The Archaeal SoxABCD Complex Is a Proton Pump in Sulfolobus acidocaldarius*

(Received for publication, October 22, 1996, and in revised form, January 23, 1997)

Michael Gleißner Dagger , Ulrike Kaiser §, Emmanouil Antonopoulos and Günter Schäfer Dagger par

From the Dagger  Institute für Biochemie, Medizinische Universität Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany, § Institut für Physikalische und Theoretische Chemie der Universität Erlangen-Nürnberg, Egerlandstrasse 3, 91058 Erlangen, Germany, and  Klinikum der Johann Wolfgang Goethe-Universität, Gustav-Embden-Zentrum der Biologischen Chemie, Laboratorium für Mikrobiologische Chemie, 60590 Frankfurt/M., Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The thermoacidophilic archaeon Sulfolobus acidocaldarius expresses a very unusual quinol oxidase, which contains four heme a redox centers and one copper atom. The enzyme was solubilized with dodecyl maltoside and purified to homogeneity by a combination of hydrophobic interaction and anion exchange chromatography. The oxidase complex consists of four polypeptide subunits with apparent molecular masses of 64, 39, 27, and 14 kDa that are encoded by the soxABCD operon (Lübben, M., Kolmerer, B., and Saraste, M. (1992) EMBO J. 11, 805-812). The optical spectra and redox potentials of the SoxABCD complex have been characterized, and the absorption coefficients of the contributing cytochromes a587 and aa3 were determined. The EPR spectra indicate the presence of three low spin and one high spin heme species, the latter associated with the binuclear heme CuB site. Standard midpoint potentials of the cytochrome a587 heme centers were determined as +210 and +270 mV, respectively. The maximum turnover of the complex (1300 s-1 at 65 °C) was found to be about three times greater than that of the previously studied isolated cytochrome aa3 subunit alone (Gleissner, M., Elferink, M. G., Driessen, A. J., Konings, W. N., Anemüller, S., and Schäfer, G. (1994) Eur. J. Biochem. 224, 983-990). With N,N,N',N'-tetramethyl-1,4-phenylenediamine as a reductant, the SoxABCD complex reconstituted into liposomes generates a proton motive force. A new method is described by co-reconstitution of SoxABCD with a Sulfolobus Rieske FeS-protein (SoxL), allowing energization by cytochrome c. It is based on the finding that this Rieske protein can equilibrate electrons between cytochrome c and quinones reversibly (Schmidt, C. L., Anemüller, S., Teixeira, M., and Schäfer, G. (1995) FEBS Lett. 359, 239-243). With this system, generating no scalar protons, the stoichiometry of proton translocation could be determined. A net H+/e- ratio >1 was determined, identifying the SoxABCD complex as a proton-pumping quinol oxidase. According to structural analysis, the cytochrome aa3 moiety of the complex does not contain the signature of a H+ pumping channel as identified in Rhodobacter sphaeroides or Paracoccus denitrificans. Therefore, for H+ translocation, a mechanism different from that in typical heme-copper oxidases of the aa3 or bo3 type is discussed.


INTRODUCTION

Terminal oxidases of all oxygen-respiring organisms are members of a superfamily of membrane-residing heme-copper oxidases (1-3). Depending on the specific organism, either reduced quinones or cytochrome c may serve as electron donors for the reduction of oxygen to water. The reaction is coupled to the electrogenic translocation of protons across the membrane, thus serving as a primary energy converter. Independent of their complexity and subunit composition, all terminal oxidases appear to have closely related core structures with respect to subunits I, II, and III. This clearly emerges from the three-dimensional structures recently resolved on an atomic scale of the enzymes from Paracoccus denitrificans (4) and from beef heart (5, 6), respectively. Subunit I carries two heme centers and CuB. The latter forms the binuclear heme-copper site for binding and reduction of oxygen. Thus, a high spin heme with variable coordination geometry is always present as well as a hexacoordinated low spin heme acting as the primary electron acceptor. Only in cytochrome c oxidases, subunit II bears a binuclear CuA site, which is missing in subunit II of quinol oxidases (2, 7). The functional role of subunit-III in electron transport is unknown.

In previous reports, we described the properties of the first archaebacterial terminal oxidase isolated in functional form (8-11). This quinol oxidase from the extreme thermoacidophilic archaeon S. acidocaldarius has been reconstituted into archaebacterial tetraether lipid vesicles, which were shown to develop a proton motive force across the membrane upon energization by reductant pulses (12). Although generation of a steady state membrane potential and/or a pH gradient could be verified with this system, the proton to electron stoichiometry (H+/e-) could not be determined simply because of the random orientation of the quinol oxidase molecules in the membrane and the lack of a membrane-impermeable reductant permitting unidirectional energization. Moreover, by genetic analysis it was demonstrated that in the membrane, the terminal oxidase of Sulfolobus is composed of a complex including an additional cytochrome a (cytochrome a587) (13) and, in contrast to the previously investigated "single entity" form (8), may combine functional properties of terminal oxidases with those of respiratory complexes III (for review see Refs. 14-16). On the basis of its unusual structure, it has been speculated that this respiratory complex, termed SoxABCD (according to its subunit composition), may exhibit proton pumping activity by a Q cycle-like mechanism (13), which would be an extraordinary novelty for terminal oxidases.

The necessity of an efficient proton-translocating device in the membrane of S. acidocaldarius is obvious, because this archaebacterium thrives at 75-80 °C (17) and pH values between 2 and 3, maintaining a large pH gradient across the plasma membrane. In this work, we report on a method for isolation of the intact SoxABCD complex from membranes of S. acidocaldarius as well as for its successful reconstitution as a proton pump into archaeal lipid vesicles. This has been achieved by co-reconstitution with the SoxL Rieske-FeS protein from Sulfolobus (18), thereby permitting construction of a system that allowed energization of the membranes by reductant pulses from only one side; thus, the problem of randomized orientation of pumps could be overcome, and H+/e- stoichiometries could be determined. Based on comparative sequence analyses, the data suggest that the single entity form of the enzyme is unlikely to pump protons, whereas the integrated SoxABCD complex is a proton pump very likely operating by an alternate pumping mechanism, eventually involving a Q cycle.


EXPERIMENTAL PROCEDURES

Bacterial Growth and Membrane Preparation

S. acidocaldarius (DSM 639) was grown heterotrophically in a mineral salt medium (17) as described by Anemüller and Schäfer (8) at the Gesellschaft für Biotechnologic Forschung (Braunschweig, Germany). Cells were suspended in 50 mM MES,1 1 mM EDTA, pH 5.5, at 4 °C, centrifuged for 15 min at 7.800 × g, resuspended in the same buffer containing 50% (v/v) glycerol, and stored frozen at -70 °C. Cells were disrupted, and membranes were prepared essentially as described by Anemüller and Schäfer (8). For chaotropic preextraction, the membranes were diluted to a concentration of 7.5 mg of protein in 50 mM Tris-Cl, 30 mM Na4P2O7, pH 7.5, at 20 °C. After 1 h, the suspension was centrifuged (120,000 × g, 1 h, 20 °C), and the membrane pellets were finally resuspended in 50 mM Tris-Cl, pH 5.5, to give a protein concentration of 45 mg/ml and stored at -20 °C. Typically, these preparations yield 30-40 mg of membrane protein/liter of culture at a concentration of 0.8-1.1 nmol of heme a/mg of protein.

Hydrophobic Interaction Chromatography

The preextracted membranes (800 mg) were suspended to a protein concentration of 8.5 mg/ml in a buffer containing 50 mM Tris-Cl, 500 mM ammonium sulfate (AS), pH 7.3, and solubilized using 20 mM n-dodecyl-beta -D-maltoside (1.2 g of DM/1 g of protein) for 1.5 h at 20 °C. Unsolubilized components were removed by centrifugation at 120,000 × g for 1 h at 20 °C. All of the following purification steps were performed at 4 °C. A saturated AS solution was slowly added to the supernatant until 50% saturation. This solution was applied onto tandem columns containing propyl-agarose (Sigma P-5268; empty  = 1.5 cm, length = 30 cm) and hexyl-agarose (Sigma H-1882, empty = 2.5 cm, length = 6 cm). Both columns had been equilibrated with 50% saturated AS, 25 mM Tris-Cl, 0.5 mM DM, pH 7.3, at 0.5 ml/min. After loading, they were washed with the above buffer to remove less tightly bound material. The hexyl-agarose column, which contained only a-type cytochromes, was eluted with 30% saturated AS, 25 mM Tris-Cl, 1 mM DM, pH 7.3, at a flow rate of 0.5 ml/ml. This intensely green fraction was desalted by dialysis twice against 3 liters of 25 mM Tris-Cl, pH 7.3, overnight, and concentrated by ultrafiltration on a PM-30 membrane (Amicon, Beverly, MA) to 3-5 ml.

Isolation of SoxABCD

The concentrated cytochrome fraction containing approximately 30 mg of SoxABCD was applied to FPLC Mono-Q (10 mm × 10 cm, Pharmacia, Germany), which was equilibrated with 25 mM Tris-Cl, 0.5 mM DM, pH 7.3. Bound proteins were eluted with a step gradient of MgSO4 in the same buffer at a flow rate of 1 ml/min. Peak fractions of SoxABCD determined by optical spectroscopy were collected, concentrated to a heme concentration of 60-70 nmol of heme a/ml by ultrafiltration on a PM-30 membrane, and stored at -70 °C.

Reconstitution of SoxABCD into Tetraetherlipid Vesicles

Tetraetherlipids from S. acidocaldarius were isolated and fractionated from freeze-dried cells as described by Elferink et al. (19) and stored under nitrogen at 4 °C until use. The liposomal forming lipid fraction was dried by rotary evaporation and dispersed in 50 mM KH2PO4, pH 6.5 at 15 mg/ml in the presence of 45% (w/w) n-octyl-beta -D-glucopyranoside. The suspension was sonicated using a probe-type sonicator (Branson Sonifier, 40 watts, 30% duty cycle) until a clear solution was obtained and centrifugated for 10 min at 13,000 × g. Sonication was performed at 0 °C, and the sample was flushed with nitrogen. Purified SoxABCD (20 nmol of heme a) was added to the tetraether lipid suspension to a final concentration of 2.5 nmol/ml and mixed extensively. The suspension was dialyzed four times at room temperature against a 500-fold volume of 50 mM KH2PO4, pH 6.5. Proteoliposomes were stored at -70 °C. Before use, a small sample of the proteoliposomes was slowly thawed at room temperature and extruded through 200-nm polycarbonate filters (Avestin, Ottawa, Canada), using a small volume extrusion apparatus (LiposoFastTM Basic, Avestin). This technique has the advantage of producing monolayer vesicles of homogeneous size with a small range (20, 21).

For proton pumping experiments, SoxABCD was co-reconstituted with a purified Rieske-FeS protein from S. acidocaldarius, which was isolated as described by Schmidt et al. (18). Tetraetherlipid from Ther- moplasma acidophilum was isolated as described (22) and suspended by sonication (see above) to 20 mg/ml in 45% (w/w) n-octyl-beta -D-glucopyranoside, 75 mM HEPES-KOH, 14 mM KCl, pH 7.4. SoxABCD (3 nmol) and Rieske-FeS protein (6 nmol) were added to the tetraether lipid suspension and dialyzed at room temperature in a dialysis cassette (Slide-A-LyzerTM, Pierce, catalog number 66425, cut-off 10 kDa) according to the following protocol: 5 h in 200 volumes of 75 mM HEPES-KOH, 14 mM KCl, 0.1% n-octyl-beta -D-glucopyranoside, pH 7.4; 17 h in 200 volumes of 75 mM HEPES-KOH, 14 mM KCl, pH 7.4; 5 h in 200 volumes of 50 mM HEPES-KOH, 24 mM KCl, 15 mM sucrose, pH 7.4; 2 × 7 h in 300 volumes of 1 mM HEPES-KOH, 45 mM KCl, 44 mM sucrose, pH 7.4. Monolayer vesicles were prepared by using the extruder (see above).

Protein and Heme a Determination

Protein concentrations were determined by the modified Lowry method in the presence of detergents (23) using bovine serum albumin as a standard. Polyacrylamide gel electrophoresis was carried out in the presence of SDS by the Laemmli procedure (24) on 15% gels. Proteins were visualized by Coomassie Brilliant Blue staining. Heme A concentrations were determined utilizing the pyridine hemochrome method as described (25).

Oxidase Activity

Quinol oxidase activity was measured spectrophotometrically at various temperatures by oxidation of N,N,N',N'-tetramethyl-1,4-phenylenediamine (TMPD) or caldariella quinol as substrate in an air-saturated 10 mM citrate buffer, pH 6.5 (12). Cytochrome c oxidase activity was measured spectrophotometrically by following horse heart cytochrome c oxidation at 540-550 nm using a differential extinction coefficient (epsilon 550-540) of 19.5 mM-1 cm-1. Horse heart cytochrome c (Boehringer Mannheim) was reduced with NaBH4, and excess NaBH4 was removed by aliquots of 0.1 N HCl.

Spectroscopic Methods

Absorption spectra were recorded either in a Kontron Uvikon 810 or a HP 8452-A diode array spectrophotometer at room temperature. EPR spectra were recorded with an X-band Bruker ER 200 D-SRC spectrometer equipped with an ESR 910 continuous flow helium cryostat from Oxford Instruments. Fluorescence measurements were performed using a fluorometer type SLM 4800S.

Quasielastic Light Scattering

Particle size distribution of the isolated detergent-oxidase complex was determined by using an ALV 5000TM system (Langen (Hessen), Germany) operating at 632.8 nm (neon-helium laser) and 50 milliwatts. Protein solutions were diluted in 25 mM Tris-Cl, pH 7.3 to give a final concentration of 10 µM and centrifuged for 30 min at 13,000 × g to remove any aggregated material. Measurements were performed at 20 °C.

Spectroelectrochemistry

Purified SoxABCD complex in 25 mM Tris-Cl, 250 mM KCl, 0.2 mM dodecyl maltoside, pH 7.3 was concentrated to approximately 1 mM using Microcon ultrafiltration cells (100 kDa). The optically transparent thin layer electrochemical (OTTLE) cell (path length set to 5-10 µm) used for redox titration was described previously (26). The gold grid working electrode was surface-modified by dipping it in a saturated solution of pyridine-3-carboxyaldehyde thiosemicarbozon as described by Hill et al. (27). After 15 min, excess pyridine-3-carboxyaldehyde thiosemicarbozon was thoroughly removed with deionized water, and the gold grid was dried. The reaction mixture consisted of 1 mM purified SoxABCD complex in 25 mM Tris-Cl, 0.2 mM DM, 250 mM KCl, pH 7.3, as conducting electrolyte. To accelerate the redox reaction, mediators were added to a final concentration of 40 µM: 1,1'-ferrocenedicarboxylic acid, potassium ferrocyanide, 1,1'-dimethylferrocene, tetrachloro-p-benzoquinone, 2,6-dichlorophenolindophenol, hexamineruthenium(III) chloride. 6-8 µl of the reaction mixture were sufficent to fill the cell. The potential in the spectroelectrochemical cell was controlled by a potentiostat built to our design (Sevenich-Gimbel type I). All potentials quoted refer to the silver, AgCl, 3 M KCl electrode (for potentials versus normal hydrogen electrode, add 208 mV). Spectra were recorded in a remodeled Cary 14 spectrophotometer at room temperature. The photometer and the potentiostat were controlled by data acquisition and treatment software (MSPEK) developed by S. Grzybek (Institut für Physikalische Chemie, Erlangen, Germany).

Potentiometric Titrations

A series of potentials in the range of 100-365 mV were applied to the spectroelectrochemical cell. After equilibration of the cell contents at each applied potential, a spectrum was recorded in the range of 380-700 nm. A plot of the amplitude of the difference bands against the applied potential allowed us to fit the experimental data to a calculated Nernst curve yielding the characteristic data of different cofactors: the midpoint potentials (Em) and the numbers (n) of electrons transferred.


RESULTS

Oxidase Preparation

S. acidocaldarius cells synthesize heme a- and heme b-containing cytochromes, exhibiting alpha -bands in reduced minus oxidized difference spectra at 562-566, 587, and 605 nm. They do not, however, synthesize c-type cytochromes (11). The cytochrome composition of the membranes changes according to oxygen tensions, and the heme a/b ratio ranges from 2.2 ± 0.4 at low oxygen to 4.8 ± 1.2 at high oxygen. Cytochrome b558/566 is only expressed at low oxygen, and its function is unknown. The absorption peak at 587 nm is the most prominent one at any oxygen tension. At least two electron transport components with different redox potentials but with identical optical absorptions contribute (28); one is the soxG gene product of the SoxM gene cluster (29), and the other is the SoxC component of the SoxABCD complex (13). Both are b-type apocytochromes, hosting, however, heme a.

To isolate the intact SoxABCD terminal oxidase complex, purified cytoplasmic membranes were washed with Na4P2O7, which removed loosely bound proteins, such as the F1 component of the ATP synthases or residual cell wall polypeptides. For solubilization of the cytochromes, several commonly used ionic and nonionic detergents like n-octyl glycoside, Chaps, CHAPSO, Mega-10, and Thesit were tested, which had either little solubilizing efficiency or led to spectral impairments. Most of the membrane-bound cytochromes (over 75%) could be subilized with dodecyl maltoside at a ratio of 1.2:1 (w/w; detergent/protein). This detergent does not induce any spectral changes of a- and b-type cytochromes and does not influence the oxidase activity.

The intact SoxABCD complex was purified by combining hydrophobic interaction with anion exchange chromatography. Crucial for the efficiency of purification was the use of two hydrophobic interaction columns in a tandem array. At high ionic strength, most of the solubilized membrane proteins such as the Rieske-FeS proteins and all b-type cytochromes remain bound to propyl-agarose, whereas the SoxABCD complex remains bound to hexyl-agarose, which is more hydrophobic. This hexyl-agarose fraction displays two alpha -bands in the difference spectrum (see below) and shows high oxidase activity.

Further purification was achieved by FPLC anion exchange on a Mono-Q column; a typical elution profile is shown in Fig. 1. The substantial amount of SoxABCD eluted at 35 mM MgSO4, with an overall yield of about 31% based on activity with TMPD, but only about 1% with reference to total membrane protein (Table I).


Fig. 1. Elution profile of the Mono-Q 10/10 column. The column was loaded with cytochromes and enriched by the preceding hydrophobic interaction chromatography. The SoxABCD was eluted by a stepwise gradient of MgSO4 in 25 mM Tris-Cl, 0.5 mM DM, pH 7.3. The MgSO4 concentration is shown by the dashed line------, absorbance at 278 nm (protein); ----, absorbance at 426 nm (heme). Sample volume was 3-5 ml, and flow rate was 1 ml/min.
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Table I.

Purification protocol of the SoxABCD complex from S. acidocaldarius


Fractions Membranes (after P2 extraction) Detergent extract Propyl-/Hexyl-agarose Mono-Q

Protein (mg) 700 295 37 7.2
Heme a contenta (nmol) 885 625 416 182
Heme b contenta (nmol) 264 243
TMPD oxidase (units) 3950 3030 2230 1260
Cytochrome c oxidase (units) 593
TMPD specific activity (units/mg) 5.6 10.3 60.3 175
Cytochrome c specific activity (units/mg) 0.85
Purification factorb 1 1.7 8.9 20
Yield (TMPD activity)c (%) 97 74 55 31

a Determined from pyridine ferrohemochrome spectrum.
b Purification factor is based on the specific heme a content.
c Yield is based on crude membranes (not P2-extracted).

The final preparation contains only a-type hemes, as revealed by pyridine hemochrome analysis (c.f. Fig. 3A). Interestingly, it resembles the previously described single entity form of this oxidase complex (8) by the presence of a highly stabilized EPR-detectable semiquinone species. This sharp signal at g = 2.002 (not shown) is present in each state of the preparation and has been assigned to partially reduced tightly bound caldariella quinone. From spin integration of the EPR signals, the single entity aa3 preparation, and especially the respective quinol oxidase from Acidianus ambivalens (30), were found to contain presumably stoichiometric amounts of bound quinone.2 However, attempts to quantify caldariella quinone by chemical extraction according to conventional methods (31, 32) fail due to instability of this thiophenoquinone under the respective assay conditions. In addition, it is insoluble in ethanol/H2O mixtures as normally used for its spectroscopic determination. Other quinol oxidases have definitely been shown to contain superstoichiometric amounts of tightly bound quinones (33-35). It has even been proposed that tightly bound quinones are a general feature of quinol oxidases (34).


Fig. 3. Difference spectra (dithionite-reduced minus air-oxidized) of SoxABCD in 50 mM KH2PO4, 75 mM NaCl, 0.5 mM DM, pH 6.5, at room temperature. Protein concentration was 1.3 µM. Inset, upscaled alpha -bands with the differential extinction coefficients (in parentheses) referring to heme a. Dotted trace, pyridine hemochrome spectrum of the respective preparation.
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It should be added that also the preparations of the Sulfolobus Rieske FeS-protein used for reconstitution (see "Experimental Procedures" and below) routinely contain the same radical species also ascribed to some tightly bound quinone.3

Subunit Composition and Size

The polyacrylamide gel electrophoresis of purified SoxABCD under usual conditions shows only three protein bands (Fig. 2, lane 1). The diffuse band with an apparent molecular mass of 38 kDa is heterogeneous and contains both SoxB (subunit I) and SoxC (cytochrome a587). It has previously been shown that this protein band cross-reacts with antisera against both subunits, which was further confirmed by protein sequencing (16). The presence of isobutyl alcohol (1%, v/v) allowed us to separate these associated peptides into two distinct bands of 39 (SoxB) and 64 kDa (SoxC), respectively (Fig. 2, lane 4), which could be verified by immunoblotting. The 27- and 14-kDa bands correspond to SoxA (subunit II) and SoxD, respectively (16), thus demonstrating the presence of all four polypeptides encoded by the sox operon (13). Analytical gel filtration (high pressure liquid chromatography) of the purified SoxABCD with its associated lipid and detergent molecules gave an estimated molecular mass of 280 ± 20 kDa. The identical elution profiles of the oxidase monitored at A278 (protein) and at A426 (heme a) suggested that the complex is homogenous. A predicted molecular mass on the basis of an equimolar 1:1 stoichiometry of all subunits (SoxA-D) of 144.2 kDa (without cofactors) and associated detergents in a roughly 1:1 weight ratio (16) suggest that the isolated detergent-protein complex is monomeric.


Fig. 2. SDS-polyacrylamide gel electrophoresis (15%) of purified SoxABCD (8 µg) in the absence (lane 1) and presence (lane 4) of 1% (v/v) isobutyl alcohol. Lanes 2 and 3, molecular weight marker proteins (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45.0; carboanhydrase, 31 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa).
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Interestingly, the oligomeric state of the finally isolated SoxABCD preparation depends critically on the ionic strength during the detergent solubilization, which could be confirmed by light scattering. When the membrane solubilization was carried out at low salt concentration, an oligomeric state of the oxidase was obtained with an apparent molecular mass of 570 ± 40 kDa. The high ionic strength applied in this work probably prevented the electrostatic surface charge attraction between monomers.

Optical Spectra

Difference spectra (reduced minus oxidized) of the purified SoxABCD (Fig. 3A) partially resemble those of the mitochondrial cytochrome c oxidase. The absorbance peak at 605 nm is typical of the aa3 center, which is located in SoxB (subunit I). The alpha peak at 587 nm is atypical for other terminal oxidases and results from two low spin hemes bound to the SoxC component. The pyridine hemochrome spectrum (Fig. 3C) as well as the Soret maximum at 440 nm indicate that the oxidase complex contains only cytochrome a. A constant alpha -band ratio (A587/A605) of 1.72 ± 0.06 of different preparations demonstrates the reproducibility of the purification procedure. The differential absorption coefficients (based upon pyridine hemochrome) are 20.6 mM-1 cm-1 (587-545 nm) for heme a587 and 12.1 mM-1 cm-1 (605-630 nm) for heme a605 (Fig. 3A). The latter value corresponds to the heme a of other cytochrome c oxidases (36, 37), whereas the extinction coefficient of the heme a587 is unusually high. In low temperature difference spectra (77 K) the absorbance peak at 587 nm is downshifted to 582 nm (with a shoulder at 573 nm).

The absolute spectra of both air-oxidized and dithionite-reduced SoxABCD, along with their apparent molar absorptivities are given in Fig. 4 and indicate a characteristic fine structure in the Soret region. The spectrum of the oxidized complex shows maxima at 426 nm for the Soret band and 601 nm for the alpha -band region. The shoulder at 587 nm results from partial reduction of the air-oxidized sample. In the presence of ferricyanide and catalytic amounts of TMPD, a symmetric alpha -peak appears at 598 nm. A ratio of A278 to A426 of 1.25 ± 0.05 was obtained for the purified complex; higher values are reported for other bacterial oxidases (36). In the reduced state, the Soret maximum shifted to 436 nm, and two alpha -band maxima at 587 and 603 nm were observed.


Fig. 4. Absolute spectra of oxidized (air) and reduced (dithionite) SoxABCD in 50 mM KH2PO4, 75 mM NaCl, 0.5 mM DM, pH 6.5, at room temperature. Inset, upscaled alpha -bands with the absorption coefficients (in parentheses) referring to heme a. Protein concentration was 1.3 µM.
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The CO difference spectrum of the reduced SoxABCD (Fig. 5) is very similar to the corresponding spectrum of the single entity cytochrome aa3 (8) with the alpha -peak at 596 nm and the beta -peak at 548 nm caused by the a3-CO complex. The Soret region has its maximum at 432 nm and its minimum at 447 nm. The concentration of heme a3, which was calculated from this spectrum using a differential (432-447 nm) absorption coefficient of 82 mM-1 cm-1 (38), was approximately 25% of the total heme concentration determined by the pyridine hemochrome method in agreement with a ratio of 3:1 heme a/heme a3.


Fig. 5. CO difference spectrum (reduced/CO minus reduced) of purified SoxABCD. After reduction of the oxidase with dithionite, CO was bubbled for 30 s through the sample. For details, see the legend to Fig. 3.
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Spectroelectrochemistry and Potentiometric Titrations

Five different redox centers are present in the intact terminal oxidase: two heme a centers from cytochrome a587 (SoxC) plus the centers heme a and heme a3/CuB from cytochrome aa3 (SoxB). All heme centers could be detected by spectroelectrochemical redox titrations at different wavelengths. A titration at 605 nm (not shown) yields values of 200 and 400 ± 30 mV, confirming previous titrations with the single entity form of the enzyme (8); these latter values of 220 mV (heme a) and 370 mV (heme a3), respectively, had been obtained by both chemical redox titrations and EPR titration. Here for the first time the midpoint potentials of the heme a centers of cytochrome a587 have been determined. An example of a spectroelectrochemical redox titration is shown in Fig. 6, evaluating the absorbance change at 587 nm. Under the applied conditions, two redox transitions within cytochrome a587 could be detected with an apparent midpoint potential of Em1 = +210 ± 10 mV and Em2 = +270 ± 10 mV. Each heme a587 contributed about 50% to the total absorbance change in the respective alpha -band. Interference by the 605-nm transition is less than 10%. Values in the range of n = 0.9 for the slope of the Nernst plot indicate that one electron is transferred during each redox transition. Deviations from the theoretical value of n = 1 are presumably due to a narrow distribution around a mean value of the midpoint potential, which is even observed for ultrapure proteins.4 Thus four redox cofactors can be differentiated by potentiometric titrations in accordance with the four heme centers and the prediction from the polypeptide structure of the SoxABCD complex.


Fig. 6. Spectroelectrochemical redox titration of the purified SoxABCD. The measurements were performed in 25 mM Tris-Cl, pH 7.3, 250 mM KCl containing 0.2 mM DM using mediators. The absorbance difference, Delta A587-566, was used to detect specifically the cytochrome a587 of the oxidase. The protein concentration was approximately 1 mM. For details see "Experimental Procedures." Potentials are shown versus H2/H+ electrode at pH 7.
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EPR Spectroscopy of Hemes

The EPR spectra in the air-oxidized state of purified single entity cytochrome aa3 and of the SoxABCD complex of S. acidocaldarius are presented in Fig. 7. The former (spectrum A) exhibits one low spin ferric signal at gzyx = 3.04, 2.21, and 1.45, arising from heme a605, which is identical to that of mitochondrial oxidases (39). The EPR spectrum of SoxABCD (Fig. 7B), however, differs considerably from that of single entity cytochrome aa3; instead of one gz value at 3.04, there are three different low spin ferric heme signals at gz = 2.93, 2.80, and 2.75. While these gz signals are partially overlapping, three gy signals are clearly resolved at 2.21, 2.28, and 2.37. The missing gz value at 3.04 in spectrum B suggests that the local microenvironments around heme a605 are different between the two oxidase preparations (see "Discussion"). The EPR spectra of both cytochrome aa3 and SoxABCD complex exhibit in the air-oxidized state a large high spin heme signal at gmax = 6.10, which is higher than that of the low spin heme signals.


Fig. 7. EPR spectra of air-oxidized single entity cytochrome aa3 (A) and SoxABCD (B). The protein concentration was 35 µM for A and 50 µM for B, respectively. The spectra were recorded in 25 mM Tris-Cl, 0.5 mM DM, pH 7.3. EPR conditions were as follows: temperature, 20 K; microwave power, 20 milliwatts; microwave frequency, 9.4317 GHz; modulation amplitude, 20 G; modulation frequency, 100 kHz; sweep width, 0.2-0.32 tesla.
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SoxABCD is a quinol oxidase; accordingly, a CuA site in subunit II (SoxA) is absent. Neither the typical EPR signals nor the 830-nm absorption band in the spectra of the oxidized enzymes could be detected; this is in line with predictions from the amino acid sequence of subunit II (13). A sharp signal at g = 2 (not shown) originates from a stabilized radical most likely from partially reduced tightly bound caldariella quinone (see above).

Catalytic Properties

The purified SoxABCD oxidase does not react with cytochrome c or blue copper proteins such as halocyanine or azurine as electron donors. However, considerable turnover numbers were found with TMPD or caldariella quinol, confirming that in vivo the enzyme is acting as a quinol oxidase. Table II summarizes the kinetic data. Detergent-solubilized SoxABCD exhibits a significantly lower activity with the artificial electron donor TMPD than when reconstituted into tetraether lipids from S. acidocaldarius, which more closely resemble the natural membrane environment. This activation could not be observed with caldariella quinol, although it is most probably the natural substrate. Hence, the amphiphilic character of TMPD allows its rapid diffusion into the lipid membrane or into detergent micelles. In contrast, the lipophilic caldariella quinol had to be applied in a Triton-stabilized micellar form (see Ref. 8 for details). This decreases the mobility of caldariella quinol toward the binding site of the oxidase in liposomes. Nevertheless, apparent Km values within the same range were found for both forms of the enzyme (Table II). Due to the limited availability of caldariella quinol, TMPD was used as a suitable electron donor for routine assays.

Table II.

Catalytic properties of the SoxABCD complex

Experimental details of the enzyme assays are described under "Experimental Procedures."


SoxABCD Substrate KM(app) Turnover kcat/KM

µM s-1 M/s
In detergent micelle (dodecyl-beta -D-maltoside) QCal 36 393  (50 °C) 1.1 107
TMPD 67 157  (40 °C) 2.3 106
In liposomes (tetraether lipids) QCal 26 239  (50 °C) 9.2 106
TMPD 46 404  (40 °C) 8.7 106

Determinations of the oxidase activity in lipid vesicles were performed in the presence of valinomycin (400 nM) and nigericin (500 nM). The oxidation of TMPD and caldariella quinol was completely inhibited by cyanide (2 mM). The highest activity of the SoxABCD complex reconstituted into liposomes (1300 s-1 at 65 °C) was approximately 3 times greater than the maximum activity found for the reconstituted single entity form of cytochrome aa3 (530 s-1) in previous investigations under the same conditions (12). An activation energy of 59.6 kJ/mol was determined within a temperature range from 30 to 75 °C.

Generation of Proton Motive Force

By reconstitution of the single entity cytochrome aa3 into liposomes, both a Delta Psi and a Delta pH could be measured when the oxidase was energized by TMPD/ascorbate (12). Delta pH measurements were performed with a fluorescent pH indicator entrapped inside the liposomes; the outside pH was kept constant. Fig. 8 compares the generation of proton motive force between liposomes containing either the single entity form of the oxidase (Fig. 8A) or the integrated SoxABCD complex (Fig. 8B). The addition of the electron donor TMPD/ascorbate caused a slow internal alkalinization of the former; a pH gradient equivalent to 32 mV was generated by the cytochrome aa3 liposomes. Valinomycin, by dissipation of the electrical potential, increased the Delta pH to 79 mV. With proteoliposomes containing the integrated SoxABCD complex, a rapid internal alkalinization was detected already in the absence of valinomycin up to 58 mV; after its addition, the Delta pH stabilized at 85 mV. In both cases, it was completely collapsed when nigericin was added. With identical amounts of terminal oxidase in terms of cytochrome aa3 present in each type of liposomes (adjusted by the 605-nm absorption) the activity of the integrated SoxABCD complex was again considerably higher than that of the single entity cytochrome aa3; this is clearly illustrated by the time scale of the individual traces in Fig. 8.


Fig. 8. Determination of Delta pH, generated by cytochrome aa3 (A) and SoxABCD (B) in proteoliposomes by measurements of pyranine fluorescence. Proteoliposomes, composed of S. acidocaldarius lipid with entrapped pyranine (100 µM) were diluted 20-fold in air-saturated 50 mM KH2PO4, pH 6.5, and energized by the addition of ascorbate (5 mM) and TMPD (100 µM) at 50 °C. Valinomycin (400 nM) and nigericin (500 nM) were added to dissipate Delta Psi and Delta pH, respectively. a, valinomycin added immediately after TMPD. b, initial reaction in the absence of valinomycin. Calibration was performed by the addition of HCl and KOH aliquots to uncoupled vesicles after each individual experiment. A conversion factor Z of 64.1 at 50 °C was used to express Delta pH (-ZDelta pH) in mV.
[View Larger Version of this Image (22K GIF file)]


Thus, it could be established that the SoxABCD complex can generate a significant proton motive force under steady state conditions. However, the experiments do not allow us to distinguish between vectorial proton pumping and a "chemical" proton gradient produced by scalar processes. As such, the release of protons from ascorbate on the outside and the proton consumption by water formation inside the vesicles must be taken into account.

Demonstration of Proton Pumping

The determination of H+/e- stoichiometries with quinol oxidases reconstituted in liposomes is generally hampered in two ways: 1) the reconstitution procedure usually leads to a random orientation of oxidase molecules in the liposome membrane, and 2) both orientations can be energized by TMPD due to its significant membrane permeability leading to proton translocation in both directions. Thus, it was necessary to construct a system overcoming these problems.

It has been shown that in isolated form one of the two Rieske-FeS proteins from Sulfolobus membranes (18, 40) can readily equilibrate electrons between cytochrome c and ubiquinone analogs. Further, we have demonstrated that the reduced Rieske-FeS protein can be reoxidized by the detergent-solubilized SoxABCD complex, which inherently contains bound caldariella quinone. On that basis, an artificial electron transport system was established by co-reconstitution of the Sulfolobus Rieske-FeS protein with the SoxABCD complex into tetraether lipid vesicles (Fig. 9A). This Rieske-FeS·oxidase complex showed a significant turnover (Vmax = 187 s-1) with reduced cytochrome c (80 µM) as substrate and displayed Michaelis-Menten kinetics. An apparent Km of 11.6 ± 0.3 µM was found. The reaction was coupled to stoichiometric oxygen uptake and was 100% cyanide-inhibited. A respiratory control ration of 2.9-3.3 could be measured (Fig. 9B). The orientation of incorporated complexes was determined spectrophotometrically, resulting in 65% accessibility (on average) to reduction by cytochrome c (cyanide present), indicating that 35% of the oxidase complexes were inversely oriented; full reduction was achieved by membrane-permeable TMPD.


Fig. 9. Oxidation of cytochrome c by co-reconstituted SoxABCD with Rieske-FeS protein. The cytochrome c (horse heart) concentration was 30 µM in 700 µl of 10 mM citrate, pH 6.5. The arrows indicate the addition (2 µl) of proteoliposomes (2.5 nmol of SoxABCD/5 nmol of Rieske protein/ml). A, dependence of the reaction rate on protein concentration. Cyanide (1.5 mM) produced full inhibition. The dashed line shows the autoxidation of cytochrome c in the presence of oxidase-free liposomes. B, cytochrome c oxidation activity of coupled and uncoupled proteoliposomes (respiratory control index = 3.2). All measurements were performed at 40 °C.
[View Larger Version of this Image (17K GIF file)]


The co-reconstituted system has two advantages; it can be unidirectionally energized by pulses of reduced cytochrome c, and it does not release scalar protons. Thus, it could be used for single turnover experiments monitoring the appearance of protons by an outside optical probe (41-43). Fig. 10 shows a typical experiment, demonstrating the rapid transient acidification following addition of 1.9 nmol of reduced horse heart cytochrome c (Fig. 10B). The subsequent slow alkalinization is caused by back-diffusion and equilibration with the intravesicular pH, which became alkaline due to H+ consumption by water formation. In fully uncoupled vesicles, only the respective alkalinization was observed (Fig. 10C). For quantitation of proton ejection and/or alkalinization, the system was calibrated by HCl aliquots after each experiment. In the coupled system, net H+/e- > 1 was routinely determined; on average, a net H+/e- of 1.2 ± 0.03 was found, definitely identifying the co-reconstituted SoxABCD complex as a vectorial proton pump.


Fig. 10. Proton pumping by the co-reconstituted SoxABCD with archaeal Rieske-FeS protein in proteoliposomes. 0.1 ml of proteoliposomes (0.189 nmol of SoxABCD) and valinomycin (3 µM) were added to 1.2 ml of a 60 mM Phenol Red solution containing 44.6 mM KCl, 44 mM sucrose and adjusted to pH 7.4 at 25 °C. At the points indicated, 1.9 nmol of reduced cytochrome c (horse heart) adjusted to the same pH were added, and the resulting pH changes were followed by absorbance of Phenol Red at 558-504 nm using an HP 8452 diode array photometer. A, control; the addition of cytochrome c in the absence of proteoliposomes produced no artifactual absorbance change. B, immediate acidification of the extravesicular medium was observed after energization. C, after uncoupling of the proteoliposomes with 5 µM nigericin, a second addition of cytochrome c leads to rapid alkalinization of the medium. The calibration bar shows the absorbance change caused by the addition of 1 nmol of H+ in the presence of nigericin.
[View Larger Version of this Image (24K GIF file)]


A series of control experiments was performed to ascertain the validity of the proton translocation experiment (41-43) with the following results. First, in the absence of proteoliposomes, the addition of reduced cytochrome c to the assay mixture did not induce any absorption change (Fig. 10A). Second, the reduction of solubilized Rieske-FeS protein with cytochrome c did not cause a redox-dependent pH change, indicating that the observed acidification is neither due to proton release after binding of cytochrome c to the Rieske protein nor to a redox-Bohr effect. Third, fully uncoupled liposomes gave only the proton uptake due to water formation with a H+/e- ratio of -1.08 ± 0.04, perfectly in agreement with the chemical reaction stoichiometry. Finally, identical experiments performed with cytochrome c oxidase from P. denitrificans (a kind gift from Prof. B. Ludwig, University of Frankfurt, Frankfurt, Germany) showed an analogous proton ejection with a net H+/e- of 0.6 ± 0.05.


DISCUSSION

The thermoacidophilic archaebacterium S. acidocaldarius thrives at pH ~2 and has been shown to conduct respiration-coupled proton ejection (44), thereby keeping its cytosolic pH near neutral. Two terminal oxidases have been identified in its plasma membrane (8, 45, 46) and genetically characterized (13, 29). Both are sharing an unusual complexity compared with other procaryotic cytochrome c or quinol oxidases (for a review see Refs. 47 and 48). While the supercomplex "SoxM" (45) could not be isolated in catalytically sufficiently active form, the SoxABCD complex described here has been identified in previous works as a highly active quinol oxidase, using the genus-typical caldariella quinol (49) as its natural substrate. Here we have demonstrated for the first time that this integrated SoxABCD complex represents a respiratory proton pump, while evidence can be derived from structural properties that its cytochrome aa3 moiety itself has no proton pumping capacity (see below).

Sulfolobus cytochrome aa3 is the product of the soxB gene and was studied previously in purified form as so-called single entity quinol oxidase (8, 50). Its spectroscopic properties essentially resemble those of other aa3-type terminal oxidases with the exception that resonance-Raman spectra indicate only very weak, if any, hydrogen bonding of the formyls of heme a and heme a3 (51) as well as an equilibrium between 6cHS and 5cHS heme a3.

In the integrated SoxABCD complex cytochrome a587, the product of the soxC gene (13), forms an additional di-heme redox component also hosting the archaetypical heme As (52). Thus, a total of five redox-active metal sites are present, comprising four heme a sites and CuB. Subunit II of the qinol oxidase is devoid of metal sites (in contrast to CuA in cytochrome c oxidases) but may be involved in tight as well as in reversible binding of caldariella quinone (34, 53).

While recent resonance-Raman studies of the integrated SoxABCD complex confirm the features found for isolated SoxB even in a more pronounced form (54), significant differences of the EPR spectra could be detected (Fig. 7B). Three different low spin signals were identified with down-shifted gz and gy values as compared with SoxB. The shifted g values may reflect an increase in electron donation by axial histidine. This has been shown by extensive studies of low spin hemes in both model compounds and hemoproteins (55, 56). The effect cannot be caused by deprotonated imidazole, because in that case the signals of histidine-imidazolate derivatives would occur in the MCD spectrum of SoxABCD, which is not the case (57). MCD spectra are very sensitive to the chemical nature of axial heme ligands (55, 58). Therefore, one or both of the histidine ligands of heme a605 in SoxABCD most likely form a stronger hydrogen bond with a neighboring amino acid side chain than the corresponding hydrogen bond in isolated SoxB. In fact, in model compounds of low spin hemes, strong hydrogen bonds have shown an effect similar to the deprotonation of the histidine ligands (59, 60). Furthermore, the strong hydrogen bond can also explain the red shift of the Soret band of SoxABCD relative to SoxB; the increased basicity of the histidine ligands may allow the optical transition of the heme to occur at lower energy.

As reported elsewhere, MCD spectra support the conclusion that in SoxABCD one of the two low spin hemes in its cytochrome a587 moiety (SoxC) displays a His-Met ligandation (57). This can also be readily derived from the primary sequence of SoxC, which resembles an apocytochrome b, however with the option to use Met instead of His at one of the heme binding motifs. This observation together with the redox titration reported here solves an interesting problem. Actually, two pools of cytochromes absorbing at 587 nm but exhibiting different redox potentials have been detected in membranes of S. acidocaldarius (11, 28, 47). The present study unequivocally assigns the high potential heme a587 to the SoxC gene product as a component of the SoxABCD terminal oxidase complex. The span between the midpoint potentials of its hemes (+210 and +270 mV) may be of significant functional importance for the process of proton pumping (see below). From this assignment it follows also that the previously described low potential cytochrome a587 with an average midpoint potential of about +80 to +100 mV (11) corresponds to the product of the soxG gene (29) which is a component of the SoxM alternate oxidase complex.

The preparation of the SoxABCD complex reported here allows a complete separation from the SoxM complex by hydrophobic interaction chromatography as revealed by removal of all b-type cytochromes during this step (Table I); it yields a 20-fold enrichment of the purified product (based on heme a content), indicating an ~5% abundance of the complex in the intact membrane. Despite the lack of cytochrome c (11, 14) Sulfolobus membranes oxidize cytochrome c from horse heart in a cyanide-sensitive manner. Since it was shown that the membrane-residing Rieske-FeS proteins of Sulfolobus can accept electrons from cytochrome c (18, 61), it is very likely that this activity is artifactual; as shown previously (61), the Rieske-FeS centers are reoxidized by the terminal oxidases also in intact membranes under aerobic conditions. Consequently, the cytochrome c oxidase activity vanishes during the purification of the SoxABCD quinol oxidase.

The catalytic activity of the detergent-solubilized complex is highest with caldariella quinone and comes close to a purely diffusion-controlled reaction, as documented by the kcat/Km value (Table II). Compared with the purified SoxB single entity quinol oxidase, a significantly higher maximum turnover could be measured under various conditions. A reasonable explanation may be that the quinone binding site is much better preserved in the integrated complex than in the single entity form. Anticipating the mechanism discussed below, the presence of cytochrome a587 may be necessary to provide a completely intact substrate binding site. Conformational differences resulting from polypeptide interactions within the complex are suggested also by the above mentioned differences of the EPR spectra between isolated cytochrome aa3 (SoxB) and SoxABCD. Although they are functionally classified as quinol oxidases, none of the classical inhibitors interacting with quinol binding sites, either in bc1 complexes or with quinol oxidases of the bo type, were found to inhibit this enzyme (data not shown). However, the persisting appearance of a strong g = 2 radical signal in oxidase samples "as prepared" suggested the presence of a tightly bound partially reduced quinone.

In fact, recent studies of various quinol oxidases support the view of tightly bound quinones as being a general feature of these membrane enzymes (33-35, 53). As in case of SoxABCD, in the resting state of the preparations, a highly stabilized quinone radical was observed also in Escherichia coli bo3 oxidase (35). From hyperfine structure in the EPR spectrum, the presence of a second quinone molecule in a distance of ~1.5 nm was postulated. Interestingly, also for the Qo site of bc1 complexes, a dual occupancy by quinones has been postulated (62), forming a quinhydrone state that represents a rather stable semiquinone radical as well as a perfect single electron donor/acceptor system. Thus, similar structures may be postulated to function in the SoxABCD complex, which in analogy to bc1 contains a di-heme cytochrome structurally homologous to cytochrome b.

Regarding the co-reconstituted liposomal system, the observed catalytic turnover is considerably below that of real cytochrome c oxidases (about 1/5-1/3). This is not surprising in a totally artificial system employing a quasireversed electron flow from reduced cytochrome c via a Rieske-FeS protein to SoxABCD-bound caldariella quinone. Although the Sulfolobus Rieske protein (SoxL (18)) presumably has no genuine cytochrome c binding domain, a sufficient rate of redox equilibration between both components is obviously possible (18). The back-transfer of electrons from the Rieske protein to quinone is thermodynamically compensated for by the large free energy change of the quinol oxidase reaction catalyzed by the integrated SoxABCD complex. As revealed by the results illustrated in Fig. 10, the catalytic activity of the system is high enough to cope with the rate of diffusional proton back-flow into the liposomes, and thus the proton pumping capability of the co-reconstituted system could be demonstrated.

Actually, it was the predominant aim of this investigation to examine whether or not the terminal oxidase complex of Sulfolobus could perform respiration-coupled proton pumping exceeding purely chemical charge separation. Although this could be shown, for interpretation of the presented data a discussion of the reconstituted model system as well as of structural properties of cytochrome aa3 from S. acidocaldarius is of crucial importance. Can subunit SoxB itself, the cytochrome aa3 moiety of the complex, act as a proton pump? With a high degree of certainty, the answer is no. Sequence alignments strongly suggest that the core structure of the catalytic subunit I of all heme-copper oxidases has been conserved throughout all aerobic organisms (47, 48, 63, 64). Besides the six invariant histidine residues in helices II, VI, VII, and X involved in heme-iron and CuB binding, a number of other residues have been identified by site-directed mutagenesis to be intimately linked with proton pumping (41, 65, 66). Convincing evidence has been presented that two separate proton transfer pathways exist in subunit I of proton pumping heme-copper oxidases: one for protons consumed for water formation at the binuclear reaction center (chemical H+) and one for vectorially translocated protons (pumped H+) (41, 67, 68). This is strongly supported by the recently presented three-dimensional structures of the enzymes from P. denitrificans (4) and from beef heart (4-6). The residues on helix IIX delineating the putative chemical proton channel are present in SoxB (as in all other oxidases), whereas the critical residues for the pumping channel are missing. These are the -NX10DX6N- motif in the loop connecting transmembrane helices II and III and a highly conserved glutamic acid of the -GHPEVY- motif in transmembrane helix VI. In proton pumping oxidases, irrespective of whether cytochrome c or quinoles serve as electron donors, the aspartate at the interhelical connection is located at the putative entrance to the pumping channel, while the glutamic acid appears to be involved in proton conduction at the output side of a mechanism involving the so-called histidine cycle for pumping (4, 69, 70). In SoxB this very glutamate is replaced by apolar valine, and the essential residues of the loop motif are replaced by nonprotonatable ones. Thus, a proton pumping activity of SoxB is indeed very unlikely. Actually, multiple alignments and phylogenetic analysis strongly suggest that two lines of oxidases already separated during early evolution (for details see Refs. 47, 48, and 71), with and without the essential residues for the H+ pumping channel, respectively. Nevertheless, recent mutagenesis experiments with R. sphaeroides have shown that alternate intermediate pathways may be used for pumped protons; however, the crucial aspartate motif (loop II-III) at the H+ uptake side was indispensable (72).

Since proton pumping by the integrated SoxABCD complex has been shown in the present work, new mechanisms involving SoxC, the diheme cytochrome a587, have to be considered. In analogy to the function of b-type cytochromes in bc1 complexes, it had been hypothesized that the SoxABCD complex might involve an intramolecular Q cycle (13). The applied reconstituted vesicle system contains tightly bound quinone (as discussed above) but does not contain an excessive quinone pool as required for steady state proton translocation by a Q cycle mechanism in native membranes. This, however, is not necessary in single turnover experiments as described here, presumably involving direct reduction of tightly bound quinone of the integrated Rieske-FeS·SoxABCD complex. Operation of a Q cycle is characterized by a H+/e- >1 (theoretically 2). Taking into account that experimentally measurable H+/e- ratios usually fall short due to proton leaks or insufficient coupling of reconstituted liposomal systems, values of >1.2 H+/e- as found in the above experiments are well in line with a Q cycle. In this context, it is important to notice that by the applied method scalar protons are not produced outside the lipid vesicles, and only pumped protons are monitored.

Possible proton pumping mechanisms of terminal ubiquinol oxidases have been controversially discussed (7, 73-75). The linkage of quinol oxidation to proton transduction has been hypothesized also in alternate models to involve redox loops between two quinone binding sites at the terminal oxidase (7). The net pumping stoichiometry for the proposed Q redox loops, however, would be H+/e- = 1, which is below the ratio of a Q cycle and the above observed results with SoxABCD of >1, respectively.

Of course, any details of such a mechanism remain speculative as long as no high resolution structure of this particular enzyme complex or related enzyme complexes becomes accessible. The two hemes of SoxC separated by 60 mV between their midpoint potentials could indeed assume a similar role as b-type cytochromes for disproportionation of Qbardot radicals. However, nothing particular is known about the Q binding site(s) on SoxABCD, especially whether equivalents to the Qi and Qo sites on bc1 complexes exist; this issue may be approached in further studies as well as the search for site-specific inhibitors or quinone analogs.

A rigorous alternate assumption could be that cytochrome a587 of SoxABCD simply serves as an electron storage device facilitating the transition of electrons from a two-electron donor (quinol) into a single electron pathway of the terminal oxidase. In that case, a novel pumping mechanism would have to occur, which appears unlikely in view of the conserved structure of oxidase subunits I (4, 5) and of the observed pumping stoichiometry.

In conclusion, therefore, it should be emphasized that the measured stoichiometry does not definitely prove the existence of a Q cycle-like mechanism, but it excludes the mechanism of "classical" aa3- or bo3-type cytochrome c oxidases.


FOOTNOTES

*   The work was supported by Deutsche Forschungsgemeinschaft Grant Scha125/17-3 (to G. S.) and a grant from the European Commission.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed. Fax: 0049-451-5004068; E-Mail: SCHAEFER@PHYSIK.MU-LUEBECK.DE.
1   The abbreviations used are: MES, 4-morpholineethanesulfonic acid; AS, ammonium sulfate; DM, dodecyl-beta -D-maltoside; Delta pH, transmembrane pH gradient; Delta Psi , transmembrane electrical potential; Em, midpoint potential; MCD, magnetic circular dichroism; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; FPLC, fast protein liquid chromatography.
2   S. Anemüller, personal communication.
3   C. L. Schmidt, unpublished results.
4   S. Grzybek, and W. Mäntele, unpublished observation.

ACKNOWLEDGEMENTS

We thank Dr. C. L. Schmidt (Lübeck) for advice with preparations of the Sulfolobus Rieske protein. The collaboration of Dr. E. Antonopoulos (Frankfurt) is acknowledged for generous help with purification and supply of the tetraether lipids from T. acidophilum for the co-reconstitution experiments. Our thanks are due to Prof. W. Mäntele (Erlangen) and Ulrike Kaiser for cooperation with spectroelectrochemical titrations and also to Dr. M. Saraste and Dr. M. Lübben (EMBL) for the gift of antibodies against subunits of the SoxABCD complex.


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