Sulfolobus aconitase, a regulator of iron metabolism?

The aconitase of Sulfolobus solfataricus, a hyperthermophilic crenarchaeon, was cloned and heterologously expressed in Escherichia coli. Enzymic analyses and EPR measurements indicated clearly that the iron-sulphur cluster of the thermophilic aconitase was already inserted in the mesophilic host. The enzyme was purified to a specific activity of approx. 44 units/mg and to 90% homogeneity. The enzymic parameters of the recombinant aconitase turned out to be in the same range as the respective values for the previously characterized native enzyme from the closely related S. acidocaldarius. Based on its primary sequence, the recombinant aconitase is closely related to bacterial A-like and to eukaryotic iron regulatory protein-like proteins. Specific aconitase activities in cytosolic extracts of S. acidocaldarius were found to be decreased markedly in iron-starved compared with iron-repleted cells. However, no differences in aconitase levels between iron-starved and iron-supplemented cells could be detected by immunostaining.

Investigations of iron uptake in Halobacterium salinarum.

The iron transport in the extremely halophilic Euryarchaeon Halobacterium salinarum JW5 was investigated. Experiments to detect endogenous siderophores from H. salinarum failed, but it was able to utilize exogenous siderophores. Measurement of the uptake of (55)Fe and [(14)C]citrate gave evidence only for the accumulation of iron. Two additional membrane proteins could be detected in iron-starved cells, one in iron-repleted membranes and one that is up-regulated there. Respiratory rates of iron-starved membranes after the addition of succinate and NADH differed considerably from iron-repleted ones. Furthermore, both types of membrane exhibited different degrees of inhibition by cyanide.

Characterization of a non-haem ferritin of the Archaeon Halobacterium salinarum, homologous to Dps (starvation-induced DNA-binding protein).

An iron-rich protein was isolated from the Archaeon Halobacterium salinarum sharing a sequence identity of 35% with the starvation-induced DNA-binding protein, DpsA, of Synechecoccus sp. PCC 7942. It consists of 20 kDa subunits, forming a dodecameric structure. The protein exhibits a ferric iron loading of up to 103 Fe ions/mol of holoprotein. CD spectra are consistent with an alpha-helical contribution of 58%. The UV/visible spectrum provides no evidence for the presence of haem groups. This protein exhibits features of a non-haem-type bacterial ferritin although it shares only little sequence homology with non-haem bacterial ferritin.

Ferredoxins from the archaeon Acidianus ambivalens: overexpression and characterization of the non-zinc-containing ferredoxin FdB.

Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus ambivalens have been sequenced; the sequences share 86% similarity. Whereas the deduced protein sequence of the ferredoxin FdA clearly contains a zinc-binding motif, the corresponding sequence of the FdB is devoid of this motif. Thus far, only the zinc-containing ferredoxin, FdA, from A. ambivalens has been chemically and functionally characterized from its native source. Using RT-PCR and Northern blot analysis, we show that both ferredoxins are expressed by A. ambivalens under either anaerobic or aerobic growth conditions. The zinc-free ferredoxin, FdB, was overexpressed in E. coli and purified to homogeneity. Using EPR spectroscopy, we could demonstrate that FdB contains one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+) cluster. The reduction potential of the [3Fe-4S](1+/0) cluster was determined as -235+/-10 mV, at pH 6.5, by EPR-monitored redox titration. The high melting temperature of 108+/-2 degrees C of FdB determined by CD spectroscopy reveals that it is not the binding of the Zn2+ that induces the extreme thermostability of these ferredoxins.

First expression and characterization of a recombinant CuA-containing subunit II from an archaeal terminal oxidase complex.

The branched respiratory chain of the archaeon Sulfolobus acidocaldarius contains a supercomplex, SoxM, consisting of a bc1-like subcomplex and a terminal oxidase moiety, including a subunit II analogous polypeptide, SoxH. However, the latter component has never been identified in preparations of SoxM. We demonstrate the presence of an mRNA transcript by Northern analysis. We succeeded in cloning and expressing the respective gene with truncated N-terminus by deleting a 20 AS membrane anchor, which resulted in a water-soluble purple copper protein, which was further characterized. The recombinant subunit II of the SoxM complex contains a correctly inserted binuclear CuA cluster as revealed by UV/vis and EPR spectroscopy. The protein is highly thermostable and displays a redox potential of +237 mV. In recombinant form, the metal interacts with cytochrome c as an artificial electron donor; the physiological electron donor is still unknown, since S. acidocaldarius does not contain any c-type cytochromes. The purple copper center of SoxM shows an interesting pH dependency with a pKa at 6.4, suggesting protonation of the Cu-ligating histidines. Further lowering the pH causes a reversible transition into another cluster form with concomitant liberation of one copper. It may thus provide a model for the study of cluster rearrangements in response to pH.

Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius.

The first archaeal aconitase was isolated from the cytosol of the thermoacidophilic Sulfolobus acidocaldarius. Interestingly, the enzyme was copurified with an isocitrate lyase. This enzyme, directly converting isocitrate, the reaction product of the aconitase reaction, was also unknown in crenarchaeota, thus far. Both proteins could only be separated by SDS gel electrophoresis yielding apparent molecular masses of 96 kDa for the aconitase and 46 kDa for the isocitrate lyase. Despite of its high oxygen sensitivity, the aconitase could be enriched 27-fold to a specific activity of approximately 55 micromol x min(-1) x mg(-1), based on the direct aconitase assay system. Maximal enzyme activities were measured at pH 7.4 and the temperature optimum for the archaeal enzyme was recorded at 75 degrees C, slightly under the growth optimum of S. acidocaldarius around 80 degrees C. Thermal inactivation studies of the aconitase revealed the enzymatic activity to be uninfluenced after one hour incubation at 80 degrees C. Even at 95 degrees C, a half-life of approximately 14 min was determined, clearly defining it as a thermostable protein. The apparent K(m) values for the three substrates cis-aconitate, citrate and isocitrate were found as 108 microM, 2.9 mM and 370 microM, respectively. The aconitase reaction was inhibited by the typical inhibitors fluorocitrate, trans-aconitate and tricarballylate. Amino-acid sequencing of three internal peptides of the S. acidocaldarius aconitase revealed the presence of highly conserved residues in the archaeal enzyme. By amino-acid sequence alignments, the S. acidocaldarius sequence was found to be highly homologous to either other putative archaeal or known eukaryal and bacterial sequences. As shown by EPR-spectroscopy, the enzyme hosts an interconvertible [3Fe--4S] cluster.

Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements.

The hyper-thermostable Fe-superoxide dismutase from the Archaeon Acidianus ambivalens: characterization, recombinant expression, crystallization and effects of metal exchange.

An iron-containing superoxide dismutase (SOD; EC 1.15.1.1) of the hyperthermophilic archaeon Acidianus ambivalens (Aa-SOD) has been purified and characterized and the gene has been cloned and sequenced. The SOD from the facultatively aerobic member of the crenarchaeota could be expressed in E. coli. Both, the native as well as the heterologously overproduced protein turned out to have extraordinarily high melting temperatures of 128 degrees C and 124.5 degrees C, respectively. To the best of our knowledge, this is the highest directly measured melting temperature of a native protein. Surprisingly, neither the native nor the recombinant superoxide dismutase displays 100% occupation of the metal coordination sites. Obviously it is not the incorporation of a metal ion that confers the extreme thermostability. Expression of the superoxide dismutase in the presence of different metals such as Fe, Co, Ni, Mn and Cu offered the possibility of studying the hitherto unknown cofactor preference of iron-superoxide dismutase. The recombinant enzyme displayed the highest preference for incorporation of cobalt although iron is used as the natural cofactor. Spectroscopic analysis by EPR, atomic absorption and UVNis spectroscopy as well as activity measurements and differential scanning calorimetry of the metal substituted superoxide dismutases were performed. However, the superoxide dismutase of A. ambivalens is active only with iron but may incorporate other metals equally well in the catalytic center without loss of conformational stability or heat tolerance. The co-form of the enzyme could be crystallized.

Functional characterization of an extremely thermophilic ATPase in membranes of the crenarchaeon Acidianus ambivalens.

A plasma membrane-bound adenosine triphosphatase with specific activities up to 0.2 micromol min(-1) (mg protein)(-1) at 80 degrees C was detected in the thermoacidophilic crenarchaeon Acidianus ambivalens (DSM 3772). The enzymatic activity exhibited a broad pH-optimum in the neutral range with two suboptima at pH 5.5 and 7.0, respectively. Sulfite activation resulted in only one pH optimum at 6.25. In the presence of the divalent cations Mg2+ and Mn2+ the ATPase activity was maximal. Remarkably, the hydrolytic rates of GTP and ITP were substantially higher than for ATP. ADP and pyrophosphate were only hydrolyzed with small rates, whereas AMP was not hydrolyzed at all. Both activities could be weakly inhibited by the classical F-type ATPase inhibitor N,N'-dicyclohexylcarbodiimide, whereas azide had no influence at all. The classical inhibitor of V-type ATPases, nitrate, also exerted a small inhibitory effect. The strongly specific V-type ATPase inhibitor concanamycin A, however, showed no effect at all. The P-type ATPase inhibitor vanadate had no inhibitory effect on the ATPase activity at pH 7.0, whereas a remarkable inhibition at high concentrations could be observed for the activity at pH 5.5. Arrhenius plots for both membrane bound ATPase activities were linear up to 95 degrees C, reflecting the enormous thermostability of the enzyme.

A novel Rieske iron-sulfur protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum: sequencing of the gene, expression in E. coli and characterization of the protein.

The crenarchaeon Pyrobaculum aerophilum is with an optimal growth temperature of 100 degrees C one of the most thermophilic organisms known to possess an aerobic respiratory chain. The analysis of DNA sequences from the Pyrobaculum genome project lead to the identification of an open reading frame potentially coding for a Rieske iron-sulfur protein. The complete gene (named parR) was cloned and sequenced. The deduced amino acid sequence displays unusual amino acid exchanges and a so far unknown sequence insertion. The N-terminus shows similarities to bacterial signal sequences. Several forms of the gene were expressed in E. coli in order to verify the classification as a Rieske protein and to facilitate biophysical studies. Soluble, thermo-stable proteins with correctly inserted iron-sulfur clusters were expressed from two versions of the gene. The delta1-23 truncated holo-protein is redox active. It displays the typical spectroscopic properties of a Rieske protein. The redox potential was determined to be +215 mV at pH 6.5 and is pH dependent above pH 7.5 revealing the influence of two protonation equilibria with pKa values of 8.1 and 9.8. Phylogenetic analysis demonstrates that the parR protein clusters together with the two other available archaeal Rieske sequences from Sulfolobus on a separate branch of the phylogenetic tree apart from the proteins from thermophilic bacteria like Aquifex and Thermus.

The unusual iron sulfur composition of the Acidianus ambivalens succinate dehydrogenase complex.

The succinate dehydrogenase complex of the thermoacidophilic archaeon Acidianus ambivalens was investigated kinetically and by EPR spectroscopy in its most intact form, i.e., membrane bound. Here it is shown that this respiratory complex has an unusual iron-sulfur cluster composition in respect to that of the canonical succinate dehydrogenases known. The spectroscopic studies show that center S3, the succinate responsive [3Fe-4S]1+/0 cluster of succinate dehydrogenases, is not present in membranes prepared from aerobically grown A. ambivalens, nor in partially purified complex fractions. On the other hand, EPR features associated to the remaining centers, clusters S1 ([2Fe-2S]1+/2+) and S2 ([4Fe-4S]2+/1+), could be observed. Similar findings were made in other archaea, namely Acidianus infernus and Sulfolobus solfataricus. Kinetic investigations showed that the A. ambivalens enzyme is reversible, capable of operating as a fumarate reductase - a required activity if this obligate autotroph performs CO2 fixation via a reductive citric acid cycle. Sequencing of the sdh operon confirmed the spectroscopic data. Center S3 ([3Fe-4S]) is indeed replaced by a second [4Fe-4S] center, by incorporation of an additional cysteine, at the cysteine cluster binding motif (CxxYxxCxxxC-->CxxCxxCxxxC). Genomic analysis shows that genes encoding for succinate dehydrogenases similar to the ones here outlined are also present in bacteria, which may indicate a novel family of succinate/fumarate oxidoreductases, spread among the Archaea and Bacteria domains.

The strict molybdate-dependence of glucose-degradation by the thermoacidophile Sulfolobus acidocaldarius reveals the first crenarchaeotic molybdenum containing enzyme--an aldehyde oxidoreductase.

In order to investigate the effects of trace elements on different metabolic pathways, the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius (DSM 639) has been cultivated on various carbon substrates in the presence and absence of molybdate. When grown on glucose (but neither on glutamate nor casein hydrolysate) as sole carbon source, the lack of molybdate results in serious growth inhibition. By analysing cytosolic fractions of glucose adapted cells for molybdenum containing compounds, an aldehyde oxidoreductase was detected that is present in the cytosol to at least 0.4% of the soluble protein. With Cl2Ind (2,6-dichlorophenolindophenol) as artificial electron acceptor, the enzyme exhibits oxidizing activity towards glyceraldehyde, glyceraldehyde-3-phosphate, isobutyraldehyde, formaldehyde, acetaldehyde and propionaldehyde. At its pH-optimum (6.7), close to the intracellular pH of Sulfolobus, the glyceraldehyde-oxidizing activity is predominant. The protein has an apparent molecular mass of 177 kDa and consists of three subunits of 80.5 kDa (alpha), 32 kDa (beta) and 19.5 kDa (gamma). It contains close to one Mo, four Fe, four acid-labile sulphides and four phosphates per protein molecule. Methanol extraction revealed the existence of 1 FAD per molecule and 1 molybdopterin per molecule, which was identified as molybdopterin guanine dinucleotide on the basis of perchloric acid cleavage and thin layer chromatography. EPR-spectra of the aerobically prepared enzyme exhibit the so-called 'desulpho-inhibited'-signal, known from chemically modified forms of molybdenum containing proteins. Anaerobically prepared samples show both, the signals arising from the active molybdenum-cofactor as well as from the two [2Fe-2S]-clusters. According to metal-, cofactor-, and subunit-composition, the enzyme resembles the members of the xanthine oxidase family. Nevertheless, the melting point and long-term thermostability of the protein are outstanding and perfectly in tune with the growth temperature of S. acidocaldarius (80 degrees C). The findings suggest the enzyme to function as a glyceraldehyde oxidoreductase in the course of the nonphosphorylated Entner-Doudoroff pathway and thereby may attribute a new physiological role to this class of enzyme.

Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius. A novel highly glycosylated, membrane-bound b-type hemoprotein.

In this study we re-examined the inducible cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639), formerly thought to be a component of a terminal oxidase (Becker, M., and Schäfer, G. (1991) FEBS Lett. 291, 331-335). An improved purification method increased the yield of the protein and allowed more detailed investigations. Its molecular mass and heme content have been found to be 64,210 Da and 1 mol of heme/mol of protein, respectively. It is only detectable in cells grown at low oxygen tensions. The composition of the growth medium also exerts significant influence on the cytochrome b558/566 content of S. acidocaldarius membranes. The cytochrome exhibits an extremely high redox potential of +400 mV and shows no CO reactivity; a ligation other than a His/His-coordination of axial ligands appears likely. It turned out to be highly glycosylated (more than 20% of its molecular mass are sugar residues) and is probably exposed to the outer surface of the plasma membrane. The sugar moiety consists of several O-glycosidically linked mannoses and at least one N-glycosidically linked hexasaccharide comprising two glucoses, two mannoses, and two N-acetyl-glucosamines. The gene of the cytochrome (cbsA) has been sequenced, revealing an interesting predicted secondary structure with two putative alpha-helical membrane anchors flanking the majority of a mainly beta-pleated sheet structure containing unusually high amounts of serine and threonine. A second gene (cbsB) was found to be cotranscribed. The latter displays extreme hydrophobicity and is thought to form a functional unit with cytochrome b558/566 in vivo, although it did not copurify with the latter. Sequence comparisons show no similarity to any entry in data banks indicating that this cytochrome is indeed a novel kind of b-type hemoprotein. A cytochrome c analogous function in the pseudoperiplasmic space of S. acidocaldarius is discussed.

Studies of the electron transport chain of the euryarcheon Halobacterium salinarum: indications for a type II NADH dehydrogenase and a complex III analog.

The components involved in the respiratory system of the euryarcheon Halobacterium salinarum were investigated by spectroscopic and polarographic techniques. Previous results about the cytochrome composition could be verified. However, under low oxygen tension, the expression of a d-type cytochrome was detected. Membranes exerted an NADH- and succinatecytochrome-c oxidoreductase as well as an NADH and succinate oxidase activity. These activities could be blocked by the following inhibitors: 7-jodocarboxylic acid, giving evidence for the presence of a type II NADH dehydrogenase, antimycin A, and myxothiazol, indicating the presence of a complex III analog, and the typical succinate dehydrogenase (SDH) and terminal oxidase inhibitors. Complex I inhibitors like rotenone and annonine were inactive, clearly excluding the presence of a coupled NADH dehydrogenase. In addition, no [Fe-S] resonances in the region of the NADH dehydrogenase (NDH) clusters could be observed after NADH addition. One of the terminal oxidases could be shown to act as a cytochrome-c oxidase with a Km value of 37 microM and an activation energy of 23.7 kJ/mol. The relative molecular mass of the endogenous c-type cytochrome could be determined as 14.1 kD. The complex III analog could be enriched after detergent extraction with Triton X-100 and hydroxylapatite (HTP) chromatography. The partially purified complex contained a Rieske iron-sulfur cluster, b- and c-type cytochromes, and was catalytically active in the decylubiquinone-cytochrome-c oxidoreductase assay.

A succinate dehydrogenase with novel structure and properties from the hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and biophysical characterization.

The sdh operon of Sulfolobus acidocaldarius DSM 639 is composed of four genes coding for the 63.1-kDa flavoprotein (SdhA), the 36.5-kDa iron-sulfur protein (SdhB), and the 32.1-kDa SdhC and 14.1-kDa SdhD subunits. The four structural genes of the sdhABCD operon are transcribed into one polycistronic mRNA of 4.2 kb, and the transcription start was determined by the primer extension method to correspond with the first base of the ATG start codon of the sdhA gene. The S. acidocaldarius SdhA and SdhB subunits show characteristic sequence similarities to the succinate dehydrogenases and fumarate reductases of other organisms, while the SdhC and SdhD subunits, thought to form the membrane-anchoring domain, lack typical transmembrane alpha-helical regions present in all other succinate:quinone reductases (SQRs) and quinol:ifumarate reductases (QFRs) so far examined. Moreover, the SdhC subunit reveals remarkable 30% sequence similarity to the heterodisulfide reductase B subunit of Methanobacterium thermoautotrophicum and Methanococcus jannaschii, containing all 10 conserved cysteine residues. Electron paramagnetic resonance (EPR) spectroscopic studies of the purified enzyme as well as of membranes revealed the presence of typical S1 [2Fe2S] and S2 [4Fe4S] clusters, congruent with the deduced amino acid sequences. In contrast, EPR signals for a typical S3 [3Fe4S] cluster were not detected. However, EPR data together with sequence information implicate the existence of a second [4Fe4S] cluster in S. acidocaldarius rather than a typical [3Fe4S] cluster. These results and the fact that the S. acidocaldarius succinate dehydrogenase complex reveals only poor activity with caldariella quinone clearly suggest a unique structure for the SQR of S. acidocaldarius, possibly involving an electron transport pathway from the enzyme complex into the respiratory chain different from those for known SQRs and QFRs.

Biochemical and spectroscopic properties of the four-subunit quinol oxidase (cytochrome ba3) from Paracoccus denitrificans.

The ba3 quinol oxidase from Paracoccus denitrificans has been purified by a new protocol leading to significantly higher yields than previously reported (Richter et al. (1994) J. Biol. Chem. 269, 23079-23086). In an SDS PAG an additional protein band compared with the previous preparation appears, which can be identified as the major form of subunit II. All protein bands can be assigned to genes of the qox operon by N-terminal sequencing, indicating that the oxidase consists of four subunits. In addition to one heme A, one heme B, and one copper atom, the preparation contains two ubiquinone molecules per enzyme. The oxidase is further characterized by electron paramagnetic resonance (EPR), circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopy.

Two different respiratory Rieske proteins are expressed in the extreme thermoacidophilic crenarchaeon Sulfolobus acidocaldarius: cloning and sequencing of their genes.

We have isolated two genes encoding Rieske iron sulfur proteins from the genomic DNA of the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius (DSM 639). One of the genes, named soxL, codes for the previously isolated novel Rieske-I protein. The second gene (soxF) 121 codes for the Rieske-II protein associated with the second terminal oxidase of Sulfolobus. Both proteins exhibit only 24% identical residues. The Rieske-I protein shows a number of unusual features. (i) The distance between the two cluster binding sites is significantly larger than in all known proteins. (ii) An unexpected Pro --> Asp exchange in one of the cluster binding sites. (iii) It shows some resemblance to the mitochondrial and plastidic Rieske proteins insofar as the soxL gene codes for a pre-sequence which is no longer present in the mature Rieske-I protein. Both proteins cluster together on a separate branch of the phylogenetic tree. To our knowledge this is the first proven case of two significantly different Rieske proteins in a prokaryote.

Perturbation of the CuA site in cytochrome-c oxidase of Paracoccus denitrificans by replacement of Met227 with isoleucine.

Subunit II of cytochrome-c oxidase contains a redox centre, CuA, with unusual spectroscopic properties; this site consists of two copper atoms and acts as the entry point for electrons from cytochrome c. We have constructed a site-directed mutant of cytochrome-c oxidase from Paracoccus denitrificans in which the CuA site has been disturbed by replacement of Met227 with isoleucine. The purified, fully assembled enzyme complex has been investigated with various techniques including metal analysis, EPR and visible spectroscopies, steady-state and fast kinetics. The stoichiometry of the metals in the enzyme remains unchanged but a clear perturbation of the CuA site can be observed in the EPR and near-infrared optical spectra. It is concluded that in the mutant CuA is still binuclear but that the two nuclei are no longer equivalent, converting the delocalized [Cu(1.5)....Cu(1.5)] centre of the wild type into a localized [Cu(I)....Cu(II)] system. Changes in the overall kinetics of the mutant are correlated with a diminished electron transfer rate between CuA and heme alpha.

EPR characterization of an archaeal succinate dehydrogenase in the membrane-bound state.

The membrane-bound succinate dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum was characterized by EPR spectroscopy and its functional properties were determined. The highest turnover values of succinate dehydrogenase activity were observed at pH 7.4, which is somewhat above the internal pH value of T. acidophilum. The temperature optimum of the reaction was determined as 78 degrees C and the Km value for succinate using phenazine methosulfate as the electron acceptor at 53 degrees C was 0.32 mM. The membrane-bound enzyme was able to reduce the artificial electron acceptors phenazine methosulfate, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,6-dichloroindophenol. Succinate oxidation was coupled to oxygen consumption in a completely 2-n-heptyl-4-hydroxyquinoline-N-oxide-sensitive manner. In the oxidized state, T. acidophilum membranes exhibited an almost isotropic EPR spectrum with g-values at gz = 2.017, gy = 2.000, and gx = 1.968 that were assigned to a [3Fe-4S]1+ cluster (S3). Upon reduction with succinate, the membranes displayed a spectrum characteristic of 2Fe-2S clusters (S1), with g-values at gz = 2.029, gy = 1.935, and gx = 1.915. In the dithionite-reduced state, additional resonances can be observed. An axial component, with g-values at gz = 2.057, gy = 1.917, and gx = 1.917 was assigned to a [4Fe-4S]1+ cluster. The saturation behaviour of the S1 cluster was strongly altered in the dithionite-reduced form, thus indicating spin-spin interaction between the S1 center and another paramagnetic center, possibly cluster S2. In both the succinate and the dithionite-reduced membranes, parallel-mode EPR spectra displayed a resonance at g = 14, which may be due to a transition of the S = 2 multiplet of the reduced 3Fe-4S cluster. Spin quantitation yielded a relative stoichiometry of cluster S1 to cluster S3 of 1:1. The results obtained by EPR spectroscopy indicated that the characteristic iron-sulfur cluster S1 [2Fe-2S], S2 [4Fe-4S], and S3 [3Fe-4S], were also present in this archaeal succinate dehydrogenase. EPR redox titrations of T. acidophilum membranes at pH 5.5 yielded a reduction potential of +60 +/- 20 mV for cluster S3 and of +68 +/- 20 mV for cluster S1. The axial [4Fe-4S]2+/1+ center had a reduction potential of -210 +/- 20 mV.

Purification and characterization of the Rieske iron-sulfur protein from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

The previously detected Rieske iron-sulfur protein from the membranes of the thermoacidophile Sulfolobus acidocaldarius [Anemüller, S., et al. (1993) FEBS Lett. 318, 61-64] was purified to electrophoretic homogeneity and the N-terminal amino acids determined. The apparent molecular weight was estimated to be 32 kDa. The reduced protein displays a rhombic EPR spectrum with gxyz = 1.768, 1.895, 2.035. The average g-value of 1.902 is typical for nitrogen ligand-containing clusters. EPR spin quantification and the iron content indicate the presence of one [2Fe-2S] cluster. The purified protein displays ubiquinol cytochrome c reductase activity. The pH optimum of this reaction is temperature dependent and was determined to be pH 7 at 56 degrees C. The results presented in this study clearly prove that the Sulfolobus Rieske protein belongs to the family of the true Rieske iron-sulfur proteins.