Electron transport and energy conservation in the archaebacterium Sulfolobus acidocaldarius

FEMS MicrobiologyReviews75 (1990) 335-348 Published by Elsevier

335

FEMSRE 00150

Electron transport and energy conservation in the archaebacterium Sulfolobus acidocaldarius Giinter Sch~ifer, Stefan Anemtiller, R a l f Moll, W o l f g a n g Meyer a n d M a t h i a s Liibben Institut fl~r Biochemie der Medizinischen Universit,~tzu L~beck. Liibeck, F.R.G.

Key words: Archaebacteria; Electrontransport; Cytochromes; ATP-synthase; Chemiosmosis; Thermoacidophiles

1. SUMMARY The bioenergetie properties of the thermoacidophilie archaebacterium Sulfotobus aeidocaldarius are reviewed and discussed under the aspect whether this archaebacterium conserves energy by oxidative phosphorylation and how the involved catalysts ate related to those from eubacteria and eukaryotes. The thermodynamic parameters contributing to the proton-motive force and the efficiency of proton pumping are presented. The major components of the electron transport system ate identified and a novel type of heme-aa 3 containing terminal oxidase is described, oxidizing reduced caldariella quinone. The properties of an Fi-analognus ATPase and of a DCCD-binding proteolipid from the plasmamembrane of Sulfo!obus are discussed as likely components of an

Abbreviations: DCCD, dicyclohexylcarbodiimide; SDS. sodium dodecylsulfate; TCS. tetrachlorosalieylanilide; TTFB, 4,S,6,7-totrachloro-2'-t rifluoromethyl-benzimidazole; NBD-CI, 4-chloro-7-nitro-benzofurazan; pHMB, p-hydroxy-mercuribenzoate. Correspondence to: G. Schlifer, ]nstitut for Biochemie, Medizini~he Universitat, Ratzeburger Allm: 160, D-2400 IAlbeck, F.R.G.

FoFl-analogous ATP-synthase. The structural and functional properties of this and other arehaebacterial ATPases are compared to each other and with respect to evolutionary relations. 2. I N T R O D U C T I O N Long before archaebacteria were recognized as a separate kingdom of organisms [1], biochemists and microbiologists had been attracted by the unusual environmental conditions tolerated by, or even being a prerequisite for various species to survive. The investigations on halobacteria are an outstanding example which finally led to the discovery of a series of membrane proteins acting as light-driven ion pumps or signal transducers (see [2,3] for review). Based on their capability to produce a proton gradient already early studies implied the proposal that they conserve energy for ATP production by a classical chemiosmotie mechanism according to Mitchell's postulate [4]. It happened only in recent years that also with methanogeni¢ archacbacteria first steps could be presented demonstrating the coupling of methanegenesis as a driving force with the formation of ATP by chemiosmotlc principles [5-7]. Other archaebacteria like thermoaeidophilic sulfurmetabolizing bacteria were proposed to generate

0168-6445/90/$O3.50 © 1990 Federation of European MicrobiologlcalSocieties

336 ATP by fermentative pathways as an energy source to extrude protons and other ions like sulfate [8]. This latter postulate appears unlikely, however, especially under the aspect that those bacteria may grow autotrophically on sulfur, a condition which requires the generation of ATP without the access to energy-rich organic fuels. In general, the situation was challenging to investigate bioenergetie mechanisms of archaebacteria from two points of view: first, as they are extremophilic organisms, and second, phylogentically, asking the question whether or not the catalysts of energy conservation are universally related and trace back to a common origin. Our studies on Sulfolobus addocaldariar are reviewed in the following and shall contribute to these questions.

3. PROTON P U M P I N G A N D ELECTRON TRANSPORT PHOSPHORYLATION

Sulfolobus acidocaldarius (DSM 639) has been routinely grown heterotrophicaUy under aeration as reported by Brock [9] at pH 2.3 and 75-85°C. As described previously, these cens show respiration on endogenous substrates [7]; after a period of starvation under aerobic conditions respiration may be stimulated by glucose or suceinate. This respiratory activity could not be inhibited by any of the commonly used intermediate inhibitors of repiratory electron transport, whereas only terminal oxidase inhibitors like azide or cyanide did inhibit both respiration and ATP production. Significantly, there was an absolute dependency of cellular ATP content on respiration. An early indication for coupling of respiration and active proton pumping has been seen in the transitory eighbfold increase of oxygen uptake following a pulse of alkalinization [10]. Later it could be demonstrated that cells kept anaerobically for a short period of several minutes respond to oxygen pulses by a rapid extrusion of protons [11]. For these experiments the cells were set to a more alkaline pH between 5.8 and 6.5 prior to the oxygen pulse. Under these conditions "initial rates of respiration and proton extrusion were found to be proportionally related and did for example depend in the same way on the presence of vari-

Table 1 H÷/O ratios of Sulfotobusacidocafdaeti¢cells Conditions K + 5 raM

+(~H+/g/) -(~02/~t ) (nmol rain-1 mg-I) 396 30-35 501 30-35

K + 20raM K ÷ 20raM+ Valinomy¢in 738

30-35

H+/O 5.6- 6.6 7.2- 8.3 10.5-12.3

Initial rates of proton extrusion and oxygen uptake following an oxygen pulse are given. Cells were incubated at 60°C at -0.8-1.5 mg/ml protein concentration in a medium containing 5 mM glucose, I mM potassium phosphate, supplemented by potassium sulfate to give the indicated final K+-concentra~ion; when present valinomycin concentration was 5.9 nmol/mg. The data are taken from ref. 12.

ous concentrations of the respiratory inhibitor azide. Thus, preliminary estimates of H+/O-ratios could be derived ranging from 0.5 to 3 with the latter values obtained only in the presence of > 50 mM potassium and valinomycin (8 ~tM). After exhaustion of an oxygen pulse a back-flow of protons into the cells occurred, following first order kinetics. This capability of Sulfolobas acidocaldarius to pump protons actively was sensitive towards protonophorcs like TTFB, TCS or gramicidin-D; moreover, low concentrations (20-100 n m o l / m g protein) of D C C D stimulated the amplitude and the rate of proton extrusion and thus suggested a classical respiration-linked proton-motive system to operate. Nevertheless, the efficiency of proton pumping appeared rather sluggish in view of the strongly acidic growth conditions for this organism (pH 2-3). In fact, using stopped flow techniques and spectroscopic monitoring instead of electrodes allowed faster initial rate measurements of both proton extrusion and respiration [12], yielding much higher H + / O ratios as depicted from Table 1. Even in absence of valinomycin values of 6 H + / O could be measured and occasionally maximum values as high as 12 have been monitored. Though these data convincingly demonstrate the high capacity to pump protons, as one might expect for an extremely acidophilic organism, it should be emphasized that these values have been obtained only after significant reduction or even omission of ( N H ~ ) in the incubation medium, a

337 condition which is in contrast to the recommended [9] concentration in optimum culture media. A reasonable explanation would be that by the N H ~ / N H s couple a compensatory proton flux occurs, diminishing the observable netproton-extrusion; furthermore, the isosteric ammonium ion may block essential potassium channels involved in a combined action of primary proton pumps and secondary exchange systems, enabling the cells to maintain a moderate internal pH and to balance for the membrane potential generated by the massive proton extrusion. In summary, assuming a ratio of three protons from outside consumed per 1 molecule of ATP formed inside - a commonly accepted value - the H+/O-ratios would easily allow for more than one coupling site, if in Sulfolobus oxidative phosphorylation occurs via a typical /'oFl-catalysed chemiosmotic pathway. With regard to this latter question two independent studies from our laboratory [11,13] made obvious that a proton-motive force of 140-160 mV is generated under steady state conditions. The components of this proton-motive force (external pH = 3.5) are a large pH-gradient (2-3 pH units) and a small membrane potential, slightly negative inside. Only under conditions of massive proton influx caused by protonophores (uncouplers, gramicidin-D) the membrane potential turned positive inside, balancing then for a residual pH-gradient even under uncoupling conditions; therefore, the overall proton-motive force in Table 2 Components of the proton-motive force ,Ap in Sulfolobus acidocaldarlus ceils; response to protonophores and gramicidin-D Condition -Z/tpH (mV) z~ff(mY) Ap (mV) Control -134 -19 -153 TTFB (6.7) - 107 + 54 -53 TCS (6.7) - 74 + 69 -5 Gramicidin¢D (30) - 67 + 63 -4 Amounts of ionophores given in paranlhesis as (nmol/mg protein). Cells were incubated at 450C. external pH 3.5; the measurements refer to to min after additionof the respective ionophote. Data from [13] and M. Lilbban (1988) Thesis; Llniversityof Hannover. Abbreviations: TTFB= 4,5,6,7-tetrachloro-2"-trif/uoromethylbenzimidazol¢;TCS, tetrachlorosalieylanilide.

this situation approaches zero. A typical experiment is given in Table 2. In fact, the dynamics of metabolic state transitions [11,13] strongly support the function of a proton-driven chemiosmotic ADP-pbospborylation. DCCD, a typical inhibitor of proton channels of FoFt-ATP-synthases , produces a respiratory control phenomenon in Sulfolobus cells, characterized by a rapid decline of cellular ATP and a concomitant respiratory inhibition without loss in proton-motive force. Subsequent uncoupling by gramicidin-D restores maximal respiration whereas the proton-motive force is collapsed completely and cellular energy charge remains extremely low.

4. THE COMPONENTS TRANSPORT

OF

ELECTRON

Though it has been reported in earlier studies [10,14] that only a- and b-type cytochromes could be detected spectroscopically in Sulfolobus membranes, the composition of the electron transport system resisted resolution by simple methods because none of the known inhibitors acting on proor eukaryotic respiratory chains proved to be active, except azide, cyanide or CO as terminal oxidase inhibitors. Interestingly, even cyanide at 2 mM concentration caused only a partial inhibition of respiration, whereas much higher concentrations were necessary to arrest respiration completely. This was an early hint for a branched electron transport system in Sulfolobus, when taking into account additionally that reduced minus oxidized difference spectra in presence of carbon monoxide revealed a typical peak at 417 run and a through at 442 nm; both are indicative for a heme-b containing cytochrnme o which acts as an alternate terminal oxidase also in many eubacteria. Besides typical heme absorptions in the a-region at 562 nm and 604 nm (heme-b and heine an3, respectively) spectroscopic evidence for an additional cytoehrome was derived from a peak at 686.5 nm appearing in fully dithionite reduced membranes or their detergent lysatcs. Due to its response to cyanide (causing a significant increase of absorption) it has been tentatively asigned as cytochrome a I [10,15]. Its true nature remains

338

Table 3

Constitutents of the electron Ifallsporlsystem in membranes from $ulfolobas acidacaldarius NADH dehydrogenas¢(Flay.; FeS) Suceinatedehydrogcnase(Flay.; soveralFeS centers) EPR detectable FeS centers: g s 2.01; 1.94; 1.9l; 1.89 Calderiellaquinon©:E~ + 100 to ~ 106 mV Cytochromes: hemeb (o); heine a; (no heine c present); E~ +400; +220and +S7OmV; EPR: high-spinand low-spinherae-Fe; copper; associatedwith heine aa~

somewhat enigmatic, since all attempts to solubilize and purify this compound failed so far. Moreover, preparations of cytochrome oa 3 (see below) slightly tend to denature for example by freezing, accompanied by a spectral change and the occurrence of a prominent peak at 587 nm reminiscent of unbound heme-a. Table 3 briefly summarizes the major components of electron transport identified in Sulfolobus as yet. Purified membranes are capable of oxidizing NADH [10]. An NADH-dehydrogenase which is easily released from the membrane has been described [16] and is likely to be only the flavin-conraining peripheral part of a larger complex. It transfers electrons from NADH to ferricyanide or other artificial acceptors. Neither rotenone nor piericidine-A or any other inhibitor of known NADH-dehydrogenase complexes act on this enzyme. A suceinate-dehydrogenase activity could also be solubilized from the membrane in our laboratory [17], containing an irou-sulfur/flavoprotein with 66 kDa apparent molecular mass. In the membrane-bound state it catalyses the transfer of electrons from suceinate via cytochromes to oxygen and is inhibited by malonate. In sohibilized form it transfers electrons to artificial acceptors (PMS/DCPIP) as does another form of sueeinate dehydrogenase which could be found in the supernatant immediately when Sulfolobus cells are disrupted by sonication. The latter soluble enzyme has only a low specific activity and consists of two polypeptide subunits (75 and 30 kDa, respectively); it contains flavin as well as iron-sulfur centers. These exhibit microwave absorption in EPR-spec-

troscopy at g = 2.01 in the oxidized stale and g = 1.91 in the fully reduced state, respectively [17]. A series of other EPR detectable redox centers could be measured at 1 0 - 4 0 ° K which were attributed as follows. In membranes a g = 1.94 ironsulfur center is seen after reduction with suecinate. Reduction with ascorbate generates a g = 1.89 signal reminiscent of a "Rieske" iron-sulfur prorein. Hence, since a cytoehsome bcI complex is absent it remains open to asign this iron-sulfur center functionally. The high-spin heine signal at g = 6.0 and the low-spin signal at g ~ 3.0 of eytochromes could be measured in membranes as well as in purified oxidase preparations as described below. In the latter a weak copper signal appears which in part is superimposed by a free radical signal at g = 2.0 which might result from a tightly bound caldariella quinone at the terminal oxidase. Actually, a unique quinone characteristic of sulfur-metabolizing thermophiles [18] has also been found in Sulfolobas. This genuine 'ealdariella qninone' (QCal), as extracted from Sulfolobus membranes, could be incorporated into detergent micelles and investigated spectroscopically. Spectra of its reduced and oxidized state together with the reduced-oxidized difference spectrum are given in Fig. 1. The presence of pronounced maxima and minima (327 nm and 351 nm, respectively) and of an isobestie point at 341 nm allowed diroet redox titrations of the micellar solutions with dithinnite as a reductant. The determined half-reduction potential at pH 6.5 for a two-electron transfer between fully reduced and fully oxidized state is + 100 mV, slightly more positive than that of regular nhiquinones or naphtoquinoaes. It should be mentioned that in the protein-bound state this midpoint potential might even be more positive and the lifetime of a semiquinone form might be significantly increased; on this basis the free radical signal found in aa3-oxidase preparations has been tentatively explained.

5. THE TERMINAL OXIDASES Recently the purification of an aa 3 terminal oxidase has been described [191. Our original

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spectra were taken in 0.3 M phosphate buffer pH 6.5 with0.187otrilon-XlflOpresent. (G, SchSferand A. Anemliller,unpublished, 1988). purification procedure could be improved by hydroxylapatite chromatography, yielding an aa 3preparation of Sulfolobus which is free from any contaminating 587-nm hemoprotein. Fig. 2 shows the absolute spectra of the oxidized and the dithionite reduced preparation together with the reduced-oxidized difference spectrum. This aa 3oxidase is unique in several respects. The differ¢atc¢ spectrum shows the typical peak at 604 am and a Soret band at 439 nm; in presence of carbon monoxide the reduced-minus-oxidized spectra show peaks at 429 nm and 594 nm, respectively. However, the oxidase apparently contains only one single polypeptide as constituent subunit with an apparent molecular mass of 38 kDa (on SDS-PAGE). In detergent mieeUes it migrates on gelcolumns as a complex of about 120 kDa. Thus, it may be speculated that in detergent micelles it

forms a dimer under the applied non-denaturing conditions. This 38-kDa polypeptide has to host two heme-a groups, one of which is CO reactive. The midpoint potentials of the heme-a centers were titrated to be + 220 and + 370 mV, respectively. These values coincide well with those of other aa3-oxidases. In addition, two copper ions, 1 Cu/heme-a, were found. The presence of a third copper per reel could not be definitely excluded; this latter copper ion is easily removed by dialysis. Surprisingly, though no c-type cytochromes were found in Sulfolobus aeJdocaldarius, fresh membranes and crude membrane extracts were reported to oxidize eukaryotic cytochrome c in a cyanide sensitive reaction [20]. The specific activity of 0.3 U / r a g membrane protein even at 5560°C is rather low compared to other eubacterial

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cytochrome c oxidases; however, with successive purification based on increase of specific heme-a content the cytochrume c oxidizing activity became negligible [19]. Therefore it might have been due, in fact, to unspecific interactions of crude preparations at higher temperatures. As an artificial electron donor TMPD could also be used. This activity co-purified with berne-a; the specific activity, however, was not fully parallel w heme-a purification. Interestingly, with TMPD as an artificial substrate, only 6070 of the heine (measured in the Sorer region) could be reduced, while dithionite was necessary to produce full reduction. The flow of electrons from TMPD to oxygen through the aa3-system leads to the formation of water as documented by the TMPD/O2-ratio, which was reported to be 4 [21]. The reaction is 100~ cyanide sensitive.

Since cytochrome c must be excluded as a natural single electron donor, the question was open for some time what the native substrate could be for this oxidase, until the preparation turned out to readily oxidize reduced Q"~ directly. This latter activity nicely parallels the purification factor of heme-a. Thus it became likely that Sulfolobus contains a novel aa3-type of quinol oxidase rather than a cytochrome c oxidase. Fig. 3 shows an example for the direct oxidation of QCa] by Sulfolobus aa 3 oxidase in a cyanide sensitive reaction. On the left, the linear dependence on enzyme concentration under conditions of substrate saturation is given. When the present contribution went to press a preliminary report appeared describing another heme-a containing terminal oxidase from Suffolobus acidocaldarias [21a]. Surprisingly it was

341 centers were determined also in the E. cull cytochrome o, which is a functional qninol-oxidase [22]. Whether the likely alternate oxidase from Sulfolobus also directly oxidizes caldariella quimme as does the aa3-oxidase remains to be investigated.

2

6. THE AT~SYNTHASE t.

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Fig. 3. Oxidation of ca]darialla quinorte by a fraction of purified cytochrcmae aa 3 from Sulfolobusacidocaldarius. Reduced caldariellaqninone was used as a substrate immer-~eclin a buffer containing0.3 M potassium phosphate pH 6.5 and 0.18~ Ulton-Xl00;oxidation was recorded in a dual wavelength ~ectrophotometer at 351-341 nm (of. Fig. I). RifJat: where indicated by arrows 3.1 ~tg punfied aa3-oxldase [19] were added; final addition 1 mM KCN. Left: linear dependence of reductionrate on proteinconcentration. found to oxidize horse heart cytochrome c as preferential substrate; in addition this very preparation contained three polypeptide subunits. Besides that differences of the optical spectra also suggest that the organism from which it was isolated in fact may be another species, namely Sulfolotms salfataricus. Interestingly, this oxidase contains also an unusual band at 583 nm similar in size and shape to the the 586.5 nm band in membranes of Sulfolotms acidocaldarius; a clear functional attribution has not been achieved, however. It has to be added, that at an earlier stage of purification by use of the 562-nm absorption band also a b-type cytochrome could be identified in detergent extracts of membranes [19] exhibiting a rather positive redox midpoint potential of about +400 inV. This clearly supports previously mentioned spectroscopic evidence, suggesting the presence of a cytochrome o as an alternate terminal oxidase. Such an enzyme is expected to contain at least one high potential heme-b as described also for E. cali [22]. Purification of this species from Salfolobm is on its way. Preliminary spectroscopic evidence revealed the existence of more than one heme-b center in detergent sohibilized membranes (a-bands at 559 and 566 tun). In fact, two heme-b

6.1. Functional aspects Chemiosmotic ATP synthesis in eukaryotes and eubacteria driven by a proton gradient is catalysed by a unique and ubiquitously present protein complex, the so called F-type ATPaze (see [23] for review). When acting as a synthase, these enzymes dissipate the proton gradient as illustrated schematically in Fig. 4. The membrane protruding part in must cases can be easily dissociated and has the properties of an ATPase, asigned as F1; it usually consists of five different polypeptide subunits in the stoichiometry a3~3y& and contai,; three catalytic nuclcotide binding sites. This part in the functional complex is attached to the memthraneresiding F0 complex which acts as a proton- (or sodium ion- [24]) conductor and among other polypeptides contains multiple copies of a characteristic proteolipid, the small DCCD-binding polypeptide [25]. Though functional properties of whole cell systems were tempting to assume that this principle is also verified in archaebacteria, not a single in-

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pumps and an FoFl-typoATP-synthasein plasma- or organel membranes, Fo: membrane integral proton channel; FI: peripheral catalyticmoietyof ATPase/-synthase.

342 tegrated membrane complex of this type could be isolated in intact form from archaebacteria. Presumably one ot the reasons thereof is the extraordinary rigid structure and lipid composition of archaebacterial membrt, nes [26]. However, ATPases cou!d be identified and characterized which may be part of such a system [6.27-31]. One of the best investigated ATPases from archaebacteria is that of Sulfolobus acidocaldarius [32-36,41]. The study of archaebactrial ATPases is of interest from two aspects: one is the question whether or not these ATPases are a separate and novel group of ion-transporting membrane complexes; the other, linked to the former, is their phylogenetic relation to F-type or (eukaryontic vacuolar) V-type ATPases, a question pertaining to the history of evolution of the various H÷-ATPases [23,37]. Actually, a membrane-bound ATPase could be characterized in Sulfolobus [32-34]. By pyrophosphate treatment a solubilized form could be obtained [35] which shares some remarkable properties with known F~-ATPases despite some characteristic differences. Table 4 summarizes its properties. The moleeular weight determined from sedimentation constants falls closely among those of well characterized F~-ATPases and is almost identical to that from E. coli. The number of subanits of this preparation is four. It should be emphasized in this context, that the usual composition of Ft-ATPases of five sobunits is not necessarily a discriminating dogma; it simply may be an accidental result of the isolation conditions. In some cases a subunit may dissociate into the supernatant, in others it might be left on the membrane as part of an FoFl-complex (see also Table 5). The large protein subunits of Sulfolobus ATPase

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Table

4

Properties of purified Fz-analogous ATPase from Sulfolobus acidocaldarius. The data arc compiled from references [34,3S,38,39]. NBD-CI= 4-chloro-7-nitrobenzofurazan; pHMB = p-hydroxymercuryhenzoate Molecularmass Number of subunits MWof subunits Activatingmelallions Activatinganion pH-optimum

380 kDa 4 65, 51.20.12 kDa ATP-Mg;dATP-Mg Mg2+. Mnz+ Sulfite 6.25 (sulfitepresent)

Number of nucleotidesites

6

Inhibitedby: NBD-CI Mersalyl,priMP Erythrosin B Nitrate

Insensitiveto: Vanadate Azide Oligomycin BafilomyelnA1

Substrate(s)

are of comparable size as a and fl in other F-type ATPases. There is reasonable evidence for an a3/ts stoichiometry of the large protein masses, which is substantially supported by the typical appearance of the Sul/olobus ATPase molecules in high-resolution electron micrographs [38]; these show the characteristic pseudobexagonal arrangement of globular peripber~l masses as confirmed for all known F-type ATPases inspected so far (Fig. 5). In agreement with this subunit stoichiometry is the finding of a total of six nueleotide binding sites [39] in analogy to other F~-ATPases. Limited by experimental restrictions imposed by the extreme thermophifie nature of the Sulfolobus enzyme a differentiation into high- and low-affinity sites or a cooperative relation could not be ascertained as yet. Interesting differences versus - for example beef heart or E. coli F~-ATPase were found regard-

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343 ing inhibitor sensitivity (Table 4). While an inhibition by rather high concentratior, s of nitrate was taken as a classifying parameter shifting this ATPase near the V-type enzymes [37], an extremely specific and effective inhibitor of the latter, bafilomycin [40], is totally inactive with the archaebaeterial enzyme. On the other hand, azide, often considered to be a discriminating inhibitor for F-type ATPases [23,37], was also inactive in the present case. Therefore inhibitor sensitivity alone is a poor discriminator and may in many cases be a matter of gradual differences, except for vanadate; it blocks specifically catalysis by P-type ATPases which are forming a covalent phosphorylated intermediate. Thus, Sulfololms ATPase could be exehided from this latter group. 6.2. Structural relations Table 5 compares the relative molecular masses of four F-type ATPases and their constituent subunit polypeptides with hitherto described archaebacterial ATPases. With only one exception the total molecular masses fall into a narrow range, all~wing for some variations. The two large subunits (usually denoted as a and/~) appear heavier in the case of halophilic bacteria, otherwise they come very close between various organisms. Regarding the number of constituent polypeptides and their stoichiometry in the functional complex some objections are justified against published proposals [27,29,30]. The issue certainly remains a matter of debate in the near future. Restricted to

the large subunits for Sulfolobus a composition of a3fl3 appears rather certain. It would appear unlikely, in contrast, if similar enzymes from other membranes of the archaebacterial kingdom deviate significantly from this structural principle. A broad immunological study has been carried out using an antiserum against the 51-kDa (/~) subunil of Sulfolobus [35,38]. The fl-subunit has been chosen because in typical F-type ATPases the fl-sabunit carries the catalytic nuclcotide binding domain. Actually, a universal cross-reactivity against pro- and eukaryotic F1-/~ subunits as well as towards the respective subunits of archaebacteria could be established. Though this is suggestive for Sulfolobus ATPase to be also a member of the F-type class, it has to be realized that weaker but definite immunological relations exist also towards several vacuolar (V-type) ATPases, while a typical P-type ATPase did not react with the same antiserum [38]. This underlines the dose relation between both F-type and V-type ATPases, emerging also from the available primary sequences communicated lately [36,411. There is even a higher degree of homology between 0t and fl from Sulfolobus with the corresponding A and B subunits from vacuolar ATPases as extensively discussed in recent reviews [37,37a] and the references therein. On the basis of sequence comparisons the possibility has been considered that the ot-subunit in Sulfolobus may provide the catalytic nueleotide binding site like it is proposed for V-type ATPascs. However,

Table 5 Comparison of solubilized archaebacterial ATPases with solubilized F~-ATPases from eubactetial and eukaryontic sources with respect to reported total molecular mass and molecular masses of constituent subunit polypeptides. The alignraent of smaller subunits according to molecularmass dees not necessarilyimply an idet~:!calfunction in the holo-enzyme Source

Re|. tool mass

Rel. mass of subunits (kDa)

Beefheart mitochondria Spinach chloroplasts Propionigeni~ modestum Escherichia colt Methanosarcina barkeri Halobacterium halobium Halobacrerium saccharovorum Sulfolobus acidocaldarius Methanolobus tindarius

371 402 S00 382 420 300-320 350 350-380 ~ 400

55.2 58 58 56 62 86 87 65 67

51.6 S3 56 52 49 64 60 51 52

Re[erence 30,1 34 37 32 29

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6. Alignment of F1-.8-subunit partial sequences from various prO- and eukaryotic organisms. The data for Sulfolobus acidocaldariusare taken from Denda et al. [41], the other sequencestretches from Walkeret al. [S0]and Senioret al. [51].

as shown in Fig. 6 the //-subunit [41] displays significant sequence stretches highly homologous also to functional domains in ,8 from classical F-type ATPases. While the upper pand gives a consensus stretch assumed to represent part of the catalytic site on fl, the lower shows an about 30 residues onward stretch with even less gaps, in known Ft-ATPases identified as the locus where 2-azido-ADP can bind to a specific tyrosine [42]. Nevertheless, very recent results of photoaffinity labelling with 2-azido-ADP have shown that the nucleotide is convalentiy inserted into both, aand/]-sabunits; in fact, predominant labeling was found in the a-polypeptide (M. Meyring-Vos and G. Sch~ifer (1989) unpublished).

6.3. The DCCD-binding proteolipid V-type and F-type ATPases share the property to bind D C C D covalently at a small extremely hydrophobic subunit of the membrane residing F0-segment of the complex [23], the so called "protenhpid" which is present in multiple copies in the functional synthase. DCCD-binding inhibits both proton conduction and ATP-synthesis, also in Sulfolobus [43], Labdling of Sulfolobus membranes with 14C-DCCD allowed to extract and characterize a DCCD-binding protcolipid in our laboratory [131 of 7 kDa apparent molecular mass. Its N-terminal sequence immediately made obvious a significant homology to published sequences of other proteolipids, for example that

from E. coil [25] and displays an almost perfect identity with a sequence derived from nucleotide analysis of a Sulfolobus acidocaldarius Cstrain 9") gene [44]. Alignment of this predicted sequence from residue 23 to the N-terminus of the chemically determined partial sequence from our strain of Sulfolobtu (DSM 639) reveals more than 80~ similarity. In addition, from this comparison it may be conchided that the genetic sequence includes an N-terminal 22-residues elongation, presumably a signal peptide of a precursor form processed during insertion into the membrane. The predicted peptide resulting theoretically after cleavage of the proposed signal peptide not only assumes the same length as the proteolipid from E. coil, but also exhibits an almost identical hydropathy profile with a glutamic acid residue (Ghi 83) flanked by two mirror-imaged hydrophobic stretches ideally at the same position where D C C D binds to the proteolipid of E. coll, Therefore it may be assumed that this residue is embedded in the membrane in an analogous way and for the same functions in Sulfolobus, i.e. proton conduction for ATP-synthesis. Thus, despite their evident sequence homologies to V-type ATPases, functionally the archacbacterial ATPase may be classified as an F-type ATPase rather than a V-type ATPase. It has to be added that by analogous labelling procedures DCCD-binding proteolipids of 5-7 kDa have been identified also in membranes from Thermopla.sma

345

cal

/

E~S+ ]~[mV]

O,

Suecinate

Fig, 7. Tentativescheme of Ihe electron transport systemin the plasma membraneof Sulfolobusa¢idocaldorius as deduced according to standard redox potentialsof the components.The exact number and ligandcoordinationof involvediron-sulfurcenters are still unknownas are the proton-pumpingpropertiesof individualcomplexesof thisprimitivetespiratot~chain.

acidophilum (O. Richter and G. Sch~ifer (1988), unpublished) and Methanolobus rindarius [31]. No sequence data have been derived, however.

7. CONCLUSIONS AND PERSPECTIVES The above reviewed studies on the bioenergetic system of Sulfolobus acidocaldarius are appealing to conclude that even heterotrophically grown cells transduce and conserve respiratory energy by a proton-driven chemiosmotic mechanism. Nevertheless, it should not be withheld that essential details on the involved catalysts still have to be clarified. Regarding the electron transport sys~m, a primitive respiratory chain as illustrated in the scheme of Fig. 7. can be proposeP Calderiella quinone may function as a central pool for reduchag equivalents delivered from the flavin/ironsulfur protein level. It is rcoxidized by two alternate cytochrome complexes, a cytochrome o and a cytochrome aa 3. This latter terminal oxidasc complex represents a new example of aa3-oxidase~ containing only

one type of polypeptide subunits [45-47]. Whether or not additional subunits ere necessary to form the functional complex in the native membrane is a matter of debate. At any rate, it might provide an interesting object for functional studies in reconstituted systems; on the other hand for strocteral and evolutionary aspects primary sequence data will be of extreme interest and are still to come. The most attractive feature is its likely function as a QH2-oxidase. This extends the classes of aa3-cytochromes by a novel member in analogy to the o-type cytochromes [22]: type 1 oxidizing cytochrome c in organisms with a bcl-complex, and type 2 oxidizing a reduced qninone in those organisms lacking bcI and c, respectively. In this regard the terminal oxidase from the erchaebacterium Halobacterium halobium which has recently been isolated [45] is of great interest. This organism has neither bc1 nor c; but no other native substrate for this isolated Cu-free oxidase could be found as yet. Eventually it may also be a quinone oxidase, which has not been tested, however. The possible role of caldariella quinone in the terminal oxidase reaction of Sulfolobus is also suggested by the EPR detectable radical (g = 2) in the oxidase

346 preparations, which could result from a firmly bound quinone molecule in the complex, as remains to be established. Despite the fact that it has been shown that electron transport induces efficient proton extrusion, it is totally unknown which of the partial reactions is coupled to vectorial proton transiocalion across the plasmamembrane. How many pumps are there and what would their H + / e stoichiometry he? What is the role of potassium ions in regulation of internal pH and active proton extrusion? The major reason for this deficiency is the impossibility to prepare closed invetted membrane vesicles or to reconstitute single complexes in functional form into fiposomes. The obstacles to do so are the membrane structure itself (the surface layer can not be stripped off the plasma membrane), the high temperature required by the thermopinlic enzymes that is not tolerated by usual bilayet liposomes, and the probable requirement for specific lipids. This latter situation appfies equally to the ATPase. So far it neither proved possible to extract an intact ATP-synthase complex including its integral membrane part, nor to reconstitute such a complex from its partial elements. Therefore, a definite figure for the H + / A T P ratio of Sulfolobus ATP-sypthase can not be given. lndependant of those details, comparison of functional and structural properties of ATPases from all evolutionary kingdoms clearly demonstrates a ubiquitous occurrence of a unique type of catalyst for ATP-synthesis, enabling organisms to conserve energy by chemiosmotic mechanisms irrespective of the primary source of an ion-motoric force. The obvious fact that classical F-type and F-type ATPases represent strongly related families [37a,48,49] on the one hand, and the finding that arcbaebacterial and vacuolar ATPases vice versa exhibit a somehow chimeric structure on the other hand, has challenged speculations on the evolution of proton-ATPases. In recent reports [37,37a] an attractive hypothesis has been presented, which appears justified on the basis of sequence data also of the DCCD-binding proteolipid of the proton channel. At any rate, the ultimate goal of such studies can only he to understand the underlying mechanism of these enzymes,

rather than to reconstruct a hypothetical ancestor. If the assignment of V-type or F-type ATPases is restricted to a functional level, the Sulfolobus enzyme, and eventually archaebactetial ATPases in general, may be postulated to be F-type ATPases, since none of the vacuolar enzymes has been shown to exert ATP-synthesis. This is supported also by the strikingly conserved quarternary architecture of these enzymes [38]. Essential evolutionary modules found in all F-type ATPasas [50] are also present in the enzyme from Sulfolobus acidocaldarius. Thus, it can be agreed upon that V- and F-type ATPases trace back to common progenotic roots.

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cytcchrome aa 3 which lacks Cu& and Cue. J- Biochem. (Tokyo) 105, 287-292. [46] Fukumori, Y, Watanabe. K. and Yamanaka, T. 0987) Cytochrome.aa 3 from the aerobic photoheterotroph Erythrobacter Iongus: purification, and enzymatic and molecular features. J. Bloc,hem. (Tokyo) 102, 777-784. [47] Zimmermann, B.H., Nitsche, C.I., Fee, J.A., Rusnak. F. and Mtinck, E. (1988) Properties of a coppex-containing cytochrom¢ ha3: a second terminal oxidase from the extreme thermophile Thermu.vthermophilus. Proc. Natl. Acad. Sci. USA 85, 5779-5783. {48] Bowman, E3., Tenney, K. and Bowman, B.J. (1988) Isolation of genes encoding the Neurospora vacuolar ATPase. J. Biol. Chem. 263,13994-140G1. [49] Zinmiak, L., Dittriuh, P., Gosarten , J.P., Kibak, H. and Taiz~ L. (1988) The eDNA sequence of the 69 kDa subunit of the carrot vacuolar H+-ATPase. J. BioL Chem. 263, 9102-9112. [50] Walker, J.E., Feagnley, I.M., Gay, N,J., Gibson, B.W., Northrop, F.D., Powell, $3., Runswlck, M.J., Saraste, M. and Tybulewiez, V.L.J. (1985) Primary structure and subunit stoichiometry of Fl-ATPase from bovine mitochondria. J. Mol. Biol. 184, 677-701. [51] Senior, A.L. and Wise, J.G. (1983) The proton ATPase of bacteria and mitochondria. J. Membr. Biol. 73, 105-124.