System. Appl. Microbiol. 16,544-555 (1994) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Has Sulfolobus an Archaic Respiratory System? Structure, Function and Genes of its Components

GUNTER SCHAFER, STEFANANEMULLER\ RALF MOLL, MICHAEL GLEISSNER, and CHRISTIAN L. SCHMIDT

Institute of Biochemistry, Medical University of Lubeck (Germany), and " Centro de Tecnologia Quimica e Biologica, Oeiras (Portugal)

Received August 30, 1993

Summary

The electron transport system of Sulfolobus acidocaldarius has been shown to act as a respiration driven proton pump. Essentially it is composed of three functional units: the NADH-and substrate dehydro­genases, caldariella quinone as a pool of bound hydrogen, and one or two terminal oxidases catalyzing its reoxidation by molecular oxygen. The latter systems contain only FeS proteins, b- and a-type cyto­chromes; e-type cytochromes are absent. The cytochrome aa3 from Sulfolobus is the first heme-a contain­ing terminal oxidase shown to act as a quinol oxidase. Its redox centers were investigated by redox potentiometry, EPR- and Raman-resonance spectroscopy, revealing a typical binuclear heme-a/Cu center, however displaying unusual structural interaction between the formyl substituents and the protein envi­ronment; the redox potentials of low and high spin centers were titrated. A cytochrome a58

? atypical for any other species emerged to contain a low spin, low potential heme-a center, mimicing the function of a b-type cytochrome and likely to be the product of the SOX-C gene from the SOX operon (Lubben et al., 1992). The aa3 oxidase when reconstituted into liposomes is shown to generate a proton motive force. Though Sulfolobus contains no equivalent to the bel complex of classical respiratory chains, in addition to the caldariella quinol oxidase a Rieske-type FeS protein could be detected in its plasma membrane; it was isolated and characterized by EPR, strongly suggesting a participation in respiratory electron transport. Its redox potential displays significant pH dependence revealing two distinct pKs. It remains to be established whether it interacts with the aa3 oxidase or other electron carriers. A cytochrome b562 appears to be present constitutively in small amount, while cytochrome-b558 is the major heme-b containing compound in the membrane of Sulfolobus. A previously envisaged function as an alternate terminal oxidase is still questionable. Recently conditions were found allowing to modulate its expression dramatically, leading to almost complete suppression of cytochrome-b558

• Based on differential spectroscopy and redox titrations a tentative scheme of the electron transport system from Sulfolobus is proposed. Sequence comparisons are discussed with regard to the question, as to whether it resembles a primitive "archaic" precursor form of more complex "modern" respiratory systems, or whether it evolved by aquisition and adaptation of "foreign" genes.

Key words: Archaea - Sulfolobus - Respiratory Chain - Electron Transport - Cytochromes - Terminal Oxidases - Proton Pumps

Introduction

The chemiosmotic principle of energy conversion has been anticipated also for archae bacteria long before its individual components have been investigated from these

Abbreviations: TMPD = Nl,N4-tetramethyl-phenylene-l,4-diamine. DBMIB = Bibromomethyl-isopropyl-p-benzoquinone. TCBQ = 2,3,5,6-tetrachloro-1,4-benzoquinone.

extremophilic organisms in detail. It became obvious that they possess the ability to generate an ionic electrochemi­cal potential across the plasma membrane, usually of pro­tons, which would be used by a membrane residing AT­pase acting as an ATP synthase. However, when such AT­Pases, resp. -synthases, were functionally, proteinchemi­cally, and genetically analized, it became clear that they

represent a separate class of chimeric enzymes differing markedly from those commonly acting as F1Fo-ATPases in eucarya and bacteria (Schafer and Meyering- Vos, 1992). Actually, their study allowed to construct a new evolutio­nary path leading from common roots to the development of both, F-type and V-type ATPases (Kibak et al., 1992).

It remains to be established, whether or not the same holds for the components of membrane bound electron transport systems from aerobic archaea. Their study is specifically challenging not only because of their phylo­genetic position, but also - as in the case of thermoacido­philic arachaea - because of their ability to generate and to withstand an extraordinary large proton gradient across their membrane at extremely high temperatures. As an example of those we have investigated the respiratory chain of Sulfolobus acidocaldarius asking the following questions:

what is the functional significance of the respiratory system; does it contain one or more proton pumping devices?

- which components are involved and how do they in­teract? are there significant differences to respective systems or components of non-archaeal origin?

Methods and Materials

Sulfolobus acidocaldarius (DSM 639) has been grown and membranes have been prepared as described previously (Lubben et al., 1989). Isolation of cytochrome aa3, measurement of oxid­ase activity, determination of protein and basic spectroscopic techniques have been described elsewhere in detail (Anemuller and Schafer, 1990). The integral terminal oxidase complex was purified by hydrophobic interaction chromatography and gelfilt­ration (GleifSner et al., thesis work, unpublished), or by a proce­dure essentially following that of (Lubben et al., 1992). Cyto­chrome b558 was isolated as described by (Becker and Schafer, 1991).

For differential spectroscopy a Hewlett-Packard 85A diode ar­ray spectrometer or a SIGMA ZWS-II dual wavelength spec­trophotometer were used; the latter was equipped with a laborat­ory designed low-temperature device, as well as a specially con­structed multireflection cell for potentiometric titrations in dilute solutions allowing for highly improved signal/noise ratio. Redox potentiometry was conducted under an argon atmosphere in principle following the method of Dutton (Dutton, 1978).

EPR spectra were recorded with an X-band Brucker ESP 380 spectrometer equipped with a continuous flow helium cryostat from Oxford instruments (Anemiiller et al., 1992; 1993). Reso­nance-Raman spectra were measured in collaboration with D. Hildebrandt (Max Planck Institut, Miihlheim) as described previ­ously (Hildebrandt et al., 1991) using Kr+ -laser excitation at 413 nm within the Soret transition of cytochrome aa3 and accumula­tion of the signal in a multichannel analyzer attached to a diode array spectrometer with a resolution of 1 cm -\ per diode.

Isolation of archaebacterial lipids and reconstitution of oxid­ase into preformed liposomes was performed as described by Elferink; accordingly, membrane potential was followed by a TPP+ electrode and intravesicular pH was monitored by fluores­cence of included pyranine dye (Elferink et al., 1992).

Sequence alignments and Cluster analysis was performed with the "Clustal" programme package (Higgins and Sharp, 1988).

Respiratory Chain of Sulfolobus 545

Results and Discussion

Sulfolobus acidocaldarius grown aerobically on sucrose acidifies any weakly buffered medium immediately when oxygen pulses are applied to cells suspended anaerobically at near neutral pH. The resulting H+ /0 ratios depend on the conditions, especially on the availability of potassium and may assume values from 3 up to > 8 in presence of valinomycin (Schafer et al., 1990). Therefore the presence of one or even more effective proton pumps has to be assumed. Other whole cell experiments have shown that the steady state proton motive force is stored mainly in a large proton gradient (> 3 to 3.5 pH) only weakly ba­lanced by a small membrane potential (Lubben and Scha­fer, 1989). Though it was assumed that this massive pro­ton output is linked to electron transport coupled proton pumping rather than to metabolic acidification, this could not be verified with isolated membranes, because these cannot reorganize themselves to form closed vesicles serv­ing as a useful model. They contain a number of compo­nents, however, known from eucaryotic and bacterial sys­tems to be involved in redox mediated proton transloca­tion.

Attached to the plasma membrane is a NADH-dehyd­rogenase which appears to be only loosely bound; at least the catalytic flavoprotein portion dissociates easily (Wakao et al., 1987). At present it is not known whether or not it catalyzes an energy conserving oxidation of NADH; i. e. whether it is part of a complex-I analogous structure.

In addition, a succinate dehydrogenase could be isolated and characterized which certainly forms an integral mem­brane complex extractable only by detergents. It consists of 4 protein subunits (66, 31, 28, and 12,8 kDa, resp.); the largest of these contains bound FAD, another contains an FeS center with EPR spectra typical for known 4Fe-4S clusters as usually found in complex-II (Moll and Schafer, 1991). Interestingly, the peripheral flavin containing part also dissociates rather easily from the membrane at low (i. e. in this case room-) temperature.

In previous models it was assumed that both these de­hydrogenases are the main feeders of reducing equivalents into the quinone pool serving as the major hydrogen col­lector within the plasma membrane (Schafer et al., 1990). Hence, Sulfolobus contains vast amounts of ferredoxin as well as an acetyl-eoA forming pyruvate-ferredoxin oxido­reductase, which recently has been shown to be generally present in many archaebacteria (Kerscher et al., 1982; Schafer et al., 1993). The further path of electrons from ferredoxin into an oxidative sink is not clear. It may be speculated, therefore, that an additional carrier might al­low electrons to flow via the same quinone into the re­spiratory system.

The specific quinone present in Sulfolobus is caldariella quinone which has been functionally characterized. Its spectral characteristics are shown in figure 1 at various fixed reduction potentials. The resulting half reduction po­tential at room temperature was calculated as + 106 mV from such titrations. This value is rather positive as com­pared to ubiquinone, the most widely distributed respirat-

w o Z <C co a: o (I)

GJ C

546 G. Schafer et al.

O. 0

(Rtd.-Oi.)-O If renc. Spectra 01 Caldarlella-qulnone

0. 40 at vlrlous oliidatlonlrtduction-potenlials

0. 20

0 . 0

- 0 . 20

- D. GO

'" '" ,., '" '" • '" 11'1 • o o til

'" 11'1 11'1

'" '" III

WAV N G TH (",m

Figure 1. Reduced minus oxidized difference spectra of caldariella quinone. The spectra were taken in O.25M phosphate buffer at pH 6.5 and room temperature under an argon atmosphere. The redox potential was monitored by a platinum electrode and gradually adjusted by dithionite after starting registration in the oxidized state. Handling and preparation of the sample are described by Anemiiller and Schafer (1990).

ory coenzyme. The presence of a thioether substituent as well as of the attached thiophen ring not only affect the midpoint potential but also may have significant influence on the stability of the semiquinone radical. As demon­strated below this is of importance for its biological func­tion as a single electron donor for terminal oxidases.

Figure 2 shows a low temperature reduced minus ox­idized difference spectrum of Sulfolobus membranes in the alpha-band region, indicating the presence of several a­and b-type cytochromes. Heme-c is apparently absent as verified also by pyridin hemochrome analysis. The number of heme-b pigments is at least two as can be derived also from the first derivative plot. In addition to a typical heme-a band immediately attributed by analogy to cyto­chrome aa3, an unusual absorption band at 586-587nm indicates the presence of a new type of cytochrome, not found so far in any other repiratory system.

For the analysis of the cytochrome system protein chem­ical methods as well as redox titrations, visible-, EPR-, Resonance-Raman-spectroscopy, and molecular genetics have been applied. Stress was especially layed on the aa3

terminal oxidase because the reduction of oxygen by cyt aa3 is accompanied by a large change of free energy specifi­cally favouring this step for coupling to efficient proton pumplllg.

Surprisingly and in obvious contrast to known cyto­chrome c ocidases the aa3 carrying integral membrane pro­tein could be isolated with full catalytic activity displaying only a single band in Commassie stained SDS-PAGE (Anemiiller and Scharer, 1990).

Its properties are summarized in table 1. Its most promi­nent feature is the lack of any cytochrome c oxidase activi­ty while it displays full activity with reduced caldariella quinone as the best substrate. It was the first demonstra­tion of an aa3 type oxidase to act as a quinol oxidase, breaking the rule that this property is restricted to so­called cytochrome-bo complexes.

Thus, it had to be concluded, that in Sulfolobus direct oxidation of reduced quinone occurs in one step without intermediate electron carriers. Like other Q-oxidases the Sulfolobus enzyme contains no CU A but exhibits a typical binuclear heme-a3lCuB center as shown by the EPR-moni-

0.200

526.8 534.1

I

556.2

581.8

604

Figure 2. Low temperature reduced minus oxidized difference spectrum of Sulfolobus membranes. Membranes (1 mg/ml) were immersed in 0.25 M phosphate buffer pH 6.5, containing 30% glycerol. The airsaturated sample was fully oxidized by addition of a trace of ferricyanide and frozen in liquid nitrogen, using a special optical device with a 1 mm lightpath cell kept in vacuum after freezing and devitrification; the absolute spectrum was re­corded and kept as reference in a digital memory. The sample was fully reduced thereafter by dithionite, refrozen and scanned again under the same conditions. The calculated difference of both spectra is given together with a first derivative scan.

tored redox potentiometry shown in figure 3 (Anemuller et al., 1992). From the bell shaped titration curve for the high spin g = 6 resonance a strong exchange coupling of the hem-a3 center with increasing amounts of oxidized CUB becomes visible which can be fitted to two consecutive I-electron processes. The resulting quantification of the

Respiratory Chain of Sulfolobus 547

high spin (g = 6) and the low spin (g = 3.02) EPR signals [not shown] is in full agreement between the isolated enzy­me and the native membrane of Sulfolobus cells.

While these data in principal are resembling properties of other aa3 cytochromes, from Resonance-Raman spectra significant deviations were derived with respect to hy­drogen bonding of the heme a3 formyl group (Hildebrandt et al., 1991). Figure 4 shows a typical resonance spectrum in comparison to beef heart cytochrome c oxidase, both in oxidized state. The heme al+ formyl vibration is split into two bands. The heme a formyl is upshifted by 6 cm- 1

suggesting a less hydrogen bonding environment than in bovine oxidase; the intensity ratio of heme a and a3 formyl also differs from the bovine enzyme. An analogous band­split is observed in the reduced state. In addition, however, detailed investigations suggest (Heibel et al., 1993) that a high spin hexa- and a high spin penta coordinated state of heme a3 exist in an equilibrium due to a relatively weak 6th ligand resulting in facilitated dissociation.

Actually, besides the described cytochrome aa3 the SOX operon of Sulfolobus is encoding a total of four polypep­tides which are obviously cotranslated (Lubben et al., 1992). In fact, by milder methods an integrated terminal oxidase complex containing 3-4 polypeptide subunits can be isolated which acts as a Q-oxidase as well and lacks any cytochrome c oxidase activity. Nevertheless, the minimum form of the oxidase is free of cyt_aSS7

; though occasionally small traces of a 18-20 kDa polypeptide detectable by an antibody against polypeptide-A (kind gift from Dr. M. Lubben) where found it represents an easily accessible sys­tem catalyzing all essential reaction steps. Therefore its properties with respect to oxygen reduction and proton translocation were further investigated.

It was important to show that dioxygen indeed is re­duced to water by this minimum terminal oxidase and no free peroxyspecies are formed. This can be demonstrated by experiments as given in figure 5. 2,3,5,6-tetrachloro-1,4-benzoquinone (TXBQ) is a sufficiently reactive qui­none analog acting as a single electron donor which has the advantage to be reducible by ascorbate. With aliquots of ascorbate added to the Sulfolobus aa3 oxidase in pre­sence of catalytic amounts of TCBQ short cycles of oxygen

Table 1. Summarized properties of isolated cyto­chrome aa3 from Sulfolobus acidocaldarius in the single polypeptide form as described by Anemiil­ler and Schafer (1990) and Anemiiller et a1. (1992)

Cytochrome aa3 from Sulfolobus acidocaldarius an active single polypeptide quinol-oxidase (the SOX-B gene product)

app. molecular mass (deterg. micelle) mass of polypeptide: SDS-PAGE mass of polypeptide: DNA derived

vis. absorption bands (red.-oxid.) (CO/red.-red.) spectrum

cofactors/mol redox titration: optical redox titration: EPR

electron donor(s) Inhibitors

105-120 kDa 38-40 kDa 57,9 kDa

604 nm, 441 nm max: 592 nm, 429 nm min: 445 nm

1 heme a, 1 heme a3, 1 Cu 208 mV (heme a), 365 mV (heme a3) 200 mY, 370 mV

caldariella quinone; (sev. analogs) CO, cyanide, azide, sulfide

548 G. Schafer et al.

1.2~---------------------------------------,

1 o •

. ~ ! 0.8 C

0.2 Figure 3. Redoxtitration of the high spin EPR signal of the a3/Cu binuclear center of cytochrome aa3 from Sul­

O+-----r-----.---.----.----.----.----I (olobus acidocaldarius. The signal amplitudes (two tit-100 150 200 250 300 350 400 450 rations) of the g = 6 signal are displayed against Eh•

E • mV Details are explained elsewhere (Anemiiller et al. 1992).

I I I I I I I

, " /', 1, ! ! \.

/ \ ,'~, ; I \,')\ : t \,,' \ , I " \', \ \', ,

,/ , : I \ \.:' ,

/ /\/ ... \;/\ , ' 1\ \ ( I \

I I I , 'y \

" / " / \ '\ " ,," , J

C: Oa 16736

be fheart

Sulfolobus

/ /' ''''-' / .... ' ,~ - .. -.... ~ ..... .::: .... ~" - .. - - -' ",~-==~-~';;';;;;;;;;:~DIiII1 .. ~t-f!i ! ii ;.-;'~-~~~-~

Figure 4. Resonance-Raman spectrum of Sul(olobus cytochrome aa3 in the oxidized state. An extended view from 1600-1700 cm -1 is shown together with the com­ponent Lorentz bands obtained from band fitting; for comparison the data for beef heart cytochrome-c oxid­ase are given as a reference; bands are assigned by letters with formyl-[C = 0] and vinyl- [C = C]. Usually 25 scans were accumulated and averaged. For details see (Heibel et aI., 1993). 11.111 1MII 1(.111 1" III

oo,---------~----------------------_, "lAse: + 2T B -AJc: + 2TCBQ' + 211

! • 2 TCBQ + 112 ~ + HI - 2 T Q + Hl

H1Asc: + In °1

2

round: :)

E - - 0.5 m I 02/ mol 8\C c

N o 100

10min T = 29 .3

",,,hout

Figure 5. Cycles of oxygen uptake by a solution of Sulfolobus cytochrome aa3 generated by additions of 35 I-tM ascorbate ali­quots in presence of 2 I-tM TCBQ. The experiment was conducted at 25°C in phosphate buffer pH 6.5 for determination of the e - / O2 ratio as described under "methods".

consumption can be generated allowing to calculate a stoichiometry of 4e - /02 for each cycle. Thus, indeed water is the product of oxygen reduction by this minimum oxid­ase.

The question of proton pumping has been approached after successful reconstitution of the single subunit oxidase into liposomes formed from archaebacteriallipids as ex­tracted from Sulfolobus membranes (Elferink et aI., 1992; Glei{5ner et aI., 1992). Figure 6 shows typical traces of membrane potential and intravesicular pH after energiza­tion of oxidase containing liposomes by ascorbateITMPD, monitored by a TPP+ electrode or the fluorescence of in­cluded pyranine dye, respectively. The upper panel shows that the velocity of pH formation increases dramatically with temperature, whereas the signal amplitude remains unchanged. This clearly documents the impressive tight­ness of the archaebacteriallipid vesicles even at very high temperatures. Calibration of the pH signal and correction for unspecific membrane binding of TPP+ yields potentials of up to -75 mV and at pH differences of 1,1-1,2. Thus, a considerable proton motive force is generated across the vesicle membrane. This process is fully sensitive to uncou­piers and cyanide as respiratory inhibitor.

Clearly, the data for proton trans locating activity repre­sent steady state values obtained in the reconstituted sys­tem. They do not allow to calculate the H+ /0 stoichiomet­ry as a measure of the true efficiency of a proton pump. In

36 System. AppL MicrobioL VoL 16/4

Respiratory Chain of Sulfolobus 549

fact, the observed proton motive force may result simply from scalar reaction stoichiometry in a process catalyzed by asymmetric interaction of the membrane residing oxid­ase with donor and acceptor species on either side of the membrane. For the moment it is open whether or not superstoichiometric protons are translocated as required for a real pump.

However, with respect to the proposed operation of a Q-cycle-like mechanism catalized by the terminal oxidase of Sulfolobus involving both, aa3 and cytochrome a S87 , it is important to note that in the described steady state experi­ments reconstitution of this integrated terminal oxidase

o

T 1l'P+ IOmV 1

Lv

5

Ig _1_-

im [min)

V I tg

t

10

Tim [min]

15 20

Figure 6. Generation of proton motive force by Sulfolobus cyto­hrome aa3 reconstituted into monolayer liposomes from ar­chaebacterial tetra ether lipid. Upper trace: intra vesicular pH sig­nal as monitored by the fluorescence of entrapped pyranine dye (for details see Elferink et al. (1992)); additions were ascorbate (5 mM), TMPD (100 I-tM), valinomycin (200 nM), nigericin (1 mM). Lower trace: membrane potential as monitored by uptake of TPP+ with an appropriate electrode at a TPP+ concentration of 4 mM; additions were Lv = proteoliposomes, ascorbate and TMPD as above, valinomycin (400 nM) nigericin (0,5 I-tM); the latter experiment was conducted at 30 0c.

550 G. Schafer et a!.

0.0.

.03 -

.02 -

500 540

586

604

580 620 nm

complex did neither yield any higher translocation rates, nor a higher proton motive force (data not shown).

Figure 7. Absorption spectrum of the integrated a587/aa3 terminal oxidase complex from Sulfolobus acidocaldarius. The oxidized minus reduced difference spectrum was taken in 50 mM phos­phate buffer pH 7 at a protein concentration of 0.84 mg/m!.

Preparations of the integrated complex exhibit both, the absorption band of aa3 and the unusual band at 587nm, in the reduced minus oxidized difference spectrum (figure 7). Pyridine hemochrome spectra show, that only heme-a is present in this complex. Interestingly, the heme-aS8

? con­taining polypeptide tends to dissociate and usually can be found also accompanying preparations of the b-cyto­chromes. Though it has not been purified individually in homogeneous form, redoxtitrations have shown, that this is a really new species, namely a low spin, low potential cytochrome a. Figure 8 shows a redoxtitration, making obvious that halfreduction is observed at about + 60 mY, a potential typical for b-type cytochromes. The titration can be fitted also by two superimposed Nernstequations with n = 1 for two redox centers contributing by 40 and 60%, with halfreduction potentials of about 20 +/-10 mv, and 100 +/-10 mY, respectively; thus, the presence of two hemes-a with very similar reduction potential is concluded. As to its function, the low reduction potential falls close to the value of caldariella quinone, which might transfer electrons also to this pigment as it does to the heme-a center of aa3' It appears likely, that this cyto­chrome is identical to the product of the SOX-C gene identified within the SOX operon (see Lubben et aI., this volume, and Lubben et aI., 1992); an oxidase complex described by these authors contained all gene products of

100 0.0. 585,5-596 nm

90

80

70

60

50

40

30

20

10

-100 -50 0 50

REDOX-TITRATION Cyt-a 586

Sulfolobus acidocaldarius

.... - EmVolt

........ • 1===-1-1.1---11

100 150 200 250

Figure 8. Potentiometric titration of cytochrome a586• Titration was carried out in a dual wavelength spectrophotometer using a special

multireflection optical cell equipped with a PtlAg,AgCl electrode. Optical density is given as mOD, measured at 585,5 nm with 596 nm as reference wavelength. Redoxmediators were added at 10 !-1M (Dutton, 1978); the sample (partially purified membrane extract containing only b-type cytochrome and cytochrome a586

) was diluted into 250 mM phosphate buffer pH 7,0 containing 0,03% dodecylmaltoside; the redox titration was conducted under argon atmosphere in both directions, reductive and oxidative.

the SOX-operon and 4 hemes a per mole. Two of these are attributed to the aa3/Cu binuclear center; for binding of the additional 2 hemes-a only the product of the SOX-C gene remains as a candidate; according to its primary se­quence it provides all characteristics of a species with two low-spin heme binding sites.

The functional attribution of the b-cytochromes is con­siderably less consistent. At least two species can be distin­guished by differential spectroscopy; a minor compound of very low redox potential absorbing at 562 nm, and the prominent cytochrome b558 displaying a split absorption with peaks at 558 and 566 nm, respectively. At liquid nitrogen temperature these peaks shift to 553 and 561 nm. The responsible heme purifies with a polypeptide of 65-66 kDa apparent Mw in SDS gels which also contains tightly bound Cu and thus was tentatively assigned as subunit of an alternate oxidase (Becker and Schafer, 1991). Support came also from its CO binding capability generating a typical (CO + reduced) minus (reduced) difference spec­trum analogous to thos of cytochrome-o species. In addi­tion, partially purified detergent extracts containing this b­type cytochrome free of any aa3, exhibit a high-spin EPR signal at g = 6 which is also in line with this assumption. Furthermore, as seen from figure 9, a strongly positive midpoint potential of +375 mV can be titrated, allowing to reduce b558 by ascorbate. It goes fully reduced signifi­cantly before any reduction of a587 becomes visible when this is also present in such preparations.

However, severe objections have to be brought forward regarding the tentative functional assignment. The first is the complete catalytic inactivity of purified b558 prepara-

Respiratory Chain of Sulfolobus 551

tions. Neither TMPD nor reduced caldariella quinone or other Q-analogs are oxidized by isolated b558

• Even when reduced by ascorbate in membranes or in detergent ex­tracts of membranes, cytochrome b558 is not reoxidized under aerobic conditions indicating an inability to react with molecular oxgen. The second is that intact membra­nes lack to develop the typical CO difference spectrum as expected when a cytochrome-bo like alternate terminal oxidase is present. If this is correct, it appears possible that the properties of the isolated cytochrome b558 might result artifactually only from membrane des integration by deter­gent treatment.

In this context our recent observation that the expres­sion of b558 is strongly regulated by oxygen tension ap­pears of significant importance (Schmidt and Schafer, unpublished). When plenty of oxygen is supplied b558 be­comes practically totally suppressed and cells grown under these conditions only contain a-tl£e cytochromes besides a small amount of cytochrome b 2 apparently being pre­sent constitutively. For comparison figure 10 shows spect­ra of membranes obtained under different growth condi­tions. Therefore one may conclude that this cytochrome in Sulfolobus rather has the function of an emergency device becoming active specifically under micro aerobic condi­tions. At least, the true biological function of the cyto­chrome b558 of Sulfolobus is still enigmatic and doubt is cast over its function as a regular member of the respirat­ory chain. Further investigation of the regulation of b558

expression and its function is in progress. Though Sulfolobus does not contain any soluble or

membrane bound c-type cytochromes the presence of a bCl

500 Redoxtitration of b-type cytochrome from

450

E mV

-Sulfolobus

Em(1) +375 mV (60%)

Em (2) + 125 mV (40%)

400

350

300

250 •

" 200

• _150

• ...

• 100

.. -.. - -

(562-575 nm) log (ox/red) ~--~----r---~--~-r----+---~-----r--~

-2 -1,5 -1 -0,5 o 0,5 1,5 2

Figure 9. Potentiometric titration of b-type cytochromes. The redox process was monitored as in fig. 8, using 562 nm as measure wavelength with 575 nm as reference. The high potential heme corresponds to cytochrome b558/566 which displays a potential of + 387 m V when observed at 566-575 nm; the low potential heme may reflect cytochrome b562 contributing to a smaller extent to the signal. Data were treated as two superimposed I-electron processes.

552 G. Schafer et a!.

A 586

I 0.00100

586

B

I 0.00100 Dithionite

Ascorbate

I i

500 550 600 650 [nm)

Figure 10. Influence of growth conditions on expression of b-type cytochromes in Sulfolobus acidocaldarius. Difference spectra (re­duced minus oxydized) of membrane extracts in dodecylmal­to side 150 mM TRIS pH 7.5 are shown. A) growth under limited oxygen supply; 0.57 mg protein per ml; B) growth with max­imum oxygen supply; 0.51 mg protein per m!. In both cases the bottom spectrum was scanned after reduction with 5 mM ascor­bate, the top spectrum after full reduction with dithionite.

complex is excluded; EPR spectra, however, reveal the existence of a Rieske-type 2Fe-2S cluster in the membrane. Its spectral and redox properties have been recently de­scribed (Anemiiller et aI., 1993). An EPR spectrum of the purified protein is shown in figure 11 displaying g values of gx = 1.725, gy = 1.89, and gz = 2.031, respectively, which perfectly coincide with those found in the intact membrane. Like the solubilized Rieske protein the cluster can be reduced by ascorbate also in the membrane. At pH 7 its reduction potential of + 340 m V is within the range of

Rieske-type proteins involved in oxidation of ubiquinone or plastoquinone. However, while the latter show pH de­pendent reduction potentials only above pH8, a significant pH dependence of the reduction potential of the ar­chaebacterial cluster occurs in the physiological pH range with two pK values which can be fitted at pH 6,2 and 8,5. Also in contrast to known Rieske-type proteins the EPR signals of the archae bacterial cluster are not shifted by the classical Rieske type FeS inhibitor DBMIB, an analog to ubiquinone. This may be related to the fact that Sulfolobus uses caldariella quinone and the assumed quinone binding site therefore may differ significantly in structure and af­finity.

Interestingly, this 2Fe-2S center can also be reduced in situ by substrates as NADH and succinate in presence of cyanide. Thus it appears reasonable to assume a direct participation in respiration linked electron transport, and further that caldariella quinone is the physiological reduc­tant. The question of the physiological electron acceptor for this protein remains open for further investigation, however. Thus, the newly discovered Rieske-FeS like pro­tein may be part of a hitherto unknown pathway of elec­trons, or of a primitive ancestor of a bel like complex. As a functional analog to the latter the cytochrome a587/aa3 complex of Sulfolobus has been proposed. Though on the basis of reduction potentials an interaction of the new Rieske-type protein with this complex can not be ex­cluded, it should be emphasized that the SOX operon cod­ing for this terminal oxidase complex does not contain any structural gene for an iron-sulfur protein.

Comparative aspects

Sufficient sequence information for structural compari­son is available only for the aartype oxidase. Confining to the catalytically active polypeptide subunit bearing heme­aa3 and CUB in the binuclear center, alignment algorithms as used in the CLUST AL programme (see methods) de­monstrate a striking analogy of these oxidases with respect to the putative heme- and metal-binding stretches which appear to be highly conserved. Figure 12 gives selected examples including the thermophilic eubacterial oxidases from Thermus aquatieus besides the archeal oxidases from Sulfolobus and Halobacterium halobium. However, while hydrophobicity profiles of the selected examples turn out to be highly analogous (not shown) suggesting at least 12 putative membrane spanning helices (an additional N-ter­minal helix is probably present in the halo bacterial sequ­ence), the overall similarity varies over a wide range as seen from the similarity matrix of table II; these results are based on the above mentioned alignment procedure. Whereas the respective subunits from eucaryotic sources and from Paracoeeus (probably a close relative to the an­cestor of mitochondria) display an extraordinary high de­gree of conservation, the relative score for the sequence of the Sulfolobus protein is close to random level with less than 20% similarity even to the enzyme from the archae on Halobaeterium halobium (Denda et aI., 1991). Thus one is tempted to ask whether this enzyme is homologous or only

Respiratory Chain of Sulfolobus 553

2.5

g-factors

2.3 2.2 2.1 2.0 1.9 1.8

.010

o.

-.010

lli.OK 2.0 mW/ 20 dB: 1.4311 GHz 10.0C; W.

- v

I 2.025

1.890

I

)

1.755

I

240 260 280 300 320 340 360 380 400 .. 20

B[mTJ

Figure 11. EPR spectrum of enriched Rieske·type protein from Sulfolobus acidocaldarius. The sample contained concentrated peak fractions from chromatographic purification in 25 mM TRIS·buffer pH 7.5, 250 mM NaCl, and about 0.2 mM dodecylmaltoside. The spectrum was taken at 15 K and 2 mW microwave power after reduction of the sample with 5 mM ascorbate.

B. b. . .. WTAHAFVMIFFMVMP .. H.s .... IVTAHAFVMIFFMVMP .. N.c .... IITAHAILMIFFMVMP .. P.d .... MITYHGVLMMFFWIP .. R.s .... MITGHGILMMFFWIP .. H.h .... LLTSHGITMLFLFGTP .. S.a .... ALTIHGWAAMIAFVPM .. Th.ca3 .. ILTLHGATMLFFFIIQ .. Th.ba3 .. GLTLHGVLNAIVFTQL .. E.c .... IFT.AHGVIMIFFVAMP ..

102 116 -T-H---M-F- --- P-

B.b .... MMSIGFLGFI~HMFTV .. H.s .... MMSIGFLGFI~HMFTV .. N.c .... MMSIGILGFIVWSHHMYTV .. P.d .... MAAIGILGFV~HMYTA .. R. s .... MVAIGVLGFVVWAHHMYTA .. H.h .... TLAIGVLSFG~HMFTT .. S.a .... IYLLAIGTMGVWVHHLQTW .. Th. ca3 .. QMGIWLGTM~HMFT\T .. Th.ba3 .. FLLFLLLSTPVGFHHQFAO .. E.c .... TVCITVLSFIVWLHHFFTM ..

320 338 ---i--L-F-V--HH--T-

..DPILYQHLFWFFGHPEVYILILPGFG

..DPILYQHLFWFFGHPEVYI ...... .

. ....... HLFWFFGHPEVYILIIPGFG · .DPVLYQHILWFFGHPEVYIIILPGFG ..DPVLYQHILWFFGHPEVYIIVLPAFG ..DPIFWQHLFWFFGHPEVYVLVLPPMG ..NNLLWAILFWFVGHPVVYYVPEPLFG ..DPVLFQQFFWFYSHPTVYVMLLPLGI ..DPLVARTLFWWTGHPIVYFWLLPAYA ..NMMMYINLI~WGHPEVYILILPVFG

271 296 Op-- - --L-W--GHP-VY----P--G-

. .. LHDTYYVVAHFHYVLS. · .. LHDTYYVVAHFHYVL .. · .. FHDTYYVVAHFHYVLSM . .. YHDTYYVVAHFHYVMSL . . . YHDTYYVVAHFHYVMSL · .. LHDTYYVVGHFHF IV .. . .. FHNSYYVVGHFHLMIWT · .. FHDSYFVVAHFHNVLMA · .. VHNTAWVPGHFHLQVAS ... LHNSLFLIAHFHNVIIG

410 426 -H--yyvv-HFH------

Figure 12. Alignment of putative metal binding sequence stretches of polypeptide·I from various terminal oxidases of the aar or boo type. The abbreviations signify: B.b. = bos; H.s. = homo sap. ; N.c: = N.crassa; P.d. = Pn. denitr.; R.s. = Rh.sphaer.; H.h. = H. halobium; S.a. = S. acidocald.; Th. = Th. aquat. [cyt caa3, or ba3, resp.]; E.c. = E. coli. Numbering refers to the sequence of E. coli. Bold and italic letters signify residues conserved throughout; other bold letters indicate highly conserved or isomorphically replaced residues; the average consensus is given on the bottom of the four sequence segments.

554 G. Schafer et al.

1

I

Figure 13: Dendrographic clusteranalysis of relative similarity between various cytochrome aa3 subunit-I sequences as based on alignment of members from the three urkingdoms, demonstrating the complete separation of the Sulfolobus enzyme from any other clusters. Data were processed using the Clustal programm pack­age for cluster alignment. For abbreviations see Table I.

analogous to other oxidases. Without implying any phy­logenetic relations the similarity dendrogramm (this is not an evolutionary tree) of figure 13 illustrates the remote position of Sulfolobus. Including the DNA derived se­quences of terminal oxidases from Th.thermophilus (Keig­thley et al., 1992; Mather et al., 1992) would produce the interesting result (not shown) that one of these (ba3) ap­pears in a cluster with Sulfolobus, whereas the other (caa3) falls closer to clusters of the eubacterial and organell type subunits-I. In fact, one is tempted to speculate on the ques­tion whether the gene for the cytochrome aa3 from Sul­folobus is an individual or parallel product of evolution, whether it reflects a precursor of ancient terminal oxi­dases, or whether it succumbed modification after it has

been adopted from more advanced aerobic organisms dur­ing adaptation of previously anaerobic archaea to oxygen andlor to high temperature. At any rate, mutual align­ments of subunit-I clearly show 171 identically occupied aminoacid positions between H. halobium aa3 and E.coli cytochrome-bo, reducing to 122 identities when H. halobium is aligned with Sulfolobus aa3 and to only 57 identities, when all three are aligned, respectively.

Conclusions

Whereas in previous tentative schemes several alternate terminal oxidases have been hypothesized, present infor­mation on components and redox potentials of the re­spiratory system of Sulfolobus suggests only a very simple model to be proposed with certainty. Therein on the sub­strate side a number of membrane bound dehydrogenases supply electrons via flavinlFeS structures to caldariella quinone forming a pool of collected hydrogen. On the oxygen side of the latter we only put the cytochrome aS8?1 aa3 complex as a terminal quinol-oxidase. This should at least function as a minimum system capable of pumping protons out of the cytosol. A strong argument in favour of that is the finding that Sulfolobus grows happily also when no bSS8 is expressed. Thus, the position and function of this b-type cytochrome remains open requiring further in­vestigation. Also unclear is the position of the newly disco­vered Rieske-type FeS cluster. Though reducible by re­spiratory substrates its expression does not follow that of the b-type cytochromes; it rather seems to be present con­stitutively. Tentatively it would be positioned, however, on the oxygen side of caldariella quinone due to its posi­tive reduction potential. More information about its rela­tion to other redox systems is expected from localization and sequencing of its gene.

The best investigated member from the respiratory chain of Sulfolobus is cytochrome aa3' On the one hand, its characterization as a quinol oxidase was a new discov­ery. On the other hand, its low degree of similarity to

Table 2. Similarity matrix of subunit-I primary sequences of cytochrome aa3 from various sources. Sequence data were copied from PIR or Swiss-Prot library using the following symbols: ASP = Aspergilus nidulans, BOY = bovine, CHIK = chicken, HAL = Halobacterium halobium (saccharovorum), HUM = human, NEU = Neurospora crassa, PAR = Paracoccus denitr., SAC = Sulfolobus acidocaldarius (DSM 639), TRY = Trypanosoma brucei. The scores for pairwise comparison were normalized referring to the shorter of the respective sequences

SAC HAL ASP NEU BOY HUM CHIK PAR TRY

HAL 16,7 ASP 16,5 34,2 NEU 18,6 33,5 70,1 BOY 16,7 35,7 59,0 60,0 HUM 16,8 36,1 60,2 60,4 91,2 CHIK 17,1 35,1 59,0 59,6 86,6 83,0 PAR 15,3 32,5 49,5 49,5 50,5 SO,6 SO,1 TRY 15,1 27,3 38,4 38,6 40,7 39,9 40,0 32,1 ECO 15,7 28,1 32,4 33,9 36,5 37,7 34,7 32,4 26,9

other terminal oxidases as revealed by primary sequence data is striking.

If multiple aartype cytochromes representing all ur­kingdoms of life are compared by sequence alignments of the heme bearing catalytic polypeptide-I, the identities practically reduce to a few short consensus stretches de­lineating the putative metal and heme binding sites; as concluded from hydrophobicity profiles all sequences would allow the formation of 12 membrane spanning helices bearing these metal-, respectively heme-, binding sites at very similar spacial positions. However, the true essentials making these polypeptides the acting center of an efficient proton pump still remain a challenge to further investigation.

References

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Anemuller, S., Schmidt, Ch. L., Schafer, G., and Teixeira, M.: Evidence for a Rieske-type FeS center in the thermoacidophilic archae bacterium Sulfolobus acidocaldarius. FEBS Lett. 318, 61-64 (1993)

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Respiratory Chain of Sulfolobus 555

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Kibak, H., Taiz, L., Starke, T., Bernasconi, P., and Gogarten, J. P.: Evolution of structure and function of V-type ATPases. J. Bioenerg. Biomembr. 24, 415-424 (1992)

Lubben, M., and Schafer, G.: Chemiosmotic energy conversion of the archaebacterial thermo acidophile Sulfolobus acidocal­darius. J. Bacteriol. 171, 6106-6116 (1989)

Lubben, M., Kolmerer, B., and Saraste, M.: An archaebacterial terminal oxidase combines core structures of two mitochond­rial respiratory complexes. EMBO J oumal 11, 805-812 (1992)

Mather, M. W., Springer, P., Buse, G., Hensel, S., Fee, J. A.: Cytochrome oxidase genes from Thermus thermophilus; nuc­leotide sequence of the fused gene and analysis of the deduced primary structures for subunits I and III of cytochrome caa3' Unpublished; data from EMBL DNA library, accession Nr. MM84341 (1992)

Moll, R., and Schafer, G.: Purification and characerization of an archaebacterial succinate dehydrogenase complex from the plasma membrane of the thermoacidophile Sulfolobus acidocaldarius. Eur. J. Biochem. 201, 593-600 (1991)

Schafer, G., Anemuller, S., Moll, R., Meyer, W., and Lubben, M.: Electron transport and energy conservation in the archaebac­terium Sulfolobus acidocaldarius. FEMS Microbiol. Reviews 75, 335-348 (1990)

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Schafer, T., Selig, M., and Schonheit, P.: Acetyl-CoA synthase in archaea, a novel enzyme involved in acetate formation and ATP synthesis. Arch. Microbiol. 159, 72-83 (1993)

Wakao, H., Wakagi, T., and Oshima, T.: Purification and pro­perties of a NADH dehydrogenase from a thermo acidophilic archaebacterium, Sulfolobus acidocaldarius. J. Biochem. (To­kyo) 102,255-262 (1987)

Prof. Dr. Gunter Schafer, Institut fur Biochemie, Medizinische Universitat zu Lubeck, Ratzeburger Allee 160, 23538 Lubeck (Ger­many)