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Engineering a Three-cysteine, One-histidine Ligand Environment into a New Hyperthermophilic Archaeal Rieske-type [2Fe-2S] Ferredoxin from Sulfolobus solfataricus

2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês

10.1074/jbc.m305923200

ISSN

1083-351X

Autores

Asako Kounosu, Zhongrui Li, Nathaniel J. Cosper, Jacob E. Shokes, Robert A. Scott, Takeo Imai, Akio Urushiyama, Toshio Iwasaki,

Tópico(s)

Porphyrin Metabolism and Disorders

Resumo

We heterologously overproduced a hyperthermostable archaeal low potential (Em = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys42 and Cys61) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His64 → Cys variant (H64C) than the His44 → Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly 15N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation. We heterologously overproduced a hyperthermostable archaeal low potential (Em = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys42 and Cys61) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His64 → Cys variant (H64C) than the His44 → Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly 15N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation. Proteins containing Rieske-type [2Fe-2S] clusters are wide-spread in nature from hyperthermophilic Archaea and Bacteria to Eukarya and play critical electron transfer roles in various pathways such as aerobic respiration, photosynthesis, and biodegradation of various alkene and aromatic compounds (1Mason J.R. Cammack R. Annu. Rev. Microbiol. 1992; 46: 277-305Crossref PubMed Scopus (376) Google Scholar, 2Trumpower B.L. Gennis R.B. Annu. Rev. Biochem. 1994; 63: 675-716Crossref PubMed Scopus (471) Google Scholar, 3Link T.A. Adv. Inorg. Chem. 1999; 47: 83-157Crossref Scopus (108) Google Scholar, 4Berry E.A. Guergova-Kuras M. Huang L.-S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (395) Google Scholar). In contrast to regular plant- and vertebrate-type ferredoxins having complete cysteinyl ligations, the Rieske-type cluster has an asymmetric iron-sulfur core with the Sγ atom of each of the two cysteine residues coordinated to one iron site and the Nδ atom of each of the two histidine residues coordinated to the other iron site. This asymmetric ligation results in some unique redox and spectroscopic properties (for reviews, see Refs. 1Mason J.R. Cammack R. Annu. Rev. Microbiol. 1992; 46: 277-305Crossref PubMed Scopus (376) Google Scholar and 3Link T.A. Adv. Inorg. Chem. 1999; 47: 83-157Crossref Scopus (108) Google Scholar, 4Berry E.A. Guergova-Kuras M. Huang L.-S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (395) Google Scholar, 5Brugna M. Nitschke W. Asso M. Guigliarelli B. Lemesle-Meunier D. Schmidt C. J. Biol. Chem. 1999; 274: 16766-16772Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). This cluster coordination was firmly established by recent x-ray crystal structures of several different Rieske-type protein domains (6Iwata S. Saynovits M. Link T.A. Michel H. Structure. 1996; 4: 567-579Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 7Carrell C.J. Zhang H. Cramer W.A. Smith J.L. Structure. 1997; 5: 1613-1625Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 8Kauppi B. Lee K. Carredano E. Parales R.E. Gibson D.T. Eklund H. Ramaswamy S. Structure. 1998; 6: 571-586Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 9Colbert C.L. Couture M.M. Eltis L.D. Bolin J. Struct. Fold Des. 2000; 8: 1267-1278Abstract Full Text Full Text PDF Scopus (101) Google Scholar, 10Bönisch H. Schmidt C.L. Schäfer G. Ladenstein R. J. Mol. Biol. 2002; 319: 791-805Crossref PubMed Scopus (60) Google Scholar, 11Hunsicker-Wang L.M. Heine A. Chen Y. Luna E.P. Todaro T. Zhang Y.M. Williams P.A. McRee D.E. Hirst J. Stout C.D. Fee J.A. Biochemistry. 2003; 42: 7303-7317Crossref PubMed Scopus (84) Google Scholar). Two different types of Rieske clusters are observed in proteins. One type displays higher reduction potentials (Em) 1The abbreviations used are: Em, midpoint redox potential; ARF, archaeal Rieske-type ferredoxin; EXAFS, extended x-ray absorption fine structure; RR, resonance Raman; XAS, x-ray absorption spectroscopy; ORF, open reading frame. 1The abbreviations used are: Em, midpoint redox potential; ARF, archaeal Rieske-type ferredoxin; EXAFS, extended x-ray absorption fine structure; RR, resonance Raman; XAS, x-ray absorption spectroscopy; ORF, open reading frame. of approximately +150 to +490 mV and occurs in proton-translocating respiratory complexes (cytochrome bc1/b6f complexes and their archaeal homologs without c-type cytochromes), being involved in not only electron transfer but also substrate binding and oxidation at the quinol-oxidizing Qo site (2Trumpower B.L. Gennis R.B. Annu. Rev. Biochem. 1994; 63: 675-716Crossref PubMed Scopus (471) Google Scholar, 3Link T.A. Adv. Inorg. Chem. 1999; 47: 83-157Crossref Scopus (108) Google Scholar, 4Berry E.A. Guergova-Kuras M. Huang L.-S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (395) Google Scholar, 5Brugna M. Nitschke W. Asso M. Guigliarelli B. Lemesle-Meunier D. Schmidt C. J. Biol. Chem. 1999; 274: 16766-16772Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12Iwasaki T. Matsuura K. Oshima T. J. Biol. Chem. 1995; 270: 30881-30892Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 13Zhang Z. Huang L.-S. Shulmeister V.M. Chi Y.-I. Kim K.K. Hung L.-W. Crofts A.R. Berry E.A. Kim S.-H. Nature. 1998; 392: 677-684Crossref PubMed Scopus (932) Google Scholar, 14Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Crossref PubMed Scopus (1062) Google Scholar, 15Samoilova R.I. Kolling D. Uzawa T. Iwasaki T. Crofts A.R. Dikanov S.A. J. Biol. Chem. 2002; 277: 4605-4608Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The other type displays lower Em values of approximately -150 to -50 mV and has been found in a diverse group of bacterial multicomponent terminal oxygenases and soluble Rieske-type ferredoxins (1Mason J.R. Cammack R. Annu. Rev. Microbiol. 1992; 46: 277-305Crossref PubMed Scopus (376) Google Scholar, 3Link T.A. Adv. Inorg. Chem. 1999; 47: 83-157Crossref Scopus (108) Google Scholar, 8Kauppi B. Lee K. Carredano E. Parales R.E. Gibson D.T. Eklund H. Ramaswamy S. Structure. 1998; 6: 571-586Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 9Colbert C.L. Couture M.M. Eltis L.D. Bolin J. Struct. Fold Des. 2000; 8: 1267-1278Abstract Full Text Full Text PDF Scopus (101) Google Scholar, 16Cline J.F. Hoffman B.M. Mims W.B. LaHaie E. Ballou D.P. Fee J.A. J. Biol. Chem. 1985; 260: 3251-3254Abstract Full Text PDF PubMed Google Scholar, 17Kuila D. Fee J.A. Schoonover J.R. Woodruff W.H. Batie C.J. Ballou D.P. J. Am. Chem. Soc. 1987; 109: 1559-1561Crossref Scopus (42) Google Scholar, 18Gurbiel R.J. Batie C.J. Sivaraja M. True A.E. Fee J.A. Hoffman B.M. Ballou D.P. Biochemistry. 1989; 28: 4861-4871Crossref PubMed Scopus (183) Google Scholar, 19Gurbiel R.J. Ohnishi T. Robertson D.E. Daldal F. Hoffman B.M. Biochemistry. 1991; 30: 11579-11584Crossref PubMed Scopus (86) Google Scholar, 20Kuila D. Schoonover J.R. Dyer R.B. Batie C.J. Ballou D.P. Fee J.A. Woodruff W.H. Biochim. Biophys. Acta. 1992; 1140: 175-183Crossref PubMed Scopus (68) Google Scholar, 21Shergill J.K. Joannou C.L. Mason J.R. Cammack R. Biochemistry. 1995; 34: 16533-16542Crossref PubMed Scopus (37) Google Scholar, 22Dikanov S.A. Xun L. Karpiel A.B. Tyryshkin A.M. Bowman M.K. J. Am. Chem. Soc. 1996; 118: 8408-8416Crossref Scopus (82) Google Scholar, 23Link T.A. Hatzfeld O.M. Unalkat P. Shergill J.K. Cammack R. Mason J.R. Biochemistry. 1996; 35: 7546-7552Crossref PubMed Scopus (58) Google Scholar, 24Holz R.C. Small F.J. Ensign S.A. Biochemistry. 1997; 36: 14690-14696Crossref PubMed Scopus (16) Google Scholar, 25Xia B. Pikus J.D. Xia W. McClay K. Steffan R.J. Chae Y.K. Westler W.M. Markley J.L. Fox B.G. Biochemistry. 1999; 38: 727-739Crossref PubMed Scopus (38) Google Scholar, 26Couture M.M. Colbert C.L. Babini E. Rosell F.I. Mauk A.G. Bolin J.T. Eltis L.D. Biochemistry. 2001; 40: 84-92Crossref PubMed Scopus (30) Google Scholar, 27Cosper N.J. Eby D.M. Kounosu A. Kurosawa N. Neidle E.L. Kurtz Jr., D.M. Iwasaki T. Scott R.A. Protein Sci. 2002; 11: 2969-2973Crossref PubMed Scopus (28) Google Scholar). However, none of the latter class has been characterized in detail from any archaeal species. We recently found that the genomic DNA sequence of the thermoacidophilic archaeon Sulfolobus solfataricus strain P-1 (DSM 1616T) encodes an archaeal homolog of bacterial small Rieske-type ferredoxins with no consensus disulfide signature (DDBJ accession number AB047031 (27Cosper N.J. Eby D.M. Kounosu A. Kurosawa N. Neidle E.L. Kurtz Jr., D.M. Iwasaki T. Scott R.A. Protein Sci. 2002; 11: 2969-2973Crossref PubMed Scopus (28) Google Scholar)). This arf gene was found by homology search against the deduced amino acid sequence of Sulfolobus tokodaii sulredoxin, a water-soluble homolog of a high potential Rieske protein (Em,low pH ∼ +190 mV) with a consensus disulfide linkage (DDBJ accession number AB023295) 2T. Iwasaki, A. Kounosu, H. Iwasaki, T. Imai, A. Urushiyama, Y. Hayashi-Iwasaki, I. Yoda, D. Ohmori, T. Oshima, N. J. Cosper, and R. A. Scott, manuscript in preparation. 2T. Iwasaki, A. Kounosu, H. Iwasaki, T. Imai, A. Urushiyama, Y. Hayashi-Iwasaki, I. Yoda, D. Ohmori, T. Oshima, N. J. Cosper, and R. A. Scott, manuscript in preparation. (28Iwasaki T. Isogai T. Iizuka T. Oshima T. J. Bacteriol. 1995; 177: 2576-2582Crossref PubMed Google Scholar, 29Iwasaki T. Imai T. Urushiyama A. Oshima T. J. Biol. Chem. 1996; 271: 27659-27663Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 30Iwasaki T. Kounosu A. Dikanov S.A. Kawamori A. Yamauchi J. Ohta H. EPR in the 21st Century. Elsevier Science B. V., Amsterdam2002: 488-493Crossref Google Scholar) (Fig. 1). Subsequent cloning and heterologous overexpression in Escherichia coli of this S. solfataricus arf gene encoding the archaeal Rieske-type ferredoxin (ARF) (27Cosper N.J. Eby D.M. Kounosu A. Kurosawa N. Neidle E.L. Kurtz Jr., D.M. Iwasaki T. Scott R.A. Protein Sci. 2002; 11: 2969-2973Crossref PubMed Scopus (28) Google Scholar) have provided an opportunity to define the influence of surrounding amino acid residues on the electronic and structural properties of this archaeal Rieske-type [2Fe-2S] cluster in greater detail by combination of site-directed mutagenesis and multidisciplinary physicochemical analyses using EPR, resonance Raman (RR), and x-ray absorption (XAS) spectroscopies. As an initial step, we report herein the overexpression and detailed RR and XAS characterization of the archaeal low potential Rieske-type [2Fe-2S] cluster in the thermostable wild-type ARF and its ligand-substituted variant, which was hampered in previous studies with mesophilic cytochrome bc1-associated, high potential Rieske proteins due to protein instability (31Davidson E. Ohnishi T. Atta-asafo-Adjei E. Daldal F. Biochemistry. 1992; 31: 3342-3351Crossref PubMed Scopus (109) Google Scholar, 32Van Doren S.R. Gennis R.B. Barquera B. Crofts A.R. Biochemistry. 1993; 32: 8083-8091Crossref PubMed Scopus (35) Google Scholar). E. coli strain DH5α and strain HB101 (TaKaRa) used for cloning were grown in LB or terrific broth medium with 50 μg/ml ampicillin when required. Plasmids pGEMT and pGEM3Zf(+) (Promega) were used for cloning and sequencing. The expression vectors pET28a and pTrc99A were purchased from Novagen and Amersham Biosciences, respectively. DNA was manipulated by standard procedures (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Water was purified by a Milli-Q purification system (Millipore). Sulredoxin was purified from the soluble fraction of S. tokodaii strain 7 (formerly Sulfolobus sp. strain 7; JCM 10545T (34Suzuki T. Iwasaki T. Uzawa T. Hara K. Nemoto N. Kon T. Ueki T. Yamagishi A. Oshima T. Extremophiles. 2002; 6: 39-44Crossref PubMed Scopus (111) Google Scholar)) as described previously (28Iwasaki T. Isogai T. Iizuka T. Oshima T. J. Bacteriol. 1995; 177: 2576-2582Crossref PubMed Google Scholar, 35Iwasaki T. Oshima T. Methods Enzymol. 2001; 334: 3-22Crossref PubMed Scopus (9) Google Scholar). Other chemicals mentioned in this study were of analytical grade. The arf gene coding for the hypothetical ARF (ORF c06009; DDBJ accession number AB047031 (27Cosper N.J. Eby D.M. Kounosu A. Kurosawa N. Neidle E.L. Kurtz Jr., D.M. Iwasaki T. Scott R.A. Protein Sci. 2002; 11: 2969-2973Crossref PubMed Scopus (28) Google Scholar) of S. solfataricus strain P-1 (DSM 1616T) was cloned and sequenced as follows. The PCR was carried out to obtain a partial genomic fragment encoding the arf gene using S. solfataricus strain P-1 genomic DNA and the following oligonucleotide primers (designed based on the genomic DNA sequence data available for S. solfataricus strain P-2 (36She Q. Singh R.K. Confalonieri F. Zivanovic Y. Allard G. Awayez M.J. Chan-Weiher C.C. Clausen I.G. Curtis B.A. De Moors A. Erauso G. Fletcher C. Gordon P.M. Heikamp-de Jong I. Jeffries A.C. Kozera C.J. Medina N. Peng X. Thi-Ngoc H.P. Redder P. Schenk M.E. Theriault C. Tolstrup N. Charlebois R.L. Doolittle W.F. Duguet M. Gaasterland T. Garrett R.A. Ragan M.A. Sensen C.W. Van der Oost J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7835-7840Crossref PubMed Scopus (669) Google Scholar)): P1-3 primer, 5′-CCC CCA TAT GCT AGT CAG AG-3′; and P1-4 primer, 5′-CCC CGG ATC CTT AAA TTT GTA T-3′. The determined nucleotide sequence (DDBJ accession number AB047031) completely matched the gene sequence reported for the hypothetical ORF c06009 of S. solfataricus strain P-2 (36She Q. Singh R.K. Confalonieri F. Zivanovic Y. Allard G. Awayez M.J. Chan-Weiher C.C. Clausen I.G. Curtis B.A. De Moors A. Erauso G. Fletcher C. Gordon P.M. Heikamp-de Jong I. Jeffries A.C. Kozera C.J. Medina N. Peng X. Thi-Ngoc H.P. Redder P. Schenk M.E. Theriault C. Tolstrup N. Charlebois R.L. Doolittle W.F. Duguet M. Gaasterland T. Garrett R.A. Ragan M.A. Sensen C.W. Van der Oost J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7835-7840Crossref PubMed Scopus (669) Google Scholar). This gene is located shortly after another hypothetical ORF c06008 (188 amino acids) of unknown function, although it is apparently not a part of multicomponent oxygenase gene cluster (36She Q. Singh R.K. Confalonieri F. Zivanovic Y. Allard G. Awayez M.J. Chan-Weiher C.C. Clausen I.G. Curtis B.A. De Moors A. Erauso G. Fletcher C. Gordon P.M. Heikamp-de Jong I. Jeffries A.C. Kozera C.J. Medina N. Peng X. Thi-Ngoc H.P. Redder P. Schenk M.E. Theriault C. Tolstrup N. Charlebois R.L. Doolittle W.F. Duguet M. Gaasterland T. Garrett R.A. Ragan M.A. Sensen C.W. Van der Oost J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7835-7840Crossref PubMed Scopus (669) Google Scholar). The nucleotide sequence of these flanking regions were also confirmed for both strands with new sets of PCR primers designed for amplification of the whole arf-coding region and the flanking sequences (DDBJ accession number AB047031). The PCR product with expected length of 327 bp as amplified above was subcloned into an NdeI/BamHI site in a pET28a vector (Novagen), and the nucleotide sequence was determined with vector-specific T7 promoter and T7 terminator. The resultant expression vector was named pET28aARF. It contains the pET28a vector-derived leader nucleotide sequence to facilitate efficient transcription of archaeal genes in E. coli so as to produce a recombinant protein with the N-terminal hexahistidine tag for rapid protein purification. The hexahistidine tag is attached away from the cluster-binding subdomain of ARF and apparently does not disturb its redox site as judged by x-ray crystal structures of several Rieske and Rieske-type protein domains (6Iwata S. Saynovits M. Link T.A. Michel H. Structure. 1996; 4: 567-579Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 9Colbert C.L. Couture M.M. Eltis L.D. Bolin J. Struct. Fold Des. 2000; 8: 1267-1278Abstract Full Text Full Text PDF Scopus (101) Google Scholar). 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 DNASIS Version 3.6 software (Hitachi Software Engineering Co., Ltd.). The homology search against data bases was performed with the BEAUTY and BLAST network service (37Worley K.C. Wiese B.A. Smith R.F. Genome Res. 1995; 5: 173-184Crossref PubMed Scopus (228) Google Scholar). The structural alignments were constructed using Swiss-Pdb Viewer Version 3.05. 3See www.expasy.ch/spdbv/mainpage.htm. The multiple sequence alignments were performed using a ClustalX graphical interface (38Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35414) Google Scholar) with minor manual adjustments. Site-directed mutagenesis was performed by PCR mutagenesis technique using a pET28aARF vector as a long template and Ex (Expand Long Template PCR system) Taq DNA polymerase (Roche Applied Science) with new sets of the following PCR primers: for replacement of Cys42 by serine (C42S), 5′-GCT ATA GAA GCA TAT AGT CCT CAT AAG GGA AGG-3′ and 5′-CCT TCC CTT ATG AGG ACT ATA TGC TTC TAT AGC-3′; for replacement of Cys61 by serine (C61S), 5′-GAG GGA TAT AAA ATA AGG AGC GAT TTA CAC GG-3′ and 5′-CCG TGT AAA TCG CTC CTT ATT TTA TAT CCC TC-3′; for replacement of His44 by cysteine (H44C), 5′-GCA TAT TGT CCT TGT AAG GGA AGG AAT CTG G-3′ and 5′-CCA GAT TCC TTC CCT TAC AAG GAC AAT ATG C-3′; and for replacement of His64 by cysteine (H64C), 5′-GGT GCG ATT TAT GCG GAT ATG AAT ATA GTC TTG AAA ACG GTG-3′ and 5′-CAC CGT TTT CAA GAC TAT ATT CAT ATC CGC ATA AAT CGC ACC-3′. Each amplified PCR product was individually excised from an agarose gel, purified by a Quantum Prep Freeze ′N′ Squeeze spin column (Bio-Rad) and QIAquick PCR purification kit (Qiagen), ligated, and transformed into E. coli HB101 competent cells. The nucleotide sequences of the resultant vectors were confirmed for both strands. The pET28aARF vectors harboring the hypothetical arf gene and its site-directed variants of S. solfataricus strain P-1 were transformed separately into the host strain, E. coli BL21-CodonPlus(DE3)-RIL strain (Stratagene). The transformants were grown overnight at 25 °C in LB medium containing 50 μg/ml kanamycin, and the recombinant holoprotein was overproduced by induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 24 h at 25 °C in the presence of 100 μm FeCl3. The cells were pelleted by centrifugation and stored at -80 °C until use. The pET28aARF vector used for heterologous overexpression of the natural abundance, wild-type ARF and its variants in E. coli BL21-CodonPlus(DE3)-RIL strain did not produce recombinant holoprotein in significant amounts in the M9 minimal salt medium supplemented with 4–6 g/liter glucose, lactose, or glycerol, 1 g/liter 14NH4Cl, 2 mm MgSO4, 0.1 mm CaCl2, 100 μm FeCl3, and 50 μg/ml ampicillin (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Therefore, the pET28aARF vector was digested with NcoI and BamHI, and the resulting DNA fragment carrying the hexahistidine-tagged arf gene was ligated into an NcoI/BamHI site in a pTrc99A vector (Amersham Biosciences). The nucleotide sequence of the resultant vector, pTrc99AARF, was confirmed for both strands. The pTrc99AARF vector was transformed into the host strain, E. coli BL21-CodonPlus(DE3)-RIL strain (Stratagene), and the transformants were grown for 16 h at 30 °C in modified M9 salt medium supplemented with 4.5 g/liter glucose, 1 g/liter 15NH4Cl (Aldrich) (in place of 14NH4Cl), 2 mm MgSO4, 0.1 mm CaCl2, 100 μm FeCl3, 100 μm FeSO4, 50 μg/ml ampicillin, and vitamin mixture (final concentrations, 5 mg/ml of thiamine and 1 mg/ml each of biotin, choline hydrogen tartrate, folic acid, niacinamide, d-pantothenate, and pyridoxal). The recombinant 15N-labeled holoprotein was overproduced by induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 24 h at 30 °C. The cells were pelleted by centrifugation and stored at -80 °C until use. Purification of each recombinant holoprotein having a hexahistidine tag at the N terminus was performed as described previously for the S. tokodaii recombinant SdhC iron-sulfur protein (39Iwasaki T. Kounosu A. Aoshima M. Ohmori D. Imai T. Urushiyama A. Cosper N.J. Scott R.A. J. Biol. Chem. 2002; 277: 39642-39648Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) except that the heat treatment step (at 65 °C for 20–30 min) was omitted for three variants, C42S, C61S, and H44C. When required, the recombinant holoprotein was further purified by Sephadex G-75 gel filtration column chromatography (Amersham Biosciences). The purified recombinant holoprotein was stored at either 4 °C or -80 °C until use. Absorption spectra were recorded with a Hitachi U3210 spectrophotometer or a Beckman DU-7400 spectrophotometer equipped with a thermoelectric cell holder. Visible-near UV CD spectra were recorded with a JASCO J720 spectropolarimeter with 0.5-cm cells. EPR measurements were performed by using a JEOL JEX-RE3X spectrometer equipped with an ES-CT470 Heli-Tran cryostat system and a Scientific Instruments digital temperature indicator/controller Model 9650. Spin concentrations of purified recombinant proteins were estimated by double integration with Cu-EDTA (0.1 mm) and S. tokodaii sulredoxin (0.05 mm) (28Iwasaki T. Isogai T. Iizuka T. Oshima T. J. Bacteriol. 1995; 177: 2576-2582Crossref PubMed Google Scholar) as standards. RR spectra were recorded at 77 K using a Spex 750M Raman spectrometer fitted with a Spectrum-One 2048 × 512 charge-coupled device camera and a Spectra-Physics 2017 Ar+ laser (output, 500 milliwatts) by collecting 45° backscattering off the surface of a frozen sample in a glass cylindrical cell with sintered glass cap (39Iwasaki T. Kounosu A. Aoshima M. Ohmori D. Imai T. Urushiyama A. Cosper N.J. Scott R.A. J. Biol. Chem. 2002; 277: 39642-39648Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The slit width of the spectrometer was 80 μm, and a multiscan signal-averaging technique was used to improve the signal-to-noise ratio. The spectral data were processed using KaleidaGraph Version 3.05 (Abelbeck Software). Purified recombinant proteins were 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 cuvettes with a Mylar tape front window for XAS studies (27Cosper N.J. Eby D.M. Kounosu A. Kurosawa N. Neidle E.L. Kurtz Jr., D.M. Iwasaki T. Scott R.A. Protein Sci. 2002; 11: 2969-2973Crossref PubMed Scopus (28) Google Scholar). X-ray absorption spectra at the iron K-edge were recorded at 10 K at Stanford Synchrotron Radiation Laboratory, beamline 7-3, with the SPEAR storage ring operating at 3.0 GeV and 60–100 mA. A Si[220] double crystal monochromator (with one crystal detuned to 50% reflected intensity for harmonic rejection) and an energy-resolving 30-element germanium solid state array detector (provided by the National Institutes of Health Biotechnology Research Resource) were used for data collection. Energies were calibrated using an internal iron foil standard, assigning the first inflection point to 7111.3 eV. All other data collection parameters were as reported previously (40Iwasaki 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, 41Cosper N.J. Scott R.A. Hori H. Nishino T. Iwasaki T. J. Biochem. 2001; 130: 191-198Crossref PubMed Scopus (5) Google Scholar). The intensities and energies for the 1s → 3d pre-edge features of the iron K-edge XAS data were quantified using the program EDG_FIT. All spectra were fit over the range 7100–7165 eV. Pseudo-Voigt line shapes of a fixed 1:1 ratio of Lorentzian to Gaussian contribution were used to model pre-edge features and successfully reproduced the spectra. Functions modeling the background contributions to the pre-edge features were chosen empirically to give the best fit and included pseudo-Voigt functions that mimicked shoulders on the rising edge. For all complexes, a fit was considered acceptable only if it successfully reproduced the data and the second derivative of the data. The pre-edge peak area after the background subtraction was obtained by integrating over a range of 10 eV. EXAFS data analysis was performed with the EXAFSPAK software. 4See www-ssrl.slac.stanford.edu/exafspak.html. Calibration and background removal were performed according to established procedures (40Iwasaki 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, 42Scott R.A. Methods Enzymol. 1985; 117: 414-459Crossref Scopus (189) Google Scholar). Theoretical amplitude and phase shift functions were calculated using the ab initio code FEFF 8.2 (43Ankudinov A.L. Ravel B. Rehr J.J. Conradson S.D. Phys. Rev. B Condens. Matter. 1998; 58: 7565-7576Crossref Scopus (4080) Google Scholar). A scale factor of 0.9 was used to fit experimental EXAFS amplitudes with those calculated by FEFF 8.2. The potentiometric titration of the purified recombinant proteins was performed under oxygen-free argon (obtained by passing the argon gas (commercial high grade product) through an Oxyout apparatus (Osaka Sanso, Osaka, Japan)) at 25 °C and at pH 7.0 in a specially designed Thunberg-type quartz cuvette (sample volume, 2–3 ml) in the presence of the redox mediators (2,6-dichlorophenolindophenol (E0′ = +217 mV), thionine (+60 mV), methylene blue (+11 mV), indigo tetrasulfonate (-46 mV), indigo disulfonate (-116 mV), cresyl violet (-166 mV), phenosafranin (-252 mV), and benzyl viologen (-359 mV)) and 1 m NaCl essentially as described previously (29Iwasaki T. Imai T. Urushiyama A. Oshima T. J. Biol. Chem. 1996; 271: 27659-27663Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) using a platinum wire plus miniaturized Ag/AgCl/KCl (3 m) redox microelectrode (BAS Inc., Tokyo, Japan). The xanthine/xanthine oxidoreductase-mediated reduction method was performed in a specially designed Thunberg-type quartz cuvette (sample volume, 0.5 ml) under oxygen-free argon (obtained by passing the argon gas (commercial high grade product) through an Oxyout apparatus (Osaka Sanso)) at 25 °C in appropriate buffer containing 1 m NaCl as described by Massey (44Massey V. Curti B. Ronchi S. Zanetti G. Flavins and Flavoproteins 1990. Walter de Gruyter & Co., Berlin1991: 59-66Crossref Google Scholar). The latter method is based on the rapid equilibration of catalytically generated reducing equivalents between the redox center of a protein and a suitable redox dye of known potential (typically ±30 mV from that of unknown) in the presence of a low concentration of benzyl viologen or methyl viologen (∼2 μm). The reaction was started by adding a catalytic amount of milk xanthine oxidoreductase anaerobically to a Thunberg-type cell (sample volume, 0.5 ml), and the visible absorbance spectra were collected during the reduction process (in general 2–4 h). The validity of this method, as well as its application to analyze redox properties of various flavoproteins and synthetic flavin analogs, has been reported (e.g. see Refs. 44Massey V. Curti B. Ronchi S. Zanetti G. Flavins and Flavoproteins 1990. Walter de Gruyter & Co., Berlin1991: 59-66Crossref Google Scholar, 45Gassner G.T. Ballou D.P. Biochemistry. 1995; 34: 13460-13471Crossref PubMed Scopus (31) Google Scholar, 46Fraaije M.W. van den Heuve R.H.H. van Berkel W.J.H. Mattevi A. J. Biol. Chem. 1999; 274: 35514-35520Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and was also confirmed for the wild-type ARF under anaerobic conditions (by reductive titrations starting from oxidized protein with dithionite and oxidative titrations starting from dithionite-reduced protein by injecting small aliquots of air in a stepwise manner (as described in Ref. 47Beharry Z.M. Eby D.M. Coulter E.D. Viswanathan R. Neidl

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