Resolution of the Aerobic Respiratory System of the Thermoacidophilic Archaeon, Sulfolobus sp. Strain 7 I. THE ARCHAEAL TERMINAL OXIDASE SUPERCOMPLEX IS A FUNCTIONAL FUSION OF RESPIRATORY COMPLEXES III AND IV WITH NO c -TYPE CYTOCHROMES*

The aerobic respiratory system of the thermoacido- philic archaeon, Sulfolobus sp. strain 7, is unusual in that it consists of only a - and b -type cytochromes but no c -type cytochromes. In previous studies, a novel cytochrome oxidase a 583 -aa 3 subcomplex has been purified, which showed a ferrocytochrome c oxidase but no caldariellaquinol oxidase activity (Wakagi, T., Yamauchi, T., Oshima, T., Mu¨ller, M., Azzi, A., and Sone, N. (1989) Bio-chem. Biophys. Res. Commun. 165, 1110–1114). We show here that the cytochrome subcomplex could be copurified with a non-CO-reactive cytochrome b 562 as a novel terminal oxidase “supercomplex,” which also contained a Rieske-type FeS cluster at g y (cid:53) 1.89. It contained one copper and all four heme centers detectable in the archaeal membranes by the low temperature spectropho- tometry and the potentiometric titration: cytochromes b 562 ( (cid:49) 146 mV), a 583 ( (cid:49) 270 mV), and aa 3 ( (cid:49) 117 and (cid:49) 325 mV). The presence of one copper atom indicates that it contains the conventional heme presteady spectra Sulfolobus at as described in Red-shift spectra of archaeal cytochrome b 562 observed in the of (cid:59) (cid:109) M HOQNO ( and 50 (cid:109) M antimycin A ( but in of myxothiazol M m M succinate-reduced minus oxidized difference spectrum of cytochrome b 562 in the Sulfolobus comparison Sulfolobus membranes no “ferricyanide-induced extra reduction of cytochrome b at either absence ( or of A , HOQNO myxothiazol m

more respiratory terminal oxidase system(s) (1)(2)(3)(4)(5). Unlike the mitochondrial system, the organizations of the prokaryotic respiratory systems are far more complex and versatile to changes in the environmental conditions (for recent reviews, see Refs. 6 -9). Nevertheless, the prokaryotic respiratory complexes generally have much simpler subunit structures compared with the structural and functional homologues of the mitochondrial counterparts, and provide useful models for the molecular mechanics and the properties of the individual catalytic and redox centers of the energy-coupled apparatus (6, 10 -13).
The bacterial respiratory terminal oxidase segment is highly diverged with respect to the number and types of hemes (such as hemes A and O, protoheme, and siroheme (14 -16)), although recent molecular genetic studies suggest that all these types belong to two structurally related larger protein families, the respiratory heme-copper oxidase family (such as cytochrome aa 3 , bo 3 , ba 3 , caa 3 , cao 3 , cbb 3 , see Refs. 6 -9) and the cytochrome bd oxidase family (17). Of these, the heme-copper oxidases are characterized by a unique binuclear metal center composed of a five-coordinated heme and a copper (Cu B ), which is the site for reducing molecular oxygen to water, and an additional six-coordinated heme (8 -11). All these metal centers are located in subunit I of the oxidase superfamily. Two types of the electron transfer to the oxidase superfamily are known: one type involves the respiratory complex III (cytochrome bc 1b 6 f complex that functions as a quinol:cytochrome c oxidoreductase (12,18)) and donates the reducing equivalents to a hemecopper oxidase functioning as a "cytochrome c oxidase" via ferrocytochrome c, while the other type does not contain complex III but directly carries out electron transfer to an another class of a heme-copper oxidase functioning as a "quinol oxidase" via quinol (5,8).
For the aerobic archaea, several aa 3 -type respiratory hemecopper oxidases have been purified from two different species of Sulfolobales (19 -23), Desulfurolobus ambivalens (24), and Halobacterium salinarium (f. halobium) (25,26). In all cases, the cytochrome compositions are very unique in that they contain only the a-and b-type cytochromes but no c-type cytochromes (24,(27)(28)(29)(30)(31). On the basis of the absence of any c-type cytochrome, the insensitivity of a respiratory activity toward several complex III-specific respiratory inhibitors such as antimycin A (27,28,32), and a purification of a "single subunit" cytochrome aa 3 with a quinol oxidase activity (19,33), earlier studies have postulated that the archaeal aerobic respiratory chain might be very simple such that an aa 3 -type cytochrome oxidase functions as a terminal quinol oxidase (34). Neither "a 1 -like" cytochrome (a 586 in Sulfolobus acidocaldarius strain DSM 639 (27,30) and a 583 in Sulfolobus sp. strain 7 (originally named Sulfolobus acidocaldarius strain 7) 1 (28), respectively) predominant in the Sulfolobus membranes, nor non-CO-reactive b-type cytochrome were involved.
Quite recently, the molecular genetic and biochemical evidence suggest that the archaeal respiratory terminal oxidase systems are in fact more complex than previously speculated. Lü bben et al. (21,23) isolated one terminal oxidase operon (soxABCD) and other isolated genes encoding the fused subunit I ϩ III of the alternative oxidase (soxM) of S. acidocaldarius strain DSM 639, and showed that the partially purified Sox-ABCD and SoxM oxidases contain the catalytic core subunits of mitochondrial respiratory complex III (SoxC) and complex IV (SoxAB and SoxM) by immunological cross-reactivities. In addition, it appears that they contain unusual hemes such as "heme A S ," which has a hydroxyethylgeranylgeranyl side chain at position 2 of the iron porphyrin instead of the hydroxyethylfarnesyl group in heme A, beside a conventional protoheme IX (22,35). These findings suggest that the archaeal respiratory and heme biosynthetic systems may represent unique evolutionary events. However, the Sulfolobus oxidase complex preparations (20 -23) have been poorly characterized at the protein level, and leave some uncertainties for the functional assignment of the individual redox metal centers. In particular, we note that the reduced-minus-oxidized difference spectrum of the SoxABCD oxidase from S. acidocaldarius strain DSM 639 (22) is similar to that of a novel three-subunit cytochrome oxidase a 583 -aa 3 subcomplex from Sulfolobus sp. strain 7, which did not show any caldariellaquinol but a ferrocytochrome c oxidase activity (20,36). More recently, we found that the b-type cytochrome is in the upstream of the a-type cytochromes of the archaeal respiratory chain, and its partially-denatured form to be copurified with the cytochrome a 583 -aa 3 subcomplex, which, however, did not retain any caldariellaquinol oxidase activity. 2 In addition, the complexity of the archaeal aerobic respiratory chain is further empathized by the recent findings that the Sulfolobus membranes in fact contain the respiratory Rieske-type FeS clusters (37,38).
In a series of the present studies, we have carried out the purification, characterization (36,39), and reconstitution in vitro of the functionally active respiratory complexes of the chemoheterotrophically grown thermoacidophilic archaeon, Sulfolobus sp. strain 7, in order to reveal the overview of the archaeal aerobic respiratory system and to discuss them in terms of the unique evolutionary and phylogenetic status of thermoacidophilic archaea. In this paper, we show that the active respiratory terminal oxidase segment of Sulfolobus sp. strain 7 contains one non-CO-reactive b-type cytochrome (b 562 ) and two different a-type cytochromes (a 583 and aa 3 ), in addition to one copper and a Rieske-type FeS cluster (38), which, as a whole, function as an active caldariellaquinol oxidase supercomplex. In addition, evidence is presented that cytochrome b 562 , which recently appeared to be in the upstream of and tightly associated with the a-type cytochromes, 2 is probably functionally equivalent to cytochrome b H of the respiratory complex III (cytochrome bc 1 -b 6 f complex), while cytochrome a 583 (36) may correspond to the c-type cytochrome of the c-aa 3 or c-bb 3 type heme-copper oxidase (8,40,41). Furthermore, the purified terminal oxidase supercomplex was reconstituted in vitro for the first time together with caldariellaquinone and the cognate respiratory complex II (39).

Materials
Antimycin A, HOQNO, 3 and myxothiazol are from Sigma, and stigmatellin was purchased from Fluka. MEGA-9 and MEGA-10 were purchased from Dohjin (Kumamoto, Japan), sodium cholate from Sigma, sucrose monolaurate (SM-1200) from Mitsubishi Kasei Co. (Japan), and lauryl maltoside (n-dodecyl ␤-D-maltoside) from Boehringer Mannheim, respectively. Hydroxylapatite (Bio-Gel HTP) was from Bio-Rad. Heme A, protoheme, heme O, and heme A S were prepared by HCl/acetone extraction from bovine heart cytochrome aa 3 , hemoglobin (Sigma), Escherichia coli membranes (14), and Thermus thermophilus membranes (35,42), respectively. Caldariellaquinone was isolated from the membrane of Sulfolobus sp. strain 7 as described previously by De Rosa et al. (43). Horse heart cytochrome c was purchased from Sigma. Water was purified by the Milli-Q purification system (Millipore). Other chemicals mentioned in this study were of analytical grade.

Organism, Cell Culture, and Membrane Preparation
Sulfolobus sp. strain 7 was cultivated aerobically and chemoheterotrophically at pH 2.5-3 and 75-80°C as described previously (28,44), and was harvested in the early to middle exponential phase of growth and stored at Ϫ80°C until use. The cells were suspended in 200 ml of 100 mM Tris-Cl buffer, pH 7.5, containing 0.2 mM PMSF and 10 mM EDTA (ϳ4 ml/g of cells (wet weight)), and disrupted with a French press (Otake Works, Tokyo) at 1500 kg/cm 2 twice. The membranes pelleted by ultracentrifugation with a Beckman 45Ti rotor at 130,000 ϫ g for 120 min at 15°C were composed of two layers. The upper soft layer was carefully collected and resuspended in 50 mM Tris-Cl buffer, pH 7.5, containing 0.2 mM PMSF and 10 mM EDTA, then pelleted by ultracentrifugation at 130,000 ϫ g for 90 min at 20°C. The membrane fraction was washed once more by resuspending in the same buffer and collected by ultracentrifugation; the pellet thus obtained was suspended in 15 mM Tris-Cl buffer, pH 7.3, containing 20% (v/v) glycerol and 0.2 mM PMSF at ϳ20 mg of protein/ml, and used for the subsequent purification of the archaeal respiratory complexes (see below). Alternatively, the membranes were suspended in 40 mM potassium phosphate buffer, pH 6.8, containing 0.2 mM PMSF at ϳ20 mg of protein/ml, then stored at Ϫ80°C until use. The pH value of 6.8 was preferably used in the present studies, because it is the optimal value for the succinate-dependent oxygen uptake and succinate:ubiquinone-1 oxidoreductase activities of the archaeal membrane fraction.

Solubilization and Purification of the Sulfolobus Membranebound Cytochromes as a Supercomplex
All purification steps were carried out at 4°C, except for Step 2 at room temperature, and following the absorption at 280 and 423 nm of each fraction at different purification steps.
Step 1: Cholate/MEGA-9 Extraction-To the washed membrane suspension was added 50 mM potassium phosphate buffer, pH 6.8, containing 5% (w/v) sodium cholate, 1% (w/v) MEGA-9, 10% (v/v) glycerol, and 0.6 mM PMSF. The concentration of MEGA-9 to the amount of protein was critical for avoiding destruction of interactions among cytochromes to the minimal extent. The detergent/protein mixture was gently stirred for 12 h at 4°C, and was ultracentrifuged at 130,000 ϫ g for 90 min at 4°C.
Step 2: Hydroxylapatite Column Chromatography-The solubilized material was directly applied to a hydroxylapatite column (2.0 ϫ 31 cm) which had been equilibrated with 0.5% cholate, 0.1% MEGA-9, 10 mM succinate, 1 mM PMSF, and 10 mM potassium phosphate buffer, pH 6.8. The column was washed with 100 ml of the same detergent buffer, followed by 500 ml of the same buffer containing a linear gradient of 10 -200 mM potassium phosphate, pH 6.8. It was then washed with 100 ml of 0.5% cholate, 0.1% MEGA-9, 10 mM succinate, 1 mM PMSF, and 200 mM potassium phosphate buffer, pH 6.8. Finally, the Sulfolobus cytochromes of both a-and b-types were concomitantly eluted with the same buffer containing 500 mM potassium phosphate.
Step 3: Sucrose Density Gradient-The pooled fractions after Step 2 were subsequently loaded on a sucrose density gradient (20 -70%, w/v) containing 0.5% cholate, 0.6% MEGA-9, 25 mM potassium phosphate buffer, pH 6.8, and the tubes of a Beckman SW 28Ti rotor were spun at 28,000 rpm for 30 h at 4°C. The greenish band near the bottom of a tube (at ϳ55-60% sucrose, w/v) was collected by a tubing, and was checked for purity by SDS-PAGE. When required, this step was repeated for a higher purity. The pooled fractions were combined and stored as the purified cytochrome supercomplex at Ϫ80°C until use.

Analytical Methods
Absorption spectra were recorded with either a Shimadzu MPS-2000 or UV-3000 spectrophotometer, or a Hitachi U-3210 spectrophotometer equipped with a thermoelectric cell holder. EPR measurements were carried out using a JEOL JEX-RE1X spectrometer equipped with an Air Products model LTR-3 Heli-Tran cryostat system, in which temperature was monitored with a Scientific Instruments series 5500 temperature indicator/controller.
The oxidation and reduction potentials of the Sulfolobus membranebound cytochromes were measured in a Thunberg-type cell similar to that described by Dutton (45) under anaerobic conditions with continuous flow of N 2 gas while stirring. The medium used was 60 mM potassium phosphate buffer, pH 6.8, with the following redox mediators: 40 M 2,3,5,6-tetramethylphenylenediamine, 100 M EDTA-Fe, 10 M phenazine methosulfate, 3 M pyocyanin, and 20 M menadione. Ambient redox potentials (E h ) were monitored with a Pt-Ag/AgCl electrode (Type PS-165F, Toa Electronic Ltd., Tokyo, Japan). Desired potentials were attained by adding a small volume of ferricyanide or dithionite solution, and obtained absorption spectra were recorded with a Shimadzu UV-3000 spectrophotometer equipped with a Fujitsu FM 16␤ HD-II personal computer. All midpoint redox potentials stated in the text were calculated from titration curves using a fitting program (written at the Department of Biology, Tokyo Metropolitan University).
Test for the presence of a cyanide-sensitive branched respiratory chain in Sulfolobus sp. strain 7 was carried out polarographically at 55°C, as a function of HOQNO and KCN concentrations, with a Clarktype oxygen electrode (Oxygen analyzer MP-1000, Iizima Products, Tokyo, Japan) equipped with temperature-controlled cells. The standard reaction mixture contained 480-1440 g of membrane proteins/ml of 40 mM potassium phosphate buffer, pH 6.8, in a total volume of 2.1 ml, and the reaction was initiated by addition of 5 mM succinate.
Presteady state reduction of the membrane-bound cytochromes b 562 and a 583 in the presence of 5 mM KCN was monitored at 562-552 and 583-573 nm, respectively, with a Shimadzu UV-3000 dual wavelength spectrophotometer at room temperature.
A ferrocytochrome c oxidase activity was measured spectrophotometrically at 50°C with horse heart cytochrome c (Sigma) as an electron donor, as described previously (20).
The in vitro reconstitution experiments of the archaeal aerobic succinoxidase chain were carried out polarographically at 50°C with a Clark-type oxygen electrode (Oxygen analyzer MP-1000, Iizima Products, Tokyo, Japan) equipped with temperature-controlled cells. The standard reaction mixture contained 0.5% (w/v) cholate, 0.6% (w/v) MEGA-9, 20% (w/v) sucrose in 25 mM potassium phosphate buffer, pH 6.8, in a total volume of 2.1 ml; for the inhibitor studies, the reaction mixture also contained 5 mM succinate, 22.1-44.3 g of the purified respiratory complex II (the preparations obtained after Step 5b were used because of a slightly superior purity (39)), 7 M caldariellaquinone, and the purified cytochrome supercomplex. The concentration of caldariellaquinone in the mixture was kept low because of its very hydrophobic nature and limited availability.
The succinate-driven reduction behavior of cytochromes in the terminal oxidase supercomplex in the in vitro reconstitution system was monitored spectrophotometrically at 20°C with a Beckman DU7400 spectrophotometer; the second-order finite derivative spectrum of the 5 mM succinate-reduced minus oxidized difference spectra of the terminal oxidase supercomplex was recorded every 30 s (the average scanning time, ϳ1-2 s), in 25 mM potassium phosphate buffer, pH 6.8, containing the purified respiratory complex II, 7 M caldariellaquinone, 0.6% (w/v) MEGA-9, and 0.5% (w/v) cholate.
Protein was measured by the BCA assay (Pierce Chemical) with bovine serum albumin as a standard. Types of hemes were determined as described by Sone and Fujiwara (15), using hemes A, A S , and O, and protoheme IX as standards (14,35). The heme contents were determined by the pyridine hemochromogen method using hemes prepared from bovine heart cytochrome aa 3 and hemoglobin for calibration (35,47). Metal content analysis was carried out by inductively coupled plasma atomic emission spectrometry with a Seiko SPS 1500 VR instrument.
Polyacrylamide gel electrophoresis in the presence of SDS was carried out according to Laemmli (48) on 13 or 16% gels after treating proteins with 2% SDS in the presence of 5% 2-mercaptoethanol at 100°C for 2 min, and proteins were visualized by silver staining (Daiichi Pure Chemicals). Fig. 1A shows the low temperature (77 K) reduced-minus-oxidized difference spectra of the membrane suspension prepared from Sulfolobus sp. strain 7 cells (the early to middle exponential phase of growth) at pH 6.8. Three prominent absorption peaks are observed in the ␣-band region, of which two at 582 and 603 nm (583 and 603 nm at room temperature, respectively) are derived from the a-type cytochromes (a 583 and aa 3 ), and the other at 558 nm is from a b-type cytochrome (b 562 ), respectively. Of these, cytochrome b 562 is not CO-or cyanide-reactive, but sensitive to acidification of the archaeal membranes at pH 3 (in 50 mM citrate buffer; the pH value corresponding to the outside (P side) of the membranes) and a high concentration of chaotropic ion such as 1.5 M LiCl. In these cases, an irreversible blue-shift of the ␣-band from 562 to 559 nm was observed, together with a partial loss of the protoheme content. 2 These results suggest that the b-type cytochrome is not located proximal to the periplasmic side of the membrane. As opposed to the case of S. acidocaldarius strain DSM 639 (30), cytochrome b 562 does not exhibit any "split ␣-peak" even in the low temperature secondorder derivative absorption spectrum (Fig. 1A). In fact, the optical redox titration of cytochrome b 562 at pH 6.8 and at room temperature shown in Fig. 2 suggested that it titrates with a single midpoint potential of ϩ146 mV (n ϭ 1), which is sufficiently more positive than that of the caldariellaquinone/quinol pair (approximately ϩ100 mV (19)) and those of the mitochondrial respiratory complexes II (succinate dehydrogenase complex (49,50)) and III (cytochrome bc 1 complex (12, 51, 52)). Recent cyanide titration experiments with the intact membranes suggested that cytochrome b 562 is in the upstream of the a-type cytochromes (20,36), and that the electron transfer from cytochrome b 562 to a 583 is probably rate-limiting in the succinoxidase respiratory chain of Sulfolobus sp. strain 7. 2 Cytochrome a 583 is the most prominent component among the membrane-bound cytochromes of Sulfolobus sp. strain 7 (28), which exhibits a highly unusual ␣-band in the redox difference spectra: at 583 nm at room temperature, and at 582 nm with a small shoulder at ϳ570 nm at 77 K, respectively (Fig. 1A). This is due probably to the unusual ligand environments around a heme a S center (36), rather than the presence of unusual chromophore. It is relatively stable against external acidification at pH 3-4, where no shift of the absorption peak at 583 nm was observed (data not shown), and is not reactive to CO-or cyanide. 2 Cytochrome a 583 is titrated with a single midpoint potential of ϩ270 mV (n ϭ 1; see Fig. 2), which is similar to those of some bacterial cytochromes c and cytochromes c 1 of cytochrome bc 1 complexes (51,53). It is notable that an analogous a-type cytochrome of S. acidocaldarius strain DSM 639, namely "cytochrome a 586 ," has a midpoint redox potential of ϳϩ90 mV (54), which most likely indicates their functional differences (36). On the other hand, the component with the ␣-band at 603 nm is typical for the aa 3 -type cytochrome oxidase, which is the only CO-and cyanide reactive component 2 and has previously been copurified with cytochrome a 583 (20,36). As in the cases of conventional cytochrome aa 3 , it consists of two different midpoint redox potentials of ϩ117 mV (the ␣ peak at 603 nm) and ϩ325 mV (the ␣ peak at 606 nm), respectively (both with n ϭ 1 oxidation-reduction curves; Fig. 2). They are slightly different from the corresponding values reported for the "single polypeptide" form of S. acidocaldarius cytochrome aa 3 (ϩ220 and ϩ370 mV; Ref. 19).

Optical and Redox Properties of the Membrane-bound Cytochromes of Sulfolobus sp. Strain 7-
As in the case of a close relative of the archaeon, S. acidocaldarius strain DSM 639 (35), the elution profiles of the reverse-phase column chromatography and pyridine hemochromogen spectra of whole hemes extracted from the archaeal membranes suggested the presence of only heme A S and protoheme in a ratio of ϳ3:1, and the absence of siroheme (28), heme A, or heme O (data not shown). 4 Concomitant Elution of the a-and b-Type Cytochromes-In the previous purification of the archaeal cytochrome oxidase a 583 -aa 3 subcomplex in the presence of 0.5% (w/v) MEGA-9 plus 1% (w/v) MEGA-10 (20), a considerable amount of cytochrome b 562 was found to coelute with the a-type cytochromes from a hydroxylapatite column (36). In the course of purification of cytochrome b 562 , it was found that the presence of cholate and of the lower content of MEGA-10 (typically less than ϳ0.25% (w/v)) was very effective for avoiding a loss of the protoheme content to the minimal extent (data not shown); under these conditions, the b-type cytochrome was always concomitantly eluted from a gel filtration column together with the a-type cytochromes as a cytochrome subcomplex. 2 Therefore, the Sulfolobus membrane was solubilized with MEGA-9 plus cholate at pH 6.8 to test for the presence of an active cytochrome FIG. 2. Redox titration of the membrane-bound cytochromes from Sulfolobus sp. strain 7. The potentiometric titration (E ϭ Ϫ100 to ϩ500 mV) of the membrane-bound cytochromes of Sulfolobus sp. strain 7 was carried out at pH 6.8 and at room temperature according to Dutton (45), and the normalized height of the ␣-bands at 562, 583, and 604 nm at desired potentials are plotted. For further details, see "Experimental Procedures." Curves are Nernst equations for n ϭ 1 processes, with E m,6.8 ϭ ϩ146 mV (cytochrome b 562 ; dash), ϩ270 mV (cytochrome a 583 ; dot-dash), and ϩ117 and ϩ325 mV (cytochrome aa 3 ; solid line).
FIG. 1. The low temperature optical spectra of the membranes of Sulfolobus sp. strain 7. A, dithionite-reduced minus oxidized difference spectrum at 77 K of the membrane-bound cytochromes of Sulfolobus (ϳ9 mg/ml) in 60 mM potassium phosphate buffer, pH 6.8. The low temperature second-order finite derivative spectrum (top) of the archaeal membranes suggests the absence of any split ␣-band in the b-type cytochrome region. B, 5 mM succinate-reduced minus oxidized difference spectrum at 77 K of the archaeal membrane-bound cytochromes. Upon addition of 5 mM succinate to the Sulfolobus membranes at room temperature in the absence of any respiratory inhibitor, cytochrome b 562 was partially reduced (ϳ65% in the case of this figure) while the bulk of the a-type cytochromes remained in the oxidized state.
oxidase "supercomplex" composed of the a-and b-type cytochromes. Fig. 3 shows a typical elution profile of the MEGA-9/cholate extract of the archaeal membrane from a hydroxylapatite column chromatography. The a-and b-type cytochromes coeluted from the column with a relatively high concentration of potassium phosphate buffer (a small amount of cytochromes could be also detected in the flow-through fractions of the column chromatography together with an NADH:dye oxidoreductase activity (44)). When the membrane was solubilized with a higher concentration of MEGA-9 in the presence of 1% cholate, some amounts of the partially-dissociated cytochromes were found as a broad band around 150 -200 mM potassium phosphate buffer, beside fractions 70 -75 (data not shown). Since cytochrome b 562 eluted concomitantly with the a-type cytochromes around 150 -200 mM potassium phosphate buffer containing 0.5% MEGA-9 plus 1% MEGA-10 (36), the elution profile shown in Fig. 3 indicates that the a-and b-type cytochromes form a stable supercomplex under these conditions. This was further supported by the elution profiles of the sucrose density gradient centrifugation in the presence of MEGA-9 and cholate (Fig. 4A).
Properties of the Sulfolobus Terminal Oxidase Supercomplex- Fig. 4B shows the subunit pattern of the purified cytochrome oxidase supercomplex from Sulfolobus sp. strain 7 on SDS-PAGE, after the preparative 20 -70% (w/v) sucrose density gradient step (Fig. 4A). The purified supercomplex (five different preparations) reproducibly contained 6 subunits with apparent molecular masses of 50, 40, 37, 28, 24, and 14 kDa (Fig. 4B, lane 1). In addition to these bands, two weakly stained polypeptides with apparent molecular masses of 22 and 19 kDa were detected in several preparations. As described in the accompanying paper (36), purified cytochrome aa 3 (Fig. 4B,  lane 2) and cytochrome a 583 -aa 3 subcomplex (data not shown) are shown to correspond to the 40-kDa subunit (subunit I), and the 40-kDa (subunit I), 24-kDa (subunit II), and 14-kDa subunits (subunit III) of the cytochrome supercomplex, respectively. Assignment of a subunit that carries the heme b 562 center is ambiguous because of the extreme lability of the protoheme cofactor, but a tentative candidate is the 37-kDa subunit. 2 The 28-kDa subunit of the purified supercomplex, which is only weakly stained by Coomassie Brilliant Blue (Fig.  4A), was not detected in the highly purified three-subunit cytochrome a 583 -aa 3 subcomplex (36); since this subunit is roughly equivalent in size to the membrane-bound Rieske FeS protein from S. acidocaldarius strain DSM 639 (ϳ30 kDa; Ref. 55), it is tentatively assigned to a putative FeS protein carrying a tightly bound Rieske FeS center (see below; cf. Ref. 38). Interestingly, no subunit equivalent in size to the cognate soluble Rieske [2Fe-2S] protein, named sulredoxin (38), could be detected in the purified supercomplex (data not shown), indicating that sulredoxin may not be constituent of the cytochrome supercomplex. The purified supercomplex was sensitive to certain detergents, and could be partially subdivided by conventional column chromatography in the presence of 0.1% lauryl maltoside. The resulting materials, however, were a partially-dissociated cytochrome subcomplex with a lower protoheme content, and an inactive "copper-free" cytochrome aa 3 similar to that reported for H. salinarium cytochrome aa 3 (25) (data not shown). Typical purification data of the Sulfolobus cytochrome supercomplex is summarized in Table I. Fig. 5 shows the low-temperature reduced-minus-oxidized difference spectrum (at 77 K) of the purified supercomplex, which appears to be identical to that in the membrane-bound state (see Fig. 1A). This is further supported by the EPR analysis of the air-oxidized form of the purified supercomplex, which shows the presence of all high-and low-spin hemes detectable in the archaeal membrane (36). Fig. 6 shows the EPR spectra of the ascorbate-reduced form of the cyanide-inhibited purified supercomplex. It elicits the EPR signal characteristic of the reduced Rieske-type FeS cluster at g z,y,x ϭ ϳ2.02, 1.89, and ϳ1.79 (Fig. 6, A and B; cf. Ref. 38). This "g y ϭ 1.89" EPR signal could also be detected in the membrane-bound state together with the overlapping EPR signal of an additional reduced Rieske-type FeS cluster at g y ϭ 1.91 (38), which was also reduced by ascorbate (Fig. 6C) and succinate (Fig. 6D). These data clearly demonstrate that the purified supercomplex of Sulfolobus sp. strain 7 contains a Rieske-type FeS cluster, which commonly exists in respiratory complexes III (12,51). In this connection, it is notable that the membranes of S. acidocaldarius strain DSM 639 also contained Rieske-type FeS cluster(s) (37), which may be a constituent of the archaeal terminal oxidase complex (23, 55). On the other hand, it remains to be clarified whether the g y ϭ 1.91 Riesketype FeS center of Sulfolobus sp. strain 7 represents the redox effect of the membranous caldariellaquinone/quinol pool on the g y ϭ 1.89 Rieske FeS center of the supercomplex (cf. Ref. 56), or another Rieske [2Fe-2S] protein such as sulredoxin (38) (Fig. 6E).
The metal content analyses by the inductively coupled plasma atomic emission spectrometry suggest that the purified supercomplex (after extensive dialysis against a detergent/ buffer containing 5 mM EDTA) has the Fe:Cu ratio of ϳ4.7:1 (or ϳ6:1.3), respectively. The Fe content was estimated to be ϳ8.1 nmol of Fe/mg of protein. This value is higher than the sum of the heme A S and the protoheme contents (ϳ5.1 nmol/mg and ϳ1.7 nmol/mg, respectively) estimated from the pyridine ferrohemochrome spectra, which is consistent with the presence of non-heme iron within an experimental error (Fig. 6). In the purified supercomplex, the ratio of heme A S :protoheme was estimated to be ϳ3:1, respectively, which is approximately the same as that in the membrane (Table I). Interestingly, the partially-purified SoxM oxidase complex from S. acidocaldarius strain DSM 639 is also reported to contain heme A S and protoheme in a ratio of ϳ3:1, respectively (23). The purified supercomplex from Sulfolobus sp. strain 7 contained ϳ1 mol of Cu/6 mol of Fe, indicating that the tightly bound copper probably corresponds to Cu B of the heme-copper binuclear center. In fact, while the adventitiously bound copper occasionally contaminates the Sulfolobus cytochrome aa 3 (57), the archaeal membrane extensively dialyzed against a buffer containing 10 mM EDTA, elicited no EPR signals characteristic of the Cu A center (data not shown), which has been proposed to be a binuclear, mixed-valence copper center (purple copper) of mitochondrial and some bacterial cytochrome c oxidases (58,59). These data indicate that cytochrome supercomplex from Sulfolobus sp. strain 7 contains about three heme A S , one protoheme, and a Rieske-type [2Fe-2S] cluster per one copper (Cu B ).
Reconstitution of the Archaeal Succinoxidase Respiratory Chain-In order to investigate a functional account of purified supercomplex (b 562 -a 583 -aa 3 supercomplex), it was reconstituted along with caldariellaquinone and the cognate respiratory complex II (39). Because of our failure to incorporate purified supercomplex into phospholipid vesicles (data not shown), the reconstitution system was monitored polarographically at 50°C in 25 mM potassium phosphate buffer, pH 6.8, containing 0.5% (w/v) cholate, 0.6% (w/v) MEGA-9, and 20% (w/v) sucrose, which stabilize the cytochrome supercomplex in vitro. Fig. 7 shows that the purified enzyme can utilize caldariellaquinone effectively as an electron mediator in the reconstitution system at 50°C and at pH 6.8. The oxygen uptake was not detected in the absence of either the purified complex II, FIG. 5. The low temperature (77 K) dithionite-reduced minus oxidized difference spectra of the purified cytochrome supercomplex of Sulfolobus sp. strain 7. The second-order finite derivative (top) of the low temperature reduced minus oxidized difference spectrum of purified supercomplex at pH 6.8 (bottom) was identical to that in the membrane-bound state (Fig. 1A), and suggests the absence of any split ␣-band in the b-type cytochrome region. caldariellaquinone, or the cytochrome supercomplex, which proves that all these components are essential for a succinate oxidase activity of the in vitro reconstituted respiratory chain (Fig. 7). Under the conditions where the archaeal respiratory complex II is in excess, the estimated turnover of the cytochrome supercomplex is ϳ9 e Ϫ /s (ϳ130 -150 nmol of O 2 /min/ nmol of heme A S ) in the presence of 7 M caldariellaquinone at 50°C and pH 6.8 (Fig. 7). For comparison, the succinate oxidase activity of the archaeal intact membranes in the presence of 5 mM succinate was ϳ8 -12 nmol of O 2 /min/nmol of heme A S at 50°C and at pH 6.8, indicating the efficiency of the reconstitution system. On the other hand, it was not possible to estimate the rate of turnover of the purified complex II when the cytochrome supercomplex is in excess, because of the limited availability and the very hydrophobic nature of caldariellaquinone.
In the membrane-bound state, the archaeal succinate-dependent oxygen uptake activity was efficiently inhibited by KCN (with an apparent concentration required for the 50% inhibition (I 50 ) of ϳ16 M: 96% inhibition with ϳ100 M KCN) and a quinol oxidase inhibitor, HOQNO (with an I 50 value of ϳ8 M), when examined polarographically at 55°C and at pH 6.8 (data not shown). These data suggest that Sulfolobus sp. strain 7 contains no cyanide-resistant terminal oxidases (cf. Refs. 30 and 34 for an another species of Sulfolobus). Similarly, the succinate oxidase activity of the in vitro reconstitution system was fully sensitive to cyanide (I 50 ϭ ϳ20 M; completely inhibited by 150 M cyanide; Fig. 7A) and a hydroxyquinone analog HOQNO (I 50 ϭ ϳ1.5 M; Fig. 7B) under the experimental conditions shown in Fig. 7. Thus, the archaeal cytochrome supercomplex functions as a true respiratory terminal caldariellaquinol oxidase both in vivo and in vitro. This further supports the presence of a caldariellaquinone pool in the Sulfolobus membranes.
In the membrane-bound state, cytochrome b 562 , which is the most fragile component among the archaeal cytochromes, could be reduced to ϳ60 -75% in 10 -15 min at room temperature, upon addition of 5 mM succinate aerobically in the absence of respiratory inhibitors (viz. when the respiratory chain is active) (Fig. 1B). We therefore investigated whether cytochrome b 562 in the purified supercomplex exhibits the same reduction behavior in the in vitro reconstitution system. Fig. 8 clearly shows that cytochrome b 562 of purified supercomplex is ϳ62% reduced by 5 mM succinate in the presence of the cognate purified respiratory complex II (1.1 g) and caldariellaquinone (7 M) at room temperature in 10 min (Fig. 8, bottom), while it is not reduced by succinate in the absence of the purified respiratory complex II (Fig. 8, top). This reduction level of cytochrome b 562 compares to that in the membrane-bound state (ϳ60 -75%). Together with the potentiometric analysis and the optical properties of the membrane-bound cytochromes of Sulfolobus sp. strain 7, these data clearly suggest the molecular identity of cytochrome b 562 in the purified supercomplex and the succinate-reducible cytochrome b 562 in the intact membranes, proving that it is not significantly altered during purification. In addition, the succinate-driven reduction of cyto- In the cyanide-treated reduced membranes, the additional g y ϭ 1.91 Riesketype FeS center could be detected (C and D). The EPR spectrum of the dithionitereduced form of "sulredoxin" (a water-soluble Rieske [2Fe-2S] protein from Sulfolobus sp. strain 7 (38)) is shown for comparison (E). chrome b 562 clearly requires additional respiratory components, viz. a caldariellaquinone and the respiratory complex II (39). Since this b-type cytochrome has been shown to be in the upstream of the a-type cytochromes 2 in the terminal caldariellaquinol oxidase supercomplex containing a Riesketype FeS cluster (Fig. 6), the effects of several respiratory inhibitors (32) specific to the mitochondrial and some bacterial respiratory complexes III (cytochrome bc 1 -b 6 f complexes (12,18,51,60,61)) were further examined. In the polarographic assay using the Sulfolobus intact membranes at 55°C and at pH 6.8, myxothiazol (ϳ95 M) and stigmatellin (ϳ100 M), both of which bind at the Q o site of cytochrome bc 1 complexes and block intramolecular electron transfer from ubiquinol (QH 2 ) to the Rieske [2Fe-2S] center and electron transfer onto the heme b L center (18,32), did not inhibit the succinate-dependent oxygen uptake activity (data not shown). 5 In addition, addition of these cytochrome bc 1 inhibitors did not introduce any redshift spectrum of the membrane-bound cytochrome b 562 (data not shown; cf. Ref. 32), suggesting that they do not bind to the archaeal cytochrome supercomplex. These data suggest that the protoheme center in the Sulfolobus cytochrome supercomplex apparently does not correspond to the heme b L center of the conventional respiratory complexes III. On the other hand, HOQNO and antimycin A (ϳ100 M), which bind at the Q i site of cytochrome bc 1 complexes and block intramolecular electron transfer from the heme b H center to ubiquinone (18,32), inhibited the succinate-dependent oxygen uptake activities of the Sulfolobus membranes (only partially inhibited by ϳ100 M antimycin A; data not shown). In addition, they introduced the red-shift spectra of the membrane-bound cytochrome b 562 (Fig.  9A). These data indicate that cytochrome b 562 of Sulfolobus sp. strain 7 has some properties unexpectedly similar to those of cytochrome b H of the mitochondrial and some bacterial respiratory complexes III (12,18,32). The absence of the heme b L analogous center in the archaeal cytochrome supercomplex is further supported by the potentiometric titration of cytochromes (Fig. 2) and the absence of the "ferricyanide-induced extra reduction of cytochrome b" either in the presence or absence of ϳ200 M antimycin A (Fig. 9B).

A Novel Terminal Oxidase Supercomplex from Sulfolobus sp. Strain 7-
The results presented in this paper have shown the detailed properties of the membrane-bound cytochromes from the thermoacidophilic archaeon, Sulfolobus sp. strain 7. Four spectrophotometrically and potentiometrically distinct heme centers (cytochromes b 562 , a 583 , and aa 3 ) have been identified, all of which are shown to be copurified as a novel terminal oxidase supercomplex together with one copper (probably corresponding to Cu B of the heme-copper binuclear center) and a g y ϭ 1.89 Rieske-type FeS cluster. The presence of cholate and the absence of strong detergents (such as lauryl maltoside, MEGA-10, and SM-1200) appeared to be critical elements in the first successful purification of this cytochrome supercomplex, which is active in the in vitro reconstituted respiratory chain and is similar to the SoxM oxidase obtained from S. acidocaldarius strain DSM 639 (the latter complex, however, was apparently partially denatured during purification (23)). It 5 In the in vitro reconstitution system, however, the addition of 100 M myxothiazol induced O 2 . generation when electrons were fed into the active terminal oxidase supercomplex (Fig. 7C, top); since this phenomenon is completely suppressed in the presence of ϳ1 M HOQNO (Fig.  7C, below) and was not observed with the archaeal intact membranes, this reaction is likely based on autoxidation of caldariella-semiquinone radical due to the side effects of replacements of the protein-associated tetraetherlipids by detergent molecules.

FIG. 7. Reconstitution of the Sulfolobus succinate-oxidizing respiratory chain in vitro.
The in vitro reconstitution was monitored polarographically at 50°C in 25 mM potassium phosphate buffer, pH 6.8, containing 0.5% (w/v) cholate, 0.6% (w/v) MEGA-9, and 20% (w/v) sucrose. Caldariellaquinone (ϳ7 M in this case) was strictly required for the oxygen uptake activity of the reconstituted succinate oxidase chain (B), which is sensitive to HOQNO (B) and cyanide (A). C, the effect of myxothiazol on the in vitro reconstitution system containing 5 mM succinate, 11 g of purified complex II, 7 M caldariellaquinone, and 9.9 g of purified terminal oxidase supercomplex; when the reconstitution chain is active, myxothiazol induced O 2 . generation (top), which is suppressed by HOQNO (bottom). Keys: Ox., purified terminal oxidase supercomplex of Sulfolobus sp. strain 7; Qcal, caldariellaquinone; SDH, purified respiratory complex II of Sulfolobus sp. strain 7 (39).
probably represents the sole respiratory terminal oxidase of Sulfolobus sp. strain 7 detectable under the cultivation conditions used in the present study (see "Experimental Procedures"), although a possibility of the presence of alternative terminal oxidases which might be expressed under different growth conditions cannot be excluded at the present time. Until quite recently, most studies on the respiratory terminal oxidases of Sulfolobales have been focused only on the a-type cytochromes (19 -21), while a functional assignment of the b-type cytochromes has not been made conclusively: We have recently shown that cytochrome b 562 of Sulfolobus sp. strain 7 is not reactive toward CO or KCN, and that it is in the upstream of the a-type cytochromes in the aerobic respiratory chain, associating to the archaeal a-type cytochrome subcomplex. 2 In this paper, we show for the first time that it is an important constituent of the caldariellaquinol oxidase supercomplex from Sulfolobus sp. strain 7 with inhibitor sensitivities similar to those of the heme b H center of the conventional respiratory complexes III (cytochrome bc 1 complexes), which, together with the presence of a Rieske-type FeS center (38) (Fig. 6), further indicates that the archaeal terminal oxidase segment may be in fact a functional fusion of respiratory complexes III and IV, in spite of the absence of c-type cytochromes and protoheme centers corresponding to the heme b L center typically present in respiratory complex III. In this context, it is very intriguing that the SoxABCD oxidase complex of S. acidocaldarius strain DSM 639 contains subunit I (SoxB) and II (SoxA) homologues of the heme-copper oxidase superfamily and an apocytochrome b homologue of the mitochondrial respiratory complex III (SoxC), but apparently no Rieske-type FeS center (21,22). The purified protein contains only heme A S but no protoheme (22), and shows optical spectra very similar to those of the cytochrome a 583 -aa 3 subcomplex from Sulfolobus sp. strain 7 with a ferrocytochrome c oxidase activity but no caldariellaquinol oxidase activity (20). As far as we aware, binding of heme A S to SoxC of the SoxABCD oxidase complex has not been demonstrated conclusively. In the case of the terminal oxidase supercomplex of Sulfolobus sp. strain 7, which is rather similar to the SoxM oxidase of S. acidocaldarius (23), we suggest the presence of a SoxC homologue with at least one protoheme center (corresponding to cytochrome b 562 ).
While most bacterial respiratory complexes III and IV have been purified separately, we have mentioned the presence of cytochrome supercomplexes in Paracoccus denitrificans (40) and the thermophilic Bacillus PS3 (41), which are composed of cytochrome bc 1 and c-aa 3 complexes functioning as terminal quinol oxidases. More recently, an unusual cytochrome supercomplex has been identified in the Bradyrhizobium japonicum bacteroids (46), which is likely composed at least of cytochrome bc 1 complex and a cb-type cytochrome c oxidase, although it is currently not known whether the latter is identical to the cbb 3 -type FixN oxidase (62) or not. These results indicate that a respiratory heme-copper oxidase may form a stable complex with a cytochrome bc 1 complex. Although an archaeal aerobic respiratory system investigated so far does not involve c-type cytochromes, the presence of the terminal oxidase supercomplexes in two different species of Sulfolobus suggest unexpected complexity of an archaeal terminal oxidase system. The biochemical data shown in this paper predict conservation of certain principle features of prokaryotic cytochrome supercomplexes to a considerable extent; a heme b H center and a Rieske FeS center of respiratory complex III, all three metal centers (one heme center and one heme-copper binuclear center) bound to subunit I of the heme-copper oxidase superfamily, and an additional low-spin heme (heme a 583 center in the case of Sulfolobus sp. strain 7 (36)) which may be functionally equivalent to a Cu A site or a c-type cytochrome of the caa 3 -or cbb 3 -type cytochrome c oxidase (cf. Ref. 8).
As far as we aware, there is no conclusive evidence for the presence of a terminal oxidase supercomplex in aerobic respiratory archaea other than two different species of Sulfolobales. Nevertheless, the membranes of H. salinarium strain L-33 contain at least four different protoheme centers (31), some of which may function as a direct electron donor to the aa 3 -type terminal oxidase (25,26), raising the possibility of the presence of a terminal oxidase supercomplex also in certain other aerobic respiratory archaea. On the other hand, a cytochrome bc 1 complex analogous component seems to be missing in the major aerobic respiratory chain of the thermophilic and hyper-acidophilic archaeon, D. ambivalens, in which a Cu A -lacking cytochrome aa 3 probably functions directly as a terminal quinol oxidase (24). Since the active terminal oxidase supercomplex of Sulfolobus sp. strain 7 apparently lacks any heme b L center typically observed in the cytochrome bc 1 /b 6 f complexes (12), the incompleteness or the absence of the cytochrome bc 1 complex analogous component in the archaeal respiratory chains might be related to degrees of the adaptation to the extremely acidic environments.
Overview of the Possible Succinate Oxidase Respiratory System of Sulfolobus sp. Strain 7-The present work demonstrates the first successful in vitro reconstitution of an archaeal respiratory chain using purified components, which is sensitive to cyanide and HOQNO (Figs. 7 and 8). These data are in line with other data presented in the preceding papers (36,39), and provide additional support for the complexity of the terminal FIG. 8. Succinate-dependent reduction levels of the heme centers in the Sulfolobus terminal oxidase supercomplex during the steady-state turnover of the in vitro reconstitution system at room temperature. Second-order finite derivatives of the succinate-reduced minus oxidized difference spectra of the purified supercomplex were recorded at room temperature and at pH 6.8 either in the absence (top) or presence (bottom) of 7 M caldariellaquinone and the cognate purified respiratory complex II (11 g/ml). Scan rate, ϳ1 s/scan; recorded every ϳ90 s after addition of 5 mM succinate; the solid traces show typical results of 550 s after initiation of the reaction; the dotted trace shows the second-order finite derivative of dithionite-reduced minus oxidized difference spectrum of cytochromes in the reconstitution system (cf. Fig. 5). The succinate-dependent reduction of cytochromes (cf. Fig. 1B) required the presence of purified respiratory complex II (bottom). The succinate-driven reduction levels of the archaeal cytochromes during the steady-state turnover of the in vitro reconstitution system at room temperature (solid trace, bottom) are: cytochrome b 562 (at 562 nm), ϳ62%; cytochrome a 583 (at 583 nm), ϳ32%; cytochrome aa 3 (at 603 nm), ϳ35%.
oxidase supercomplex of Sulfolobus sp. strain 7. In addition, the minimal constituents of the archaeal succinate-oxidizing respiratory chain have proven to be the respiratory complexes II containing no heme groups, caldariellaquinone (probably supplied as the membranous caldariellaquinone/quinol pool in vivo), and the terminal oxidase supercomplex). Thus, the overall electron flow scheme of the minimal succinate oxidase respiratory chain of Sulfolobus sp. strain 7 is tentatively assigned as follows: succinate 3 the respiratory complex II (a covalently-bound flavin and three different Fe/S clusters) 3 caldariellaquinone 3 the terminal oxidase supercomplex (cytochrome b 562 plus a Rieske-type FeS cluster 3 a 583 3 aa 3 ) 3 molecular oxygen.
Evolutionary Implication-The rooted universal phyloge-netic tree based on molecular evolutionary analyses suggests that the branching of the Archaea domain is found among the earliest of living organisms (64,65), thus archaea being phylogenetically extremely diverged from the ␣-subgroup of the purple bacteria division of the Bacteria domain involving the Paracoccus and mitochondrial lines. On the other hand, the recent sequence analysis of a variety of respiratory heme-copper oxidases by Castresana et al. (66,67) indicates their monophyletic origin, although the respiratory heme-copper oxidase tree apparently does not follow the phylogeny of organisms on the basis of 16 S rRNA sequences, implying the occurrence of several gene duplication and/or lateral transfer events during the evolution of this superfamily (9, 66, 67). Thus, it has been speculated that the first gene duplication event of a common ancestor led to separate evolution of the cbb 3 -type cytochrome c oxidases (the FixN-like oxidases), which can function in the microaerobic conditions (46,62,68,69) and are likely the most closely related to the bacterial bc-type NO reductases, from the rest of the heme-copper oxidases (66,67), then, the putative second gene duplication event led to separate evolution of S. acidocaldarius SoxB and Thermus thermophilus CbaA oxidases from the rest of the archaeal and the bacterial hemecopper oxidases (9,66,67). Both the SoxB and CbaA oxidases are derived from the thermophiles that grow optimally at ϳ75°C or higher (19,21,70), indicating that they also function under the microaerobic conditions in vivo. Thus, the most ancestral type of heme-copper oxidases may be similar to an FixN-like oxidase and derived from aerobic thermophiles grown in the microaerobic conditions.
On the other hand, the presence of the ferredoxin-dependent metabolism involving 2-oxoacid:ferredoxin oxidoreductases is one of the common metabolic features in all archaeal species investigated so far (71)(72)(73)(74)(75)(76)(77), and has also been reported for several anaerobic eukarya (protozoa) which contain hydrogenosome but no mitochondria (78). This anaerobic redox system has been reasonably speculated to be of highly ancient origin because of the existence in the hyperthermophilic anaerobic archaea and bacteria (74,79), and even in the thermophilic and mesophilic aerobic archaea such as Sulfolobus, Thermoplasma, and Halobacteria (72,76). In the aerobic bacteria and eukarya with mitochondria, the NAD-dependent 2-oxoacid dehydrogenase multienzyme complexes functionally replace the 2-oxoacid: ferredoxin oxidoreductases (72,73). Since mitochondria are likely to be derived from the ␣-subgroup of the purple bacteria via the endosymbiotic process (64), these observations support the idea that the last common ancestors of the archaea and eukarya might contain a ferredoxin-dependent metabolic pathway in the cytoplasm, which typically remains in some anaerobic bacteria, anaerobic amitochondrial eukarya, and anaerobic and aerobic archaea nowadays. This further implies that their last common ancestors might be anaerobic hyperthermophiles.
This contradicts the recent proposal in literature (66) that an aerobic respiratory chain might be present in the common ancestor of archaea and bacteria, because the deepest branching point between the archaea and the bacteria domains is positioned deeper than that between the archaea and the eukarya domains (65). In addition, although the organism with the highest growth temperature in the Bacteria domain, Aquifex pyrophilus, is facultative aerobe (80), the aerobic respiration is rare in the Archaea domain, and have been found in the "fast-clock" archaea of long evolutionary lineage, such as Thermoplasma, Halobacteria, and Sulfolobales, with one exception of a hyperthermophilic archaeon, Pyrobaculum aerophilum, which grows optimally at 100°C and belongs to the order Thermoproteales (a representative of a deep and short lineage within the universal phylogenetic tree) (81). Now that the heme-copper respiratory oxidase superfamily has been shown to be apparently of monophyletic origin although their primary structures and molecular properties are considerably diverged even within the bacteria domain (8,9,66), we shall point out that further studies on the aerobic respiratory systems of the hyperthermophiles (such as Aquifex and Pyrobaculum) are certainly required for discriminating whether the archaeal respiratory terminal oxidases are indeed derived from a putative "uroxidase" present in the common ancestor of archaea and bacteria (21,66), or derived from some ancestral aerobic and possibly thermophilic bacteria via lateral transfer; in view of the unique metabolic pathways in archaea (72, 73, 75-77) and of the uncertainty in the evolutionary origin of denitrification, the latter possibility cannot be excluded conclusively at the present time. In either case, some early archaea might have certainly benefited by the presence of the aerobic respiratory chain, not only for the better bioenergetic efficiency, but also for protecting their oxygen-sensitive ferredoxin-dependent metabolic pathways from the microaerobic environments by scavenging toxic molecular oxygen and converting it into water.
In summary, the novel active cytochrome oxidase supercomplex purified for the first time from Sulfolobus sp. strain 7 appears to contain at least one protoheme and three heme A S centers, one copper (Cu B ) and a Rieske FeS cluster; of these, the protoheme center (b 562 ) is assigned to be functionally equivalent to the heme b H center of the conventional cytochrome bc 1 complex. This predicts the presence of certain bc 1 complexrelated components in some other aerobic respiratory archaea, which may be useful for discriminating the evolutionary origin of the archaeal terminal oxidase segments and the aerobic respiration further. In this connection, it will be tempting to characterize this novel terminal oxidase supercomplex further by molecular genetic studies.