolutton resulting m only one coalescent hem47 dbsorptton m,txtmum at 560 ntn (not shown) Tl11.s was not the case when rnctnbranes were solubthzed by 1% dodecylmaltoslde ,tftcr preextrCtcrton wtth 20 mM py~ophospftatc wfttch removed 7-l 5% of loo~ly mcmbr.tne ossoctatcd pt otctns (for detatls see Methods) On a first hydroxyupat~te column, a change of detergent wa\ achtcvcd togelhcr Wth pdt?liIlrctcntton of’a-type cytochromcs. Tflc! compound

second hydroxydpatitc column removed a large portton ofcontatntnattng protetn from ‘1slowly mtgrattttg broad hcme-contatntng band Actually, one of the rnaJor obstacles IS the obvtously strong assoctatton of the vartoits resptratory catrtcrb rcsulttng in a smear of componcnts over a wtdc range of fracttons. thus, an tnverse gradual dtstrtbuttott of u- and h-contamtng hcmoprotctns over a broad peak could bc Ctchtevcd. rather than a clear scp.tratton A subscqucnt gel chromatographtc step on a Supcrdex-200 colutnn had the purpose to cftongc the dctcrgcnt ags~n to a kighcr coticcntratton of S&l2 only, yiclditty two peaks, one of which tndtcetcd a htgfi 5pcc1fic cnrtchtnctit of hcmc-17 Anion cx333

Volume 291, number Z

400

450

FEBS LETTERS

500

550

wavelength

6QQ

October

1991

650

[ftml

Fig 4 CO/reduced rnmus reduced spectrum of purlfcd cytochrome-6 from Su~oiobus acrdocaldurnts Protem concentration was 0 045 mgl ml m a buffer contammg 50 mM Imldazole, 0 2% SB12, pH 7 5, d = I cm

kg 5 Low-temperature reduced rnmus oxlchzed difference spectrum of purdied cytochrome-0 The sample was oxidized by ferrlcyamde (0 6 mM), after the spectrum wds run and stored as a reference, the sample was reduced by d small gram of dlthlonltc and the spectrum scanned again at hqmd mtrogen temperature, hght path I mm, 0 8 mg proteml ml III a buffer contammg 0 2 M potassmm phosphate pM 6 5 30% glycerol

change chromatography on a Mono- Q column finally gave a fraction of&-type cytochrome, fret of any contaminating heme-a as venfied by difference spectroscopy and pyrldme hemochrome analysis Elutlon profiles of the last two separation steps are given m Fig. 2 From its clutlon volume on a calibrated S-200 gel chromatography column a molecular mass of 48 kDa was calculated for the detergent-embedded cytochrome Fig 3 shows a silver stained SDS-gel of the preparation compared to the membrane extract. The band of JO-31 kDa was identified to carry the heme-b by heme-staining of gels run m the absence of mercaptoethanol, dllowmg to prevent a complete loss of the prosthetic u-on porphyrin during SDS gel electrophoresls A smaller peptide of about 18-20 kDa was frequently observed as well as a tendency to form aggregates of about 67 kDa apparent molecular mass. It 1s unclear whether or not this polypeptide IS an additIona constituent part of the cytochrome-i) complex. Table I gives a typical purlficatlon protocol Though a high degree of enrichment was achieved, It also ~llustrates the poor yield of punfied cytochrome Obviously also some loss of heme-b occurred, especially during the 2nd and 3rd column steps. The final product shows a

typlcal cytochrome-6 reduced mmus oxidized difference spectrum at room temperature, with maxima at 427 nm, 5.58 nm and a pronounced shoulder at 566 nm. It binds carbon monoxide. yelldmg a CWreduced minus reduced difference spectrum typical for terminal oxlddses as given m Fig 4 The charactcrlstic maximum at 418 nm and troughs at 428 and 558 nm ale clearly developed, resembhng the typical spectrum of CO-binding hemecontammg cytochromes. In Fig. 5 a low-temperature difference-spectrum (dlthlomte-reduced minus OXIdlzed) IS shown, mdlcatmg the presence of two heme-b centers It parallels nicely spectra of well-characterized o-type cytochromes [19], displaying at 554 and 561 nm the same maxima m the cl-band region as found in intact Su&dobus membranes (Fig. 1). The purified cytochrome also contained tightly bound copper wlthstandmg extensive dlalysls against 10 mM EDTA From several preparations an average copper content ofO.4- 0.6 mol/mol heme- was determined, suggesting that the comparatively small 30 kDa-polypeptlde hosts two

Table I Typlcdl purihcdtion Purllicdtlon stdlc

protocol lor cytoctiromc-b

Total protcm (mg)

Spcc~fic

from S1rlJ0k1bu.sr~~rffuc~~/~~urrrtr

hcmc-h co!) tent (nmol/mg)

Purlfirdtlon

fdt,tor 11 -

me 291, number

2

FEBS

es and one copper associated with one of the hemes

xpected for an o-type terminal oxldase. Despite e similarities no significant catalytic actlvlty as a rldase or a TMPB-oxldase could be verified Allgh an almost neghglble activity with caldarlella lone could be detected, the preparation IS likely to lactlve due to the loss of either addItIona subunits, f specific hpids essential for catalytic actlvlty AddIof soybean asolectms or a crude Sulfolobus lxprd 3ct did not unprove catalytic activity It should be tloned, however, that in membrane extracts by IXtitrations the presence of high- and low-potential pe cytochromes could be estabhshed which would ie values for a functional o-type cytochl ome [6,20] WCl_JSSION he presence of b-type cytochromes m aerobically vn archaebactena has been shown for HalobacteI cutrrublurtz [21] and H halobwm [22,23]. None of e has been purified so far and, moreover, then= redox ntlals differed slgmfcantly when determined either )lutlon or m membranes, respectively Nevertheless, ast one of those might represent a terminal oxidase ancluded from Its CB-bmdmg properties Here for Rrst time lsolatlon and spectral characterization of ghly purified b-type cytochrome from a thermoaclhlhc archaebactenum, SulfalobuA acrdoculdar MS, d be demonstrated Though its heme-b content, Jer content, and CO-bmdmg properties resemble e of Q-oxldrzmg o-type cytochromcs, a corresding catalytic activity could not be preserved. An‘r dlffcrcncc to known o-type cytochromes [24] IS the apparent molecular mass of the isolated polylde carrying the heme centers In this regard it falls zr to b-type cytochxomes acting as intermediary tron carrlels However, since more than only one anal oxldase IS present m this archaebactcnal genus 7 together with the fact that a low molecular weight qumol-oxldasc was identified 191.the assumption of a lather simple o-type terminal oxldase may be fied. Actually, the pal tlally cyanide-msensltlve n-atlon at low mhlbltor concentration, sufficient to k cytochrorne-era, completely, would be m lme with concept. Besides that, it was shown m previous les [6] that a high-potential b-type cytochrome (E”, 300 mV) could be detected by redox-titration m lbrane extracts from Strlfolohus, supportmg the encc of an alternatlvc termmal oxldase apart from chrome- CI(I? In addltlon, quahtatlve observations mdlcatrng that the ratio of cr-type versus b-type cyu-omcs m SulfuluDtts WI ICSwith oxygen avallabihty )ublrshcd results) Caldallclla qumone serves as an rmedlary pool fo1 rcducmg cqulvalcnts III the

LETTERS

October

1991

membrane of Sulfolobus and may provide the specific reductant for both terminal oxldases presumably differmg m oxygen affinity. Regrettably, It was lmposslble to follow the catalytic activity during purlficatlon due to competition with the highly active aa3-oxidase. The failure to isolate the mferrcd o-type cytochrome m active form may result from the loss of spcc~fic lipids or of other constituent polypeptides for an active complex The latter IS not unhkely, taking into account an obvious tendency of Sulfoiobus membrane protems to desmtegrate under condmons necessary to resolve the extraordinary rigid membrane structure. Sequencing of the b-polypeptlde IS m progress and IS expected to allow a defimte functional assignment by comparison to known heme-b contammg cytochromes A~ktrolvkligenrenlJ Our thanks are due to Mrs Dow Mutschall for sk:llfuf techmcal awstanre

REFERENCES [I] Mall. R dnd Schafer. G (1988) FEBS Lctt 232,359-363 [2] Lubben, M and Schafer, G (1989) J Bact 171,6106-61 I6 [3] Wakao, H Wakagl, T and Oshlma, T (1987) J Blochem (Tokyo) 102, 255-262 [4] Mall. R and Schafer, G (1989) B~ol Chem Hoppe-Seyler 370, .936 PI hnemtiller, S , Lubben, M and Schafer, G (1985) FEBS Lett 193, 83-87 VI Schafer, G , Anemilller, 5 , Mall, R , Meyer, W and Llibben, M 11990) FEMS Mlcroblol Rev 75. 335-348 G . Ldbben, M and Ane&ller, S (1990) BlochIm BIO[71 ichdier, phys Acta 1018. 271-274 Anemuller, S and Schdfcr. G (1989) FEBS Lett 244,451-455 [ii Anemtiller. S dnd Schdfer, G (1990) Eur J Blochcm 191.653657 [lOI Becker, M and Schafer, G (1990) Gth EBEC Short Reports, I02 Elsewer Amsterdam 1111Brock, T D Brock, K M , Belly, R T and Welts, R L (1972) Arch Mxroblol 84. 54-68 [I21 Wllhdms, J N (1964) Arch Blochem B~ophys 107. 537-543 [I31 Thlsehus. A , Hlertcn, S and Levm 0 (1956) Arch Blochem B~ophys 65 132-155 [I41 Watters. C (1978) Andl Blochem 88 695-698 [I 51 Peterson. G L (1977) AnnI Blochcm 83, 346-356 Laemmh, U K (1970) Ndturc 227, 680-685 ;;:; Heukeshovcn, J dnd Dcrmck, R (1985) Electrophoresls 6, 103112 [I81 Wdkagl. T , Ydmauchi T , Oshm~a, T , MLillcr, M , AZI, A dnd Sonc, N (1990) Blochem B~ophys Rcs Commun IG5. 11IOII14 [I91 Klta, K 1Komshl, K dnd Anrdku, Y (1984) J Blot Chem 259, 3368-3374 [20] Jones. C W and Poole R K (1985) Methods Mtcroblol IS. 285-328 [2l] Ldnyl, J K (1968) Arch Blochcm Blophys 128, 716-724 [22] Hallberg, C dnd Bdltschcffsky, H (1981) FEBS Lctt 125, 201204 [21] Hallberg-Giddm, C dlld CdlmsJo. A (1989) Arch Blochcm Blophys 272. I TO-I36 [24] Anrdku, Y dnd Gcnn~s, Ii B (19X7) Trends Bmchcm SCI 12, 262-266

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