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Spectroscopic Investigation of Selective Cluster Conversion of Archaeal Zinc-containing Ferredoxin fromSulfolobus sp. Strain 7

2000; Elsevier BV; Volume: 275; Issue: 33 Linguagem: Inglês

10.1074/jbc.m909243199

ISSN

1083-351X

Autores

Toshio Iwasaki, Eiji Watanabe, Daijiro Ohmori, Takeo Imai, Akio Urushiyama, Minoru Akiyama, Yoko Hayashi‐Iwasaki, Nathaniel J. Cosper, Robert A. Scott,

Tópico(s)

Photosynthetic Processes and Mechanisms

Resumo

Archaeal zinc-containing ferredoxin fromSulfolobus sp. strain 7 contains one [3Fe-4S] cluster (cluster I), one [4Fe-4S] cluster (cluster II), and one isolated zinc center. Oxidative degradation of this ferredoxin led to the formation of a stable intermediate with 1 zinc and ∼6 iron atoms. The metal centers of this intermediate were analyzed by electron paramagnetic resonance (EPR), low temperature resonance Raman, x-ray absorption, and1H NMR spectroscopies. The spectroscopic data suggest that (i) cluster II was selectively converted to a cubane [3Fe-4S]1+ cluster in the intermediate, without forming a stable radical species, and that (ii) the local metric environments of cluster I and the isolated zinc site did not change significantly in the intermediate. It is concluded that the initial step of oxidative degradation of the archaeal zinc-containing ferredoxin is selective conversion of cluster II, generating a novel intermediate containing two [3Fe-4S] clusters and an isolated zinc center. At this stage, significant structural rearrangement of the protein does not occur. We propose a new scheme for oxidative degradation of dicluster ferredoxins in which each cluster converts in a stepwise manner, prior to apoprotein formation, and discuss its structural and evolutionary implications. Archaeal zinc-containing ferredoxin fromSulfolobus sp. strain 7 contains one [3Fe-4S] cluster (cluster I), one [4Fe-4S] cluster (cluster II), and one isolated zinc center. Oxidative degradation of this ferredoxin led to the formation of a stable intermediate with 1 zinc and ∼6 iron atoms. The metal centers of this intermediate were analyzed by electron paramagnetic resonance (EPR), low temperature resonance Raman, x-ray absorption, and1H NMR spectroscopies. The spectroscopic data suggest that (i) cluster II was selectively converted to a cubane [3Fe-4S]1+ cluster in the intermediate, without forming a stable radical species, and that (ii) the local metric environments of cluster I and the isolated zinc site did not change significantly in the intermediate. It is concluded that the initial step of oxidative degradation of the archaeal zinc-containing ferredoxin is selective conversion of cluster II, generating a novel intermediate containing two [3Fe-4S] clusters and an isolated zinc center. At this stage, significant structural rearrangement of the protein does not occur. We propose a new scheme for oxidative degradation of dicluster ferredoxins in which each cluster converts in a stepwise manner, prior to apoprotein formation, and discuss its structural and evolutionary implications. Protein Data Bank extended x-ray absorption fine structure x-ray absorption spectroscopy watt(s) 3-(cyclohexylamino)propanesulfonic acid Ferredoxins, small iron-sulfur (FeS) proteins in archaea, serve as water-soluble electron acceptors of acyl-coenzyme A forming 2-oxoacid:ferredoxin oxidoreductase, a key enzyme involved in the central archaeal metabolic pathways (1Kerscher L. Oesterhelt D. FEBS Lett. 1977; 83: 197-201Crossref PubMed Scopus (36) Google Scholar, 2Kerscher L. Nowitzki S. Oesterhelt D. Eur. J. Biochem. 1982; 128: 223-230Crossref PubMed Scopus (68) Google Scholar, 3Kerscher L. Oesterhelt D. Trends Biochem. Sci. 1982; 7: 371-374Abstract Full Text PDF Scopus (117) Google Scholar, 4Adams M.W.W. FEMS Microbiol. Rev. 1994; 15: 261-277Crossref PubMed Scopus (90) Google Scholar, 5Iwasaki T. Wakagi T. Isogai Y. Tanaka K. Iizuka T. Oshima T. J. Biol. Chem. 1994; 269: 29444-29450Abstract Full Text PDF PubMed Google Scholar). The 2.0-Å resolution x-ray crystal structure (PDB1 entry 1XER ; Ref. 6Fujii T. Hata Y. Wakagi T. Tanaka N. Oshima T. Nat. Struct. Biol. 1996; 3: 834-837Crossref PubMed Scopus (53) Google Scholar) of ferredoxin from Sulfolobus sp. strain 7 (JCM 10545; optimal growth conditions, pH 2.5–3 and 80 °C) showed the presence of an unexpected isolated zinc center, tetrahedrally coordinated by three nitrogen atoms from histidine residues in the N-terminal extension region (Nδ1 of His16, Nε2 of His19, and Nδ1 of His34) and one Oδ1 atom from Asp76in the FeS cluster-binding core fold (Fig. 1). A similar zinc site was also found in ferredoxin from Thermoplasma acidophilumstrain HO-62 that also contained one [3Fe-4S]1+,0cluster, and one [4Fe-4S]2+,1+ cluster (7Iwasaki T. Suzuki T. Kon T. Imai T. Urushiyama A. Ohmori D. Oshima T. J. Biol. Chem. 1997; 272: 3453-3458Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). This site was analyzed by zinc K-edge x-ray absorption spectroscopy (XAS) (8Cosper N.J. Stålhandske C.M.V. Iwasaki H. Oshima T. Scott R.A. Iwasaki T. J. Biol. Chem. 1999; 274: 23160-23168Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) and was found to be essentially identical to the zinc site inSulfolobus sp. ferredoxin. Thus, these unusual ferredoxins contain both the conventional FeS clusters and a structurally conserved, isolated zinc center. This new class of bacterial type ferredoxin, isolated from phylogenetically diverse members of several aerobic and thermoacidophilic archaea, are thus called “zinc-containing ferredoxins” (2Kerscher L. Nowitzki S. Oesterhelt D. Eur. J. Biochem. 1982; 128: 223-230Crossref PubMed Scopus (68) Google Scholar, 6Fujii T. Hata Y. Wakagi T. Tanaka N. Oshima T. Nat. Struct. Biol. 1996; 3: 834-837Crossref PubMed Scopus (53) Google Scholar, 7Iwasaki T. Suzuki T. Kon T. Imai T. Urushiyama A. Ohmori D. Oshima T. J. Biol. Chem. 1997; 272: 3453-3458Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 8Cosper N.J. Stålhandske C.M.V. Iwasaki H. Oshima T. Scott R.A. Iwasaki T. J. Biol. Chem. 1999; 274: 23160-23168Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 9Fujii T. Hata Y. Oozeki M. Moriyama H. Wakagi T. Tanaka N. Oshima T. Biochemistry. 1997; 36: 1505-1513Crossref PubMed Scopus (42) Google Scholar, 10Iwasaki T. Oshima T. FEBS Lett. 1997; 417: 223-226Crossref PubMed Scopus (10) Google Scholar). Another unexpected result of the crystal structure of air-oxidizedSulfolobus sp. zinc-containing ferredoxin was the presence of two [3Fe-4S] clusters beside one isolated zinc center (9Fujii T. Hata Y. Oozeki M. Moriyama H. Wakagi T. Tanaka N. Oshima T. Biochemistry. 1997; 36: 1505-1513Crossref PubMed Scopus (42) Google Scholar) (Fig. 1). The number and type of FeS clusters in this structure are inconsistent with our previous spectroscopic analysis of the purified protein, which suggested one [3Fe-4S]1+,0 cluster (cluster I; E ½ = −280 mV) and one [4Fe-4S]2+,1+ cluster (cluster II;E ½ = −530 mV) (5Iwasaki T. Wakagi T. Isogai Y. Tanaka K. Iizuka T. Oshima T. J. Biol. Chem. 1994; 269: 29444-29450Abstract Full Text PDF PubMed Google Scholar). Other archaeal zinc-containing ferredoxins from T. acidophilum (7Iwasaki T. Suzuki T. Kon T. Imai T. Urushiyama A. Ohmori D. Oshima T. J. Biol. Chem. 1997; 272: 3453-3458Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 8Cosper N.J. Stålhandske C.M.V. Iwasaki H. Oshima T. Scott R.A. Iwasaki T. J. Biol. Chem. 1999; 274: 23160-23168Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar),Sulfolobus acidocaldarius (11Breton J.L. Duff J.L.C. Butt J.N. Armstrong F.A. George S.J. Pétillot Y. Forest E. Schäfer G. Thomson A.J. Eur. J. Biochem. 1995; 233: 937-946Crossref PubMed Scopus (54) Google Scholar), and Acidianus ambivalens (formerly Desulfurolobus ambivalens) (12Teixeira M. Batista R. Campos A.P. Gomes C. Mendes J. Pacheco I. Anemüller S. Hagen W.R. Eur. J. Biochem. 1995; 227: 322-327Crossref PubMed Scopus (64) Google Scholar) also contain one [3Fe-4S] cluster and one [4Fe-4S] cluster, rather than two [3Fe-4S] clusters. The primary structure ofSulfolobus sp. ferredoxin showed the presence of seven cysteines that are arranged in two FeS cluster-binding motifs, one from the sequence, Cys45-Leu-Ala-Asp48-Gly-Ser-Cys51and Cys93-Pro, and the other from the sequence, Cys55-Pro and Cys83-Ile-Phe-Cys86-Met-Ala-Cys89(5Iwasaki T. Wakagi T. Isogai Y. Tanaka K. Iizuka T. Oshima T. J. Biol. Chem. 1994; 269: 29444-29450Abstract Full Text PDF PubMed Google Scholar, 13Wakagi T. Fujii T. Oshima T. Biochem. Biophys. Res. Commun. 1996; 225: 489-493Crossref PubMed Scopus (16) Google Scholar). The latter motif provides a typical binding site for a [4Fe-4S] cluster with complete cysteinyl ligation, although Cys86 does not serve as a ligand to the [3Fe-4S] cluster II in the crystal structure (Fig. 1). Because FeS clusters have a remarkable facility for interconversion under protein-bound conditions (reviewed in Ref. 14Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1531) Google Scholar), the significant discrepancy of the types of FeS clusters of Sulfolobus sp. zinc-containing ferredoxin in the crystalline and as-isolated states may represent selective degradation of the cluster II to a [3Fe-4S] form in vitro. Although the [4Fe-4S] ↔ [3Fe-4S] cluster interconversion is well known in some bacterial type ferredoxins (14Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1531) Google Scholar, 15Moura J.J.G. Moura I. Kent T.A. Lipscomb J.D. 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Biol. 1994; 237: 437-451Crossref PubMed Scopus (75) Google Scholar), there is no conclusive report demonstrating the selective cluster conversion at the cluster II site in bacterial type dicluster ferredoxins. The most extensive spectroscopic analyses of oxidative degradation process in dicluster ferredoxins have been conducted with Azotobacter vinelandiiferredoxin I with one [3Fe-4S] cluster and one [4Fe-4S] cluster (30Morgan T.V. Stephens P.J. Devlin F. Stout C.D. Melis K.A. Burgess B.K. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1931-1935Crossref PubMed Scopus (37) Google Scholar, 31Morgan T.V. Stephens P.J. Devlin F. Burgess B.K. Stout C.D. FEBS Lett. 1985; 183: 206-210Crossref PubMed Scopus (17) Google Scholar, 32Stephens P.J. Morgan T.V. Devlin F. Penner-Hahn J.E. Hodgson K.O. Scott R.A. Stout C.D. Burgess B.K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5661-5665Crossref PubMed Scopus (27) Google Scholar, 33Iismaa S.E. Vázquez A.E. Jensen G.M. Stephens P.J. Butt J.N. Armstrong F.A. Burgess B.K. J. Biol. Chem. 1991; 266: 21563-21571Abstract Full Text PDF PubMed Google Scholar, 34Hu Z. Jollie D. Burgess B.K. Stephens P.J. Münck E. Biochemistry. 1994; 33: 14475-14485Crossref PubMed Scopus (37) Google Scholar, 35Scott R.A. Penner-Hahn J.E. Hodgson K.O. Beinert H. Stout C.D. Hodgson K.O. Hedman B. Penner-Hahn J.E. EXAFS and Near Edge Structure III: Proceedings of an International Conference, Stanford, CA, July 16–20, 1984. Springer-Verlag, Berlin1984: 105-110Google Scholar). In this instance, it has been proposed that ferricyanide oxidation results in complete destruction of the [4Fe-4S] cluster, to produce an intermediate containing only one [3Fe-4S] cluster (cluster I), en route to complete disruptions of both clusters and ultimately to unfolded protein (30Morgan T.V. Stephens P.J. Devlin F. Stout C.D. Melis K.A. Burgess B.K. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1931-1935Crossref PubMed Scopus (37) Google Scholar, 31Morgan T.V. Stephens P.J. Devlin F. Burgess B.K. Stout C.D. FEBS Lett. 1985; 183: 206-210Crossref PubMed Scopus (17) Google Scholar). We have recently found conditions where the native 7Fe form ofSulfolobus sp. ferredoxin (5Iwasaki T. Wakagi T. Isogai Y. Tanaka K. Iizuka T. Oshima T. J. Biol. Chem. 1994; 269: 29444-29450Abstract Full Text PDF PubMed Google Scholar) is slowly and irreversibly converted to a stable intermediate species under aerobic conditions (10Iwasaki T. Oshima T. FEBS Lett. 1997; 417: 223-226Crossref PubMed Scopus (10) Google Scholar). Herein, we report comparisons of EPR, resonance Raman, XAS, and1H nuclear magnetic resonance (NMR) analyses of the metal centers in the native 7Fe and the intermediate forms ofSulfolobus sp. zinc-containing ferredoxin. Our spectroscopic data demonstrate the oxidative degradation of cluster II to form a [3Fe-4S] cluster, yielding a 6Fe-containing intermediate of zinc-containing ferredoxin. This will be also discussed with respect to the structure and evolution of a ferredoxin core fold module. DEAE-Sepharose Fast Flow and Sephadex G-50 gels were purchased from Amersham Pharmacia Biotech, and NMR-grade D2O was from Wako Pure Chemicals (Tokyo, Japan). Water was purified by the Milli-Q purification system (Millipore). Other chemicals used in this study were of analytical grade. Sulfolobus sp. strain 7 cells (JCM 10545), originally isolated from Beppu Hot Springs, Japan, were cultivated aerobically and chemoheterotrophically at pH 2.5–3 and 75–80 °C, and the 7Fe form of the archaeal ferredoxin was routinely purified as described previously (5Iwasaki T. Wakagi T. Isogai Y. Tanaka K. Iizuka T. Oshima T. J. Biol. Chem. 1994; 269: 29444-29450Abstract Full Text PDF PubMed Google Scholar, 10Iwasaki T. Oshima T. FEBS Lett. 1997; 417: 223-226Crossref PubMed Scopus (10) Google Scholar). The intermediate form of Sulfolobus sp. ferredoxin was obtained by artificial conversion at pH 5.0 as described previously (10Iwasaki T. Oshima T. FEBS Lett. 1997; 417: 223-226Crossref PubMed Scopus (10) Google Scholar), after removal of the unconverted 7Fe form by a DEAE-Sepharose Fast Flow column chromatography (Amersham Pharmacia Biotech) followed by a Sephadex G-50 column chromatography (Amersham Pharmacia Biotech). 2-Oxoacid:ferredoxin oxidoreductase ofSulfolobus sp. strain 7 was purified as described previously (5Iwasaki T. Wakagi T. Isogai Y. Tanaka K. Iizuka T. Oshima T. J. Biol. Chem. 1994; 269: 29444-29450Abstract Full Text PDF PubMed Google Scholar, 36Zhang Q. Iwasaki T. Wakagi T. Oshima T. J. Biochem. (Tokyo). 1996; 120: 587-599Crossref PubMed Scopus (80) Google Scholar). 2-Oxoacid:ferredoxin oxidoreductase activity was monitored with a horse heart cytochrome c reduction assay at 50 °C using purified ferredoxin an intermediate electron acceptor (2Kerscher L. Nowitzki S. Oesterhelt D. Eur. J. Biochem. 1982; 128: 223-230Crossref PubMed Scopus (68) Google Scholar, 5Iwasaki T. Wakagi T. Isogai Y. Tanaka K. Iizuka T. Oshima T. J. Biol. Chem. 1994; 269: 29444-29450Abstract Full Text PDF PubMed Google Scholar). Enzymatic reduction of Sulfolobus sp. ferredoxin with the cognate 2-oxoacid:ferredoxin oxidoreductase was conducted under anaerobic conditions at 55 °C as described previously (5Iwasaki T. Wakagi T. Isogai Y. Tanaka K. Iizuka T. Oshima T. J. Biol. Chem. 1994; 269: 29444-29450Abstract Full Text PDF PubMed Google Scholar). Absorption spectra were recorded using a Hitachi U3210 spectrophotometer or a Beckman DU-7400 spectrophotometer. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of purified apoferredoxin (made in distilled water) was performed by a Finnigan MAT VISION 2000 instrument at an accelerating potential of 5.0 kV, using a 2,5-dihydroxybenzoic acid matrix. Electron paramagnetic resonance (EPR) measurements were performed using a JEOL JES-FE3XG spectrometer equipped with an Air Products model LTR-3-110 Heli-Tran cryostat system and a Scientific Instruments series 5500 temperature indicator/controller. Spin concentrations were estimated by double integration, with 0.1 and 1 mm Cu-EDTA as standards. The spectral data were processed using KaleidaGraph version 3.05 (Abelbeck Software). Low temperature resonance Raman spectra were recorded at 77 K using 488.0-nm and 457.9-nm Ar+ laser excitation (500 mW) as described previously (7Iwasaki T. Suzuki T. Kon T. Imai T. Urushiyama A. Ohmori D. Oshima T. J. Biol. Chem. 1997; 272: 3453-3458Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 37Imai T. Urushiyama A. Saito H. Sakamoto Y. Ota K. Ohmori D. FEBS Lett. 1995; 368: 23-26Crossref PubMed Scopus (8) Google Scholar). The sample was immersed into a liquid nitrogen reservoir and the scattered light was collected at 45 degrees to the incident beam. The spectral slit width was 4 cm−1, and a multiscan averaging technique was employed. Purified zinc-containing ferredoxins in 20 mm potassium phosphate buffer, pH 6.8, were concentrated by pressure filtration with an Amicon YM-3 membrane. Further concentration was achieved by placing the samples under a stream of dry nitrogen gas. The resultant samples (∼2–3 mm), containing 30% (v/v) glycerol, were frozen in a 24 × 3 × 2-mm polycarbonate cuvette with a Mylar-tape front window for XAS studies. XAS data were collected at Stanford Synchrotron Radiation Laboratory with the SPEAR storage ring operating in a dedicated mode at 3.0 GeV (Table I), as reported previously (8Cosper N.J. Stålhandske C.M.V. Iwasaki H. Oshima T. Scott R.A. Iwasaki T. J. Biol. Chem. 1999; 274: 23160-23168Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). EXAFS analysis was performed with the EXAFSPAK software according to standard procedures (38Scott R.A. Methods Enzymol. 1985; 117: 414-459Crossref Scopus (188) Google Scholar). Curve-fitting analysis was performed as described previously (8Cosper N.J. Stålhandske C.M.V. Iwasaki H. Oshima T. Scott R.A. Iwasaki T. J. Biol. Chem. 1999; 274: 23160-23168Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 39Cosper 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). Multiple scattering models, calculated using FEFF version 7.02 (40Zabinsky S.I. Rehr J.J. Ankudinov A. Albers R.C. Eller M.J. Phys. Rev. B. 1995; 52: 2995Crossref PubMed Scopus (2698) Google Scholar), were based on bis(acetato)-bis(imidazole)-zinc(II) (41Harrocks W.D. Holmquist J.N.I.B. Thompson J.S. J. Inorg. Biochem. 1980; 12: 131Crossref PubMed Scopus (54) Google Scholar) or tetra(imidazole) zinc(II) perchlorate (42Bear C.A. Duggan K.A. Freeman H.C. Acta Crystallogr. Sect. B Struct. Sci. 1975; 31: 2713Crossref Google Scholar).Table IX-ray absorption spectroscopic data collection for iron and zinc analysisIron EXAFSZinc EXAFSSR facilitySSRLSSRLBeamline7–37–3Current in storage ring80–100 mA50–60 mAMonochromator crystalSi[220]Si[220]Detection methodFluorescenceFluorescenceDetector typeSolid-state array1-aThe 13-element Ge solid-state x-ray fluorescence detector at Stanford Synchrotron Radiation Laboratory (SSRL) is provided by the NIH Biotechnology Research Resource.Solid-state array1-aThe 13-element Ge solid-state x-ray fluorescence detector at Stanford Synchrotron Radiation Laboratory (SSRL) is provided by the NIH Biotechnology Research Resource.Scan length, min2825Scans in average1610Temperature (K)1010Energy standardIron foil, first inflectionZinc foil, first inflectionEnergy calibration (eV)7111.39660.7E 0 (eV)71209670Pre-edge background Energy range (eV)6789–70758657–9625 Gaussian center (eV)64038638 Width (eV)750750Spline background Energy range (eV)7120–7354 (4)9333–9902 (4) (Polynomial order)7354–7589 (4)9902–10134 (4)7589–7822 (4)10134–10366 (4)1-a The 13-element Ge solid-state x-ray fluorescence detector at Stanford Synchrotron Radiation Laboratory (SSRL) is provided by the NIH Biotechnology Research Resource. Open table in a new tab 1H NMR spectra were recorded on using JEOL GSX-400 spectrometer operating at 399.78 MHz Larmor frequency. The NMR sample was prepared by exchanging the buffer of purified ferredoxin into 10 mm sodium deuterium phosphate buffer, pH 7.5 (pH was uncorrected), using an Amicon ultrafiltration device with a YM-3 membrane (final concentration, ∼3 mm ferredoxin). Sweep widths of 30 KHz were used for the purified protein. 1H spectra were recorded with either a slow repetition time (2 s) using presaturation for water suppression or with a fast repetition time (100 ms) using a super water elimination Fourier transform (super-WEFT) pulse sequence (43Inubushi T. Becker E.D. J. Magn. Reson. 1983; 51: 128-133Google Scholar) with a relaxation delay (60 ms) between the π and π/2 pulse sequence for water suppression. The spectra were calibrated by referencing to the residual HDO signal, assigned to a 4.74-ppm shift at 303 K. The standard JEOL software package was used for data processing. Protein concentration of purified ferredoxins was measured as described previously (10Iwasaki T. Oshima T. FEBS Lett. 1997; 417: 223-226Crossref PubMed Scopus (10) Google Scholar). Metal content analyses were carried out by inductively coupled plasma atomic emission spectrometry with a Seiko SPS 1500 VR instrument at Tokyo Institute of Technology and a Jobin-Yvon JY 38S instrument at Rigaku Ltd. The typical yield of the purified intermediate obtained from the 7Fe form was ∼30% under the conditions described under “Experimental Procedures.” The matrix-assisted laser desorption ionization-time of flight mass spectrometry suggested that masses [M + H]1+ of these apoproteins are identical within the experimental error (data not shown). Chemical analysis by inductively coupled plasma atomic emission spectrometry suggested the iron:zinc ratio of 6.9:1.0 mol/mol in the native 7Fe form and 5.6:1.0 mol/mol in the intermediate form. The 7Fe form of Sulfolobussp. ferredoxin elicited a sharp g = 2.02 EPR signal (0.9–1.0 spin/mol) attributed to a [3Fe-4S]1+ cluster as reported previously (5Iwasaki T. Wakagi T. Isogai Y. Tanaka K. Iizuka T. Oshima T. J. Biol. Chem. 1994; 269: 29444-29450Abstract Full Text PDF PubMed Google Scholar, 8Cosper N.J. Stålhandske C.M.V. Iwasaki H. Oshima T. Scott R.A. Iwasaki T. J. Biol. Chem. 1999; 274: 23160-23168Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) (Fig. 2,trace A). The intermediate form also elicited a sharp EPR signal at g = 2.02, detectable up to 20 K, but with different lineshapes and relaxation behavior. The microwave power saturation behavior of the EPR signal at 8 K, of the intermediate form (P ½ of 36 mW assuming a single component; P ½ of 0.7 mW and 110 mW assuming two components; open squares in Fig.2 B), was also different from that of the native 7Fe form (P ½ of 60 mW; closed squares in Fig. 2 B). Under non-saturation conditions, the spin concentration of the g = 2.02 EPR signal of the intermediate was estimated to be ∼1.7 spin/mol, indicating the presence of approximately two S = 1/2 [3Fe-4S]1+ clusters. No EPR signals were detected at temperatures above 35 K, suggesting the absence of a stable radical species in the air-oxidized intermediate (cf. Refs.30Morgan T.V. Stephens P.J. Devlin F. Stout C.D. Melis K.A. Burgess B.K. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1931-1935Crossref PubMed Scopus (37) Google Scholar, 33Iismaa S.E. Vázquez A.E. Jensen G.M. Stephens P.J. Butt J.N. Armstrong F.A. Burgess B.K. J. Biol. Chem. 1991; 266: 21563-21571Abstract Full Text PDF PubMed Google Scholar, and 34Hu Z. Jollie D. Burgess B.K. Stephens P.J. Münck E. Biochemistry. 1994; 33: 14475-14485Crossref PubMed Scopus (37) Google Scholar). Upon reduction of the native 7Fe form, the sharp g = 2.02 EPR signal attributed to that of a [3Fe-4S]1+cluster was fully reduced, thus giving rise to a very broad low field resonance at g ∼ 12, which is characteristic of the reduced S = 2 [3Fe-4S]0 cluster (44Johnson M.K. Bennett D.E. Fee J.A. Sweeney W.V. Biochim. Biophys. Acta. 1987; 911: 81-94Crossref PubMed Scopus (42) Google Scholar) (Fig. 3, trace A). A rhombic EPR signal at g = 2.06, 1.94, and 1.90, attributed to the reduced S = 1/2 [4Fe-4S]1+ cluster, was detected. This signal had additional wings on the high and low field sides of the main EPR signal (g = 2.11 and 1.85), resulting from magnetic interactions with the reduced S = 2 [3Fe-4S]0 cluster. These EPR signals could be detected up to 30 K (data not shown), and no evidence for the presence of a high multiplicity S = 3/2 [4Fe-4S]1+ cluster was obtained (Fig. 3, trace A). Reduction of the intermediate under the same conditions resulted in the disappearance of most of the sharp g = 2.02 EPR signal attributed to the S = 1/2 [3Fe-4S]1+clusters, and the appearance of the broad low field resonance atg = 12 characteristic of the S = 2 [3Fe-4S]0 cluster, as the predominant species (Fig. 3,traces B, C, and D). Several weak and minor resonances, mainly consisting of the remainingS = 1/2 [3Fe-4S]1+ cluster atg = 2.02 and a very weak radical feature atg = 2 of unknown origin, were also reproducibly detected in the g ∼ 2 region (<0.1 spin/mol). All these minor species existed in a substoichiometric amount (<0.1 spin/mol), indicating minor heterogeneity in the dithionite-reduced intermediate form. Low temperature resonance Raman spectroscopy has been utilized as a probe for distinguishing between types of oxidized FeS clusters (16Conover R.C. Kowal A.T. Fu W. Park J.-B. Aono S. Adams M.W.W. Johnson M.K. J. Biol. Chem. 1990; 265: 8533-8541Abstract Full Text PDF PubMed Google Scholar, 45Johnson M.K. Czernuszewicz R.C. Spiro T.G. Fee J.A. Sweeney W.V. J. Am. Chem. Soc. 1983; 105: 6671-6678Crossref Scopus (69) Google Scholar, 46Czernuszewicz R.S. Macor K.A. Johnson M.K. Gewirth A. Spiro T.G. J. Am. Chem. Soc. 1987; 109: 7178-7187Crossref Scopus (102) Google Scholar, 47Spiro T.G. Czernuszewicz R.S. Methods Enzymol. 1995; 246: 416-460Crossref PubMed Scopus (75) Google Scholar). Hence, the properties of the air-oxidized FeS clusters of Sulfolobus sp. ferredoxin were investigated by resonance Raman spectroscopy (Fig.4, A and B). Based on extensive assignments by Spiro and co-workers (45Johnson M.K. Czernuszewicz R.C. Spiro T.G. Fee J.A. Sweeney W.V. J. Am. Chem. Soc. 1983; 105: 6671-6678Crossref Scopus (69) Google Scholar, 46Czernuszewicz R.S. Macor K.A. Johnson M.K. Gewirth A. Spiro T.G. J. Am. Chem. Soc. 1987; 109: 7178-7187Crossref Scopus (102) Google Scholar), it appears that the [3Fe-4S]1+ cluster of the native 7Fe form exhibits three primarily Fe-S bridging modes (260, 285, and 347 cm−1) and at least two Fe-S terminal modes (369 and 385 cm−1) (Fig. 4 B). A weak band at 335 cm−1 appears in the native 7Fe form and is assigned to the Fe-S bridging mode of a regular biological [4Fe-4S]2+ cluster with complete cysteinyl ligation (46Czernuszewicz R.S. Macor K.A. Johnson M.K. Gewirth A. Spiro T.G. J. Am. Chem. Soc. 1987; 109: 7178-7187Crossref Scopus (102) Google Scholar) (Fig. 4 A). In Pyrococcus furiosus4Fe ferredoxin and active aconitase, which contain a [4Fe-4S] cluster coordinated by one non-cysteinyl and three cysteinyl ligands (16Conover R.C. Kowal A.T. Fu W. Park J.-B. Aono S. Adams M.W.W. Johnson M.K. J. Biol. Chem. 1990; 265: 8533-8541Abstract Full Text PDF PubMed Google Scholar, 48Park J.-B. Fan C. Hoffman B.M. Adams M.W.W. J. Biol. Chem. 1991; 266: 19351-19356Abstract Full Text PDF PubMed Google Scholar,49Calzolai L. Gorst C.M. Zhao Z.-H. Teng Q. Adams M.W.W. La Mar G.N. Biochemistry. 1995; 34: 11373-11384Crossref PubMed Scopus (89) Google Scholar), the equivalent band was shifted to a higher frequency (16Conover R.C. Kowal A.T. Fu W. Park J.-B. Aono S. Adams M.W.W. Johnson M.K. J. Biol. Chem. 1990; 265: 8533-8541Abstract Full Text PDF PubMed Google Scholar). It has been reported that the resonance Raman spectra for biological [4Fe-4S]2+ clusters are normally much less intense than those for biological [3Fe-4S]1+ clusters(reviewed in Refs. 45Johnson M.K. Czernuszewicz R.C. Spiro T.G. Fee J.A. Sweeney W.V. J. Am. Chem. Soc. 1983; 105: 6671-6678Crossref Scopus (69) Google Scholar and 47Spiro T.G. Czernuszewicz R.S. Methods Enzymol. 1995; 246: 416-460Crossref PubMed Scopus (75) Google Scholar). Relative intensity of the Fe-S bridging mode of a [4Fe-4S]2+ cluster at 335-cm−1 band in the native 7Fe form ofSulfolobus sp. ferredoxin (Fig. 4 A) is very similar to that reported for Thermus thermophilus 7Fe ferredoxin (45Johnson M.K. Czernuszewicz R.C. Spiro T.G. Fee J.A. Sweeney W.V. J. Am. Chem. Soc. 1983; 105: 6671-6678Crossref Scopus (69) Google Scholar, 47Spiro T.G. Czernuszewicz R.S. Methods Enzymol. 1995; 246: 416-460Crossref PubMed Scopus (75) Google Scholar). A very weak Fe-S terminal mode at ∼249 cm−1 is normally seen for [4Fe-4S]2+ clusters; however, the signal in this region, for the 7Fe form of Sulfolobus sp. ferredoxi

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