Novel [2Fe-2S]-type Redox Center C in SdhC of Archaeal Respiratory Complex II from Sulfolobus tokodaii Strain 7
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m207312200
ISSN1083-351X
AutoresToshio Iwasaki, Asako Kounosu, Miho Aoshima, Daijiro Ohmori, Takeo Imai, Akio Urushiyama, Nathaniel J. Cosper, Robert A. Scott,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoThe SdhC subunit of the archaeal respiratory complex II (succinate:quinone oxidoreductase) from Sulfolobus tokodaii strain 7 has a novel cysteine rich motif and is also related to archaeal and bacterial heterodisulfide reductase subunits. We overexpressed the sdhC gene heterologously inEscherichia coli and characterized the gene product in greater detail. Low temperature resonance Raman and x-ray absorption spectroscopic investigation collectively demonstrate the presence of a [2Fe-2S] cluster core with complete cysteinyl ligation (Center C) and an isolated zinc site in the recombinant SdhC. The [2Fe-2S]2+ cluster core is sensitive to dithionite, resulting in irreversible breakdown of the Fe-Fe interaction. EPR analysis confirmed that the novel Center C is an inherent redox center in the archaeal complex II, showing unique EPR signals in the succinate-reduced state. Distinct subunit and cofactor arrangements in the S. tokodaiirespiratory complex II, as compared with those in mitochondrial and some mesophilic bacterial enzymes, indicate modular evolution of this ubiquitous electron entry site in the respiratory chains of aerobic organisms. The SdhC subunit of the archaeal respiratory complex II (succinate:quinone oxidoreductase) from Sulfolobus tokodaii strain 7 has a novel cysteine rich motif and is also related to archaeal and bacterial heterodisulfide reductase subunits. We overexpressed the sdhC gene heterologously inEscherichia coli and characterized the gene product in greater detail. Low temperature resonance Raman and x-ray absorption spectroscopic investigation collectively demonstrate the presence of a [2Fe-2S] cluster core with complete cysteinyl ligation (Center C) and an isolated zinc site in the recombinant SdhC. The [2Fe-2S]2+ cluster core is sensitive to dithionite, resulting in irreversible breakdown of the Fe-Fe interaction. EPR analysis confirmed that the novel Center C is an inherent redox center in the archaeal complex II, showing unique EPR signals in the succinate-reduced state. Distinct subunit and cofactor arrangements in the S. tokodaiirespiratory complex II, as compared with those in mitochondrial and some mesophilic bacterial enzymes, indicate modular evolution of this ubiquitous electron entry site in the respiratory chains of aerobic organisms. iron-sulfur extended x-ray absorption fine structure fumarate reductase resonance Raman succinate dehydrogenase Stanford Synchrotron Radiation Laboratory x-ray absorption spectroscopy caldariellaquinone Respiratory complex II (succinate:quinone oxidoreductase) is an iron-sulfur (FeS)1flavoprotein complex that serves as the sole membrane-bound component of the oxidative tricarboxylic acid cycle as well as one of the most important primary dehydrogenases at the electron entry site of the aerobic respiratory chain for a variety of aerobic organisms from archaea to bacteria to eukarya (1Ackrell B.A.C. Johnson M.K. Gunsalus R.P. Cecchini G. Müller F. Chemistry and Biochemistry of Flavoenzymes. 3. CRC Press, Inc., Boca Raton, FL1992: 229-297Google Scholar, 2Hägerhäll C. Biochim. Biophys. Acta. 1997; 1320: 107-141Crossref PubMed Scopus (371) Google Scholar, 3Hederstedt L. Science. 1999; 284: 1941-1942Crossref PubMed Scopus (54) Google Scholar, 4Ohnishi T. Moser C.C. Page C.C. Dutton P.L. Yano T. Struct. Fold. Des. 2000; 8: R23-R32Abstract Full Text Full Text PDF Scopus (71) Google Scholar). In general, mesophilic bacterial and eukaryal enzymes consist of three to four different subunits. The largest flavoprotein subunit (SdhA) contains the dicarboxylate active site at a covalently linked FAD via 8α-[N(3)-histidyl] linkage, and the second largest FeS protein subunit (SdhB) contains a high potential [2Fe-2S] cluster (Center S-1), a low potential [4Fe-4S] cluster (Center S-2), and a high potential [3Fe-4S] cluster (Center S-3) (1Ackrell B.A.C. Johnson M.K. Gunsalus R.P. Cecchini G. Müller F. Chemistry and Biochemistry of Flavoenzymes. 3. CRC Press, Inc., Boca Raton, FL1992: 229-297Google Scholar, 2Hägerhäll C. Biochim. Biophys. Acta. 1997; 1320: 107-141Crossref PubMed Scopus (371) Google Scholar, 4Ohnishi T. Moser C.C. Page C.C. Dutton P.L. Yano T. Struct. Fold. Des. 2000; 8: R23-R32Abstract Full Text Full Text PDF Scopus (71) Google Scholar) (see Fig. 1,A and B). The membrane anchor subunits bind quinones involved in the electron transfer reactions of the enzyme, and in some cases, they contain one or two protohemes IX as prosthetic groups (1Ackrell B.A.C. Johnson M.K. Gunsalus R.P. Cecchini G. Müller F. Chemistry and Biochemistry of Flavoenzymes. 3. CRC Press, Inc., Boca Raton, FL1992: 229-297Google Scholar, 2Hägerhäll C. Biochim. Biophys. Acta. 1997; 1320: 107-141Crossref PubMed Scopus (371) Google Scholar, 4Ohnishi T. Moser C.C. Page C.C. Dutton P.L. Yano T. Struct. Fold. Des. 2000; 8: R23-R32Abstract Full Text Full Text PDF Scopus (71) Google Scholar). Recently, the crystal structures of closely related enzymes, fumarate reductase (Frd) complexes of Escherichia coli and Wolinella succinogenes, which are key terminal enzymes in the bacterial anaerobic respiratory chains and catalyze fumarate reduction, were solved at 3.3- and 2.2-Å resolution, respectively (5Iverson T.M. Luna-Chavez C. Cecchini G. Rees D.C. Science. 1999; 284: 1961-1966Crossref PubMed Scopus (365) Google Scholar, 6Lancaster C.R.D. Kröger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (310) Google Scholar). These structures revealed the presence of a covalently bound FAD and three FeS clusters in a nearly linear cofactor arrangement with the common sequence, FAD-[2Fe-2S]-[4Fe-4S]-[3Fe-4S] (4Ohnishi T. Moser C.C. Page C.C. Dutton P.L. Yano T. Struct. Fold. Des. 2000; 8: R23-R32Abstract Full Text Full Text PDF Scopus (71) Google Scholar, 5Iverson T.M. Luna-Chavez C. Cecchini G. Rees D.C. Science. 1999; 284: 1961-1966Crossref PubMed Scopus (365) Google Scholar, 6Lancaster C.R.D. Kröger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (310) Google Scholar) (see Fig.1 B). In contrast, marked variation was noted between the membrane anchor subunits involved in the electron transfer reactions, which bind two menaquinone molecules in the E. coli complex (5Iverson T.M. Luna-Chavez C. Cecchini G. Rees D.C. Science. 1999; 284: 1961-1966Crossref PubMed Scopus (365) Google Scholar) and two protoheme centers in the W. succinogenes complex (6Lancaster C.R.D. Kröger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (310) Google Scholar), both arranged vertically relative to the cytoplasmic membranes. The aerobic respiratory chain of the hyperthermoacidophilic crenarchaeote, Sulfolobus tokodaii strain 7 (formerlySulfolobus sp. strain 7, JCM 10545 (7Suzuki T. Iwasaki T. Uzawa T. Hara K. Nemoto N. Kon T. Ueki T. Yamagishi A. Oshima T. Extremophiles. 2002; 6: 39-44Crossref PubMed Scopus (110) Google Scholar)), consists of somea- and b-type cytochromes, Rieske-type [2Fe-2S] protein, copper-binding protein, caldariellaquinone (Qcal), and several primary dehydrogenases (8Wakao H. Wakagi T. Oshima T. J. Biochem. (Tokyo). 1987; 102: 255-262Crossref PubMed Scopus (48) Google Scholar, 9Iwasaki T. Matsuura K. Oshima T. J. Biol. Chem. 1995; 270: 30881-30892Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 10Iwasaki T. Wakagi T. Isogai Y. Iizuka T. Oshima T. J. Biol. Chem. 1995; 270: 30893-30901Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 11Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 12Iwasaki T. Oshima T. FEMS Microbiol. Lett. 1996; 144: 259-266Crossref PubMed Google Scholar, 13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar, 14Iwasaki T. Oshima T. Methods Enzymol. 2001; 334: 3-22Crossref PubMed Scopus (9) Google Scholar). 2T. Iwasaki and Y. Kawarabayashi, unpublished results. 2T. Iwasaki and Y. Kawarabayashi, unpublished results. It should be noted that no c-type cytochromes are involved in the archaeal unique respiratory chain. Previously, we have solubilized and purified the archaeal respiratory complex II by following the succinate:ubiquinone-1 oxidoreductase activity from the membranes (11Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The purified enzyme consists of four different subunits (SdhA, B, C, D) with apparent molecular masses of 66, 37, 33, and 12 kDa, respectively (11Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and can be reconstituted to afford the succinate-O2respiratory chain in the presence of the cognate respiratory terminal oxidase super-complex and Qcal in vitro (9Iwasaki T. Matsuura K. Oshima T. J. Biol. Chem. 1995; 270: 30881-30892Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). ThesdhABCD gene cluster encoding the archaeal respiratory complex II has been cloned, sequenced, and initially characterized (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar) (DDBJ accession number, AB055061) (see Fig. 1 A). These studies showed that the SdhA subunit has a consensus binding site for flavin and that the SdhB subunit has one [2Fe-2S] and two [4Fe-4S] cluster binding motifs (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar). Some catalytically important residues in these subunits are well conserved. EPR analysis of the FeS centers in the purified complex revealed the presence of at least one high potential [2Fe-2S] and more than one lower potential [4Fe-4S] clusters, and the absence of any [3Fe-4S] cluster in a stoichiometric amount (11Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar). Spin coupling between the [2Fe-2S] cluster and a semiquinone radical was observed in the succinate-reduced state. Based on the reported crystal structures of bacterial Frd complexes (5Iverson T.M. Luna-Chavez C. Cecchini G. Rees D.C. Science. 1999; 284: 1961-1966Crossref PubMed Scopus (365) Google Scholar, 6Lancaster C.R.D. Kröger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (310) Google Scholar), these results suggest that the S. tokodaii SdhAB subcomplex has a similar nearly linear cofactor arrangement with the sequence FAD-[2Fe-2S]-[4Fe-4S]-[4Fe-4S] (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar). Despite these studies, little is known about the putative electron acceptor subunits, SdhC and SdhD, of the respiratory complexes II of the Sulfolobales (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar, 15Janssen S. Schäfer G. Anemüller S. Moll R. J. Bacteriol. 1997; 179: 5560-5569Crossref PubMed Google Scholar, 16Gomes C.M. Lemos R.S. Teixeira M. Kletzin A. Huber H. Stetter K.O. Schäfer G. Anemüller S. Biochim. Biophys. Acta. 1999; 1411: 134-141Crossref PubMed Scopus (36) Google Scholar, 17Lemos R.S. Gomes C.M. Teixeira M. Biochem. Biophys. Res. Commun. 2001; 281: 141-150Crossref PubMed Scopus (35) Google Scholar). Previously, we have shown that the S. tokodaii Qcal-utilizing complex II contains no heme cofactor (9Iwasaki T. Matsuura K. Oshima T. J. Biol. Chem. 1995; 270: 30881-30892Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), which was confirmed by the gene sequence analysis (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar). In place of regular membrane-spanning subunits of bacterial enzymes with bound protoheme groups, the S. tokodaii complex II has two unrelated hydrophilic subunits (see Fig. 1 A), as reported for other respiratory complexes II of the Sulfolobales (15Janssen S. Schäfer G. Anemüller S. Moll R. J. Bacteriol. 1997; 179: 5560-5569Crossref PubMed Google Scholar, 16Gomes C.M. Lemos R.S. Teixeira M. Kletzin A. Huber H. Stetter K.O. Schäfer G. Anemüller S. Biochim. Biophys. Acta. 1999; 1411: 134-141Crossref PubMed Scopus (36) Google Scholar, 17Lemos R.S. Gomes C.M. Teixeira M. Biochem. Biophys. Res. Commun. 2001; 281: 141-150Crossref PubMed Scopus (35) Google Scholar): SdhD, which has been shown to associate tightly with SdhAB to form a subcomplex (11Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), is a small hydrophilic protein (104 amino acids) of unknown function (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar); SdhC (290 amino acids) is homologous to some archaeal and bacterial heterodisulfide reductase subunit B (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar, 15Janssen S. Schäfer G. Anemüller S. Moll R. J. Bacteriol. 1997; 179: 5560-5569Crossref PubMed Google Scholar, 16Gomes C.M. Lemos R.S. Teixeira M. Kletzin A. Huber H. Stetter K.O. Schäfer G. Anemüller S. Biochim. Biophys. Acta. 1999; 1411: 134-141Crossref PubMed Scopus (36) Google Scholar, 17Lemos R.S. Gomes C.M. Teixeira M. Biochem. Biophys. Res. Commun. 2001; 281: 141-150Crossref PubMed Scopus (35) Google Scholar) and has two tandem repeat structures, each with the consensus cysteine-rich motif, -YXGC-//-CCG-//-PCSXC-, followed by short hydrophobic stretch(es), implying a pseudo 2-fold symmetrical topology (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar). Of particular interest in the Sulfolobales respiratory complex II is the SdhC subunit carrying the unique cysteine motifs of unknown function. At question is whether SdhC serves as a true membrane anchor (17Lemos R.S. Gomes C.M. Teixeira M. Biochem. Biophys. Res. Commun. 2001; 281: 141-150Crossref PubMed Scopus (35) Google Scholar) and which cofactors, if any, are bound to the unique cysteine motif. In this study, we heterologously overexpressed theS. tokodaii sdhC gene in E. coli to characterize the gene product in greater detail by various spectroscopic techniques (CD, resonance Raman (RR), x-ray absorption spectroscopy (XAS), and EPR). Our results demonstrate the presence of a novel redox "Center C" with a [2Fe-2S] cluster core structure in the archaeal SdhC family. E. coli strain DH5α and strain HB101, which were purchased from TaKaRa Biomedicals and used for cloning, were grown in Luria-Bertani (LB) or Terrific Broth medium with 50 μg/ml ampicillin when required. Plasmids pGEMT and pGEM3Zf(+) (Promega) were used for cloning and sequencing. Expression vector pET28a was purchased from Novagen. DNA was manipulated by the standard procedures (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Habor Laboratory Press, Plainview, NY1989Google Scholar). Water was purified by a Milli-Q purification system (Millipore). Other chemicals mentioned in this study were of analytical grade. Native respiratory complex II was purified from the membrane fraction ofS. tokodaii strain 7 (formerly Sulfolobussp. strain 7, JCM 10545 (7Suzuki T. Iwasaki T. Uzawa T. Hara K. Nemoto N. Kon T. Ueki T. Yamagishi A. Oshima T. Extremophiles. 2002; 6: 39-44Crossref PubMed Scopus (110) Google Scholar)) in the presence of Lubrol-PX (Nacalai Tesque) as a detergent, as described previously (11Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The entire gene sequence of the S. tokodaii respiratory complex II operon sdhABCD has been determined (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar) and deposited in the DNA Data Bank of Japan (DDBJ) (DDBJ accession numberAB055061). The sequence determination was performed by the dideoxy chain termination method with an automatic DNA sequencer, ABI model 373A and 370A (Applied Biosystems Inc.). The DNA sequence was processed with the DNASIS ver. 3.6 software (Hitachi Software Engineering Co., Ltd.). The homology search against databases was performed with the BEAUTY and BLAST network service (19Worley K.C. Wiese B.A. Smith R.F. Genome Res. 1995; 5: 173-184Crossref PubMed Scopus (228) Google Scholar). Sequence alignments were performed using a CLUSTAL X graphical interface (20Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35300) Google Scholar) with minor manual adjustments. The PCR was carried out to amplify the sdhC gene coding for the archaeal SdhC subunit, using the S. tokodaii strain 7 genomic DNA and the following oligonucleotide primers (designed based on the nucleotide sequence AB055061 (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar)): SdhC-N primer, 5′-GGG CCC GCT AGC ATG AGC TAT GCG TAT TAT-3′ and SdhC-C primer, 5′-GGG CCC CTC GAG TTA AAC TAC ACC CTT CGA-3′. The PCR product thus amplified was subcloned into an NheI/XhoI site in a pET28a vector (Novagen), and the nucleotide sequence was determined with vector-specific T7 promoter and T7 terminator. The resultant vector was named a pET28aSdhC. The pET28aSdhC vector was transformed into the host strain, E. coli BL21-CodonPlus(DE3)-RIL (Stratagene). The transformants were grown overnight at 25 °C in LB medium containing 50 μg/ml kanamycin, and recombinant protein was produced by induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 24 h at 25 °C in the presence of 100 μmFeCl3. The cells pelleted by centrifugation were stored at −80 °C until use. Purification of the recombinant SdhC was performed as follows: frozenE. coli cells were thawed, suspended in 20 mmpotassium phosphate buffer, pH 7.0, containing 0.1 mmphenylmethylsulfonyl fluoride, and disrupted by brief sonification on ice. The supernatant collected by ultracentrifugation was immediately incubated at 65 °C for 20 min, and heat-denatured proteins from the host cells were pelleted by ultracentrifugation using a Hitachi 42-135 rotor at 26,000 rpm for 15 min at 4 °C and discarded. The supernatant thus obtained was passed through a DEAE-Sepharose Fast Flow column (Amersham Biosciences) equilibrated with 20 mm potassium phosphate buffer, pH 7.2, and the column was washed with the same buffer. The flow-through fraction was then applied to a small nickel-nitrilotriacetic acid Superflow column (Qiagen) at 4 °C, and the column was extensively washed with 20 mm potassium phosphate buffer, pH 7.0, containing 300 mm NaCl and 20 mm imidazole. The recombinant holoprotein, still adsorbed as a brown band at the top of the column, was eluted with 20 mm potassium phosphate buffer, pH 7.0, containing 300 mm NaCl and 250 mm imidazole. When required, the protein was further purified by Sephadex G-75 gel filtration column chromatography (Amersham Biosciences). The purified recombinant holoprotein was stored at −80 °C until use. Recombinant SdhC was concentrated by pressure filtration with an Amicon YM-10 membrane. Further concentration was achieved by placing the samples under a stream of dry argon gas. The resultant samples (∼1 mm), containing 30% (v/v) glycerol, were frozen in 24 × 3 × 2 mm polycarbonate cuvets with a Mylar-tape front window for XAS and solid-state cryoreduction studies. XAS data were collected at 10 K at the Stanford Synchrotron Radiation Laboratory (SSRL), beamline 7-3, with the SPEAR storage ring operating at 3.0 GeV. A monochromator containing a Si[220] crystal and a 30-element germanium solid state x-ray fluorescence detector (provided by the National Institutes of Health Biotechnology Research Resource) were employed for data collection. The first inflection of the edge of zinc and iron foils (assumed to be 7111.3 and 9660.7 eV, respectively) were used for energy calibration. All other data collection parameters were as reported previously (21Cosper N.J. Stålhandske C.M.V. Saari R.E. Hausinger R.P. Scott R.A. J. Biol. Inorg. Chem. 1999; 4: 122-129Crossref PubMed Scopus (25) Google Scholar). EXAFS analysis was performed with the EXAFSPAK software (www-ssrl.slac.stanford.edu/exafspak.html) according to standard procedures (22Scott R.A. Methods Enzymol. 1985; 117: 414-459Crossref Scopus (189) Google Scholar). Curve-fitting analysis was performed as described previously (21Cosper N.J. Stålhandske C.M.V. Saari R.E. Hausinger R.P. Scott R.A. J. Biol. Inorg. Chem. 1999; 4: 122-129Crossref PubMed Scopus (25) Google Scholar). The γ-irradiation of purified SdhC (∼0.8 mm) containing glycerol (final concentration, ∼40% v/v) was performed in the XAS cuvets fully immersed in liquid nitrogen in a Dewar flask that was exposed to γ-rays in a 60Co source at the Tokyo Institute of Technology (Ookayama, Japan). The irradiation Dewar flask was refilled after every 90–120 min to maintain the samples at 77 K. Glycerol is an efficient electron hole-trapping agent (23Davydov R. Kuprin S. Gräslund A. Ehrenberg A. J. Am. Chem. Soc. 1994; 116: 11120-11128Crossref Scopus (92) Google Scholar) and is present in the irradiated samples to facilitate the solid-state cryoreduction of the FeS center at 77 K. EPR measurements were made on samples in XAS cuvets that had been exposed to a dose of ∼3 megarads and kept fully immersed in liquid nitrogen at 77 K when not in use. The resulting cryoreduced samples in the cuvets were carefully manipulated in liquid nitrogen throughout the experiments and directly transferred at 77 K to the JEOL liquid nitrogen Dewar/cavity for the EPR measurements performed with a JEOL JEX-RE3X spectrometer. The cognate soluble Rieske protein called sulredoxin (14Iwasaki T. Oshima T. Methods Enzymol. 2001; 334: 3-22Crossref PubMed Scopus (9) Google Scholar) was spontaneously γ-irradiated under the same conditions at 77 K as a positive control for EPR-monitored cryoreduction of FeS cluster at 77 K (24Iwasaki T. Kounosu A. Dikanov S.A. Kawamori A. Yamauchi J. Ohta H. EPR in the 21st Century. Elsevier Science Publishers B.V., Amsterdam2002: 488-493Crossref Google Scholar). Absorption spectra were recorded with a Hitachi U-3210 spectrophotometer or a Beckman DU-7400 spectrophotometer. CD spectra were recorded with a JASCO J720 spectropolarimeter with 0.5-cm cells. CW EPR measurements were performed by using a JEOL JES-FE3XG or JEOL JEX-RE3X spectrometer equipped with an ES-CT470 Heli-Tran cryostat system and a Scientific Instruments digital temperature indicator/controller Model 9650. The spectral data were processed using KaleidaGraph v.3.05 (Abelbeck Software) and IgorPro v.3.02 (Wave Metrics, Inc.). RR spectra were recorded at 77 K using a Spex 750M Raman spectrometer fitted with a Spectrum-One 2048 × 512 CCD camera and a Spectra-Physics 2017 Ar+ laser (output, 500 mW) by collecting 45° backscattering off the surface of a frozen sample in a glass cylindrical cell with sintered glass cap. The slit width of the spectrometer is 80 μm and the wavelength reproducibility is guaranteed by ±0.005 nm (±0.2 cm−1). Multiscan signal-averaging technique was employed to improve the signal-to-noise ratio. Protein was measured by the BCA assay (Pierce) with bovine serum albumin as a standard. The archaeal sdhABCD gene cluster of S. tokodaii strain 7 has been cloned and sequenced (13Iwasaki T. Aoshima M. Kounosu A. Oshima T. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins 1999. Agency for Scientific Publications, Berlin1999: 779-781Google Scholar) (Fig. 1). To characterize the properties of SdhC subunit in greater detail and to assign its possible function, we heterologously overproduced the sdhC gene product with a hexa-His tag at the N terminus in E. coliBL21-CodonPlus(DE3)-RIL strain. Although the isolation of the archaeal native respiratory complex II requires the presence of mild detergent for solubilization (11Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), the recombinant SdhC subunit was obtained as a water-soluble protein (data not shown). The purified soluble protein shows a brown color, indicating the presence of bound cofactor(s). Metal analysis by inductively coupled plasma atomic emission spectrometry suggested an iron:zinc ratio of 2.3:1.0 mol/mol in the purified recombinant SdhC. In accordance, the optical spectra of the air-oxidized protein shows absorption maxima at 279, 323, and 417 nm and broad shoulders at 460 and ∼570 nm (Fig.2 A, solid trace), which are reminiscent of the plant ferredoxin-type [2Fe-2S]2+ clusters. This FeS chromophore is stable under aerobic conditions at least for 2–3 days as judged by visible absorption spectroscopy but irreversibly degraded within a week, suggesting that it is much less stable than the FeS clusters in the cognate zinc-containing ferredoxin (25Iwasaki T. Watanabe E. Ohmori D. Imai T. Urushiyama A. Akiyama M. Hayashi-Iwasaki Y. Cosper N.J. Scott R.A. J. Biol. Chem. 2000; 275: 25391-25401Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Fig. 2 B shows the visible near UV CD spectra of the air-oxidized SdhC and the effect of dithionite reduction. The oxidized protein has three negative troughs at 355, 495, and ∼560 nm, a dominant positive peak at 411 nm, and a weak positive peak at around 620 nm (Fig. 2 B, solid trace). These features are similar to plant- or vertebrate-type [2Fe-2S]2+ clusters, but the positions of these dominant positive and negative features are markedly blue-shifted in SdhC, indicating some differences in the bondings and/or dipoles in the oxidized cluster environment. Upon either aerobic or anaerobic addition of dithionite, the brown color of purified protein gradually fades away due to the irreversible degradation of the FeS cluster, giving completely featureless visible absorption and CD spectra (Fig. 2, dashed traces). Addition of ferricyanide after dithionite treatment of SdhC rapidly caused aggregation and apoprotein precipitation (data not shown). The instability of the dithionite-treated cluster in SdhC makes determination of its midpoint redox potential impractical (data not shown). RR spectroscopy is a highly sensitive probe for the microenvironment of oxidized FeS clusters, and vibrational comparisons can be used to confirm cluster type and assess cluster ligation (26Han S. Czernuszewicz R.S. Spiro T.G. J. Am. Chem. Soc. 1989; 111: 3496-3504Crossref Scopus (77) Google Scholar, 27Han S. Czernuszewicz R.S. Kimura T. Adams M.W.W. Spiro T.G. J. Am. Chem. Soc. 1989; 111: 3505-3511Crossref Scopus (105) Google Scholar, 28Crouse B.R. Sellers V.M. Finnegan M.G. Dailey H.A. Johnson M.K. Biochemistry. 1996; 35: 16222-16229Crossref PubMed Scopus (69) Google Scholar). The 77 K RR spectra in the Fe–S stretching region are clearly indicative of a [2Fe-2S]2+ cluster in recombinant SdhC (Fig.3). The frequencies of the Agt mode at 324 cm−1 and the B3ut mode at 287 cm−1 have predominantly Fe–S(Cys) stretching character and can be interpreted in terms of complete cysteinyl ligation to the [2Fe-2S]2+cluster (26Han S. Czernuszewicz R.S. Spiro T.G. J. Am. Chem. Soc. 1989; 111: 3496-3504Crossref Scopus (77) Google Scholar, 27Han S. Czernuszewicz R.S. Kimura T. Adams M.W.W. Spiro T.G. J. Am. Chem. Soc. 1989; 111: 3505-3511Crossref Scopus (105) Google Scholar, 28Crouse B.R. Sellers V.M. Finnegan M.G. Dailey H.A. Johnson M.K. Biochemistry. 1996; 35: 16222-16229Crossref PubMed Scopus (69) Google Scholar). The [2Fe-2S]2+ cluster environment in SdhC is similar to that of a vertebrate-type ferredoxin with the lower frequency shifts of 2–7 cm−1 for all bands observed in the Fe–S stretching region, implying weakening of Fe–S bonds in SdhC. These shifts may reflect different compression effects of the polypeptide surroundings and hydrogen bonding to the S atoms from nearby donor groups in the protein, presumably ascribed to the unique cysteine motifs (Fig. 1 A). In conjunction with visible CD spectra in Fig. 2 B, we concluded that the FeS chromophore in recombinant SdhC has a [2Fe-2S]2+ core structure with complete cysteinyl ligation. We designated this novel center of the S. tokodaii SdhC as Center C to distinguish it from another [2Fe-2S] cluster (Center S-1 or [2Fe-2S] clu
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