担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

The electronic structure and geometry of redox-active metal cofactors in proteins are tuned by the pattern of hydrogen bonding with the backbone peptide amino acid labeling of a hyperthermophilic archaeal metalloprotein with engineered Escherichia coli auxotroph strains, and we applied this to resolve the hydrogen bond interactions with the reduced Rieske-type [2Fe-2S] cluster by two-dimensional pulsed electron spin resonance technique. Because deep electron spin-echo envelope modulation of two histidine N-14(delta) ligands of the cluster decreased non-coordinating N-15 signal intensities via the cross-suppression effect, an inverse labeling strategy was employed in which N-14 amino acid-labeled archaeal Rieske-type ferredoxin samples were examined in an N-15-protein background. This has directly identified Lys45 N-a as providing the major pathway for the transfer of unpaired electron spin density from the reduced cluster by a "through-bond" mechanism. All other backbone peptide nitrogens interact more weakly with the reduced cluster. The extension of this approach will allow visualizing the three-dimensional landscape of preferred pathways for the transfer of unpaired spin density from a paramagnetic metal center onto the protein frame, and will discriminate specific interactions by a "through-bond" mechanism from interactions which are "through-space" in various metalloproteins.

DOI： 10.1021/ja308049u

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studies by revealing structural and functional information unattainable from a crystallographic approach. However, efficient labeling of proteins with selected amino acids necessitates auxotrophic hosts, which are often not available. We have constructed a set of auxotrophs in a commonly used Escherichia coli expression strain C43(DE3), a derivative of E. coli BL21 (DE3), which can be used for isotopic labeling of individual amino acids or sets of amino acids. These strains have general applicability to either soluble or membrane proteins that can be expressed in E. coli. We present examples in which proteins are selectively labeled with C-13- and N-15-amino acids and studied using magic-angle spinning solid-state NMR and pulsed EPR, demonstrating the utility of these strains for biophysical characterization of membrane proteins, radical-generating enzymes and metalloproteins. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ymeth.2011.08.019

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

The crystal structure of the ISC-like [2Fe-2S] ferredoxin (FdxB), probably involved in the de novo iron-sulfur cluster biosynthesis (ISC) system of Pseudomonas putida JCM 20004, was determined at 1.90- resolution and displayed a novel tail-to-tail dimeric form. P. putida FdxB lacks the consensus free cysteine usually present near the cluster of ISC-like ferredoxins, indicating its primarily electron transfer role in the iron-sulfur cluster. Orientation-selective electron-nuclear double resonance spectroscopic analysis of reduced FdxB in conjunction with the crystal structure has identified the innermost Fe2 site with a high positive spin population as the nonreducible iron retaining the Fe3+ valence and the outermost Fe1 site as the reduced iron with a low negative spin density. The average g (max) direction is skewed, forming an angle of about 27.3A degrees (+/- 4A degrees) with the normal of the [2Fe-2S] plane, whereas the g (int) and g (min) directions are distributed in the cluster plane, presumably tilted by the same angle with respect to this plane. These results are related to those for other [2Fe-2S] proteins in different electron transport chains (e.g. adrenodoxin) and suggest a significant distortion of the electronic structure of the reduced [2Fe-2S] cluster under the influence of the protein environment around each iron site in general.

DOI： 10.1007/s00775-011-0793-8

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

CW EPR spectra of reduced [2Fe-2S](Cys)(3)(His)(1) clusters of mammalian mitoNEET soluble domain appear to produce features resulting from the interaction of the electron spins of the two adjacent clusters, which can be explained by employing the local spin model. This model favors the reduction of the outermost iron with His87 and Cys83 ligands, which is supported by orientation-selected hyperfine sublevel correlation (HYSCORE) characterization of the uniformly N-15-labeled mitoNEET showing one strongly coupled nitrogen from the His87 N-delta ligand with hyperfine coupling (15)a = 8 MHz. The N-14 and N-15 HYSCORE spectra also exhibit at least two different cross-peaks located near diagonal in the (++) quadrant, with frequencies similar to 2.8 and 2.4 MHz (N2), and the other similar to 4.0 and 3.5 MHz (N1), but did not show any of the larger splitting similar to 1.1-1.4 MHz previously seen with Rieske proteins. Further analysis with partially N-15(3)-His-labeled protein indicates that His87 N-epsilon cross-peaks produce resolved features (N2) in the N-14 spectrum but contribute much less than weakly coupled peptide nitrogen species to the (++) quadrant in the N-15 spectrum. It is suggested that these quantitative data may be used in future functional and theoretical studies on the mammalian mitoNEET [2Fe-2S] cluster system.

DOI： 10.1021/ja903228w

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe2S] cluster in the hyperthermophilic archaeal Rieske-type ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe3+/2+ center at the engineered local metal-binding site ( Zn2+ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/ insertion/ assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds.

DOI： 10.1074/jbc.M414051200

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

DOI： 10.1021/ja031976p

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We heterologously overproduced a hyperthermostable archaeal low potential (E-m = - 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 (Cys(42) and Cys(61)) 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 His(64) --> Cys variant (H64C) than the His(44) --> 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 wildtype, natural abundance and uniformly N-15-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.

DOI： 10.1074/jbc.M305923200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER-VERLAG TOKYO  

The taxonomic position of a thermoacidophilic crenarchaeote SulfolobuS Sp. strain 7, previously isolated from the Beppu Hot Springs in the geothermal area of Kyushu Island, Japan, was investigated by cloning and sequencing, by phylogenetic analysis of the 16S rRNA gene sequence, by DNA-DNA homology with similar species, and by biochemical characterization of the isolate. This isolate is an obligate aerobe and grows optimally at 80degreesC and pH 2.5-3 under aerobic and chemoheterotrophic growth conditions by aerobic respiration rather than simple fermentation. In conjunction with the phenotypic properties, the present phylogenetic analysis based on the 16S rRNA gene sequence and DNA-DNA hybridization experiments indicate that this isolate is related to the described Sulfolobus taxon and should be considered a novel species of the genus. We propose that this isolate is a novel species of the genus Sulfolobus that we name Sulfolobus tokodaii sp. nov. The type strain is strain 7 (JCM 10545).

DOI： 10.1007/s007920100221

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N-delta atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N-delta involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant.

DOI： 10.1074/jbc.C100664200

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Archaeal zinc-containing ferredoxin from Sulfolobus 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 similar to 6 iron atoms. The metal centers of this intermediate were analyzed by electron paramagnetic resonance (EPR), low temperature resonance Raman, x-ray absorption, and H-1 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.

DOI： 10.1074/jbc.M909243199

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Japanese Biochemical Society  

The sequence motif-specific assignment of the two distinct [2Fe-2S] clusters in rat xanthine oxidoreductase (XOR) was unequivocally established by site-directed mutagenesis of recombinant enzymes expressed in a baculovirus-insect cell system and electron paramagnetic resonance (EPR) spectroscopy. The conserved cysteine residues, including Cys-115, in the unusual C-terminal-Cys-Xaa2-Cys-//-Cys-Xaa1-Cys- motif serve as ligands to the Fe/S I center, which is probably located in close proximity to the Mo- pterin center. Other conserved cysteine residues, including Cys-43 and Cys- 51, in the N-terminal plant ferredoxin-like motif serve as ligands to the Fe/S II center, which is distantly located from the Mo-pterin center. The present sequence motif-specific assignment of the Fe/S I and II centers is discussed in the light of the structural features of XOR.

DOI： 10.1093/oxfordjournals.jbchem.a022669

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press  

The purified 2-oxoacid:ferredoxin oxidoreductase of a thermoacidophilic and aerobic crenarchaeote, Sulfolobus sp. strain 7, consists of 70-kDa α and 37-kDa β subunits, and contains one thiamine pyrophosphate (TPP), one [4Fe-4S]2+,1+ cluster, and two magnesium atoms per αβ structure. It exhibits a broad substrate specificity toward 2-oxoacids such as 2-oxoglutarate, 2-oxobutyrate, and pyruvate. The gene encoding the archaeal oxidoreductase was cloned, and the two open reading frames encoding the α (632 amino acids) and β subunits (305 amino acids), respectively, were sequenced. Careful sequence alignment revealed several consensus motifs of this enzyme family, as well as possible cofactor binding residues of the Sulfolobus enzyme. This new structural information also indicates that (i) several genetic fusions and reorganization of the early, possibly αβγδ-type enzyme similar to those from hyperthermophiles have taken place during evolution of the 2-oxoacid:ferredoxin (flavodoxin) oxidoreductase superfamily, which might have occurred in different ways in early aerobic archaea and early anaerobic bacteria, and that (ii) enzymes with different subunit compositions should have an essentially similar catalytic mechanism with one TPP and at least one [4Fe-4S] cluster as the minimal set of redox centers.

DOI： 10.1093/oxfordjournals.jbchem.a021454

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

An active respiratory complex II (succinate:quinone oxidoreductase) has been purified from tetraether lipid membranes of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, It consists of four different subunits with apparent molecular masses of 66, 37, 33, and 12 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 66-kDa subunit contains a covalently bound flavin, the 37-kDa subunit is a possible iron-sulfur protein carrying three distinct types of EPR-visible FeS cluster, and the 33- and 12-kDa subunits are putative membrane-anchor subunits, respectively. While no heme group is detected in the purified complex II, it catalyzes succinate dependent reduction of ubiquinone-1 and 2,6-dichlorophenolindophenol in the absence of phenazine methosulfate. The respiratory complex II of Sulfolobus sp, strain 7 appears to be novel in that it functions as a true succinate:caldariellaquinone oxidoreductase, although inherently lacking any heme group. This further indicates that the heme group of several respiratory complexes II may not be involved in the redox intermediates of the electron transfer from succinate to quinone.

DOI： 10.1074/jbc.270.52.30902

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The terminal segment of the aerobic respiratory chain of the thermoacidophilic archaeon Sulfolobus sp. strain 7 is an unusual caldariellaquinol oxidase supercomplex, which contains at least one b-type and three spectroscopically distinguishable a-type cytochromes, one copper, and a Rieske-type FeS center. In this paper, we report the purification and characterization of two different forms of the archaeal a-type cytochromes, namely, a three-subunit cytochrome a(583)-aa(3) subcomplex and a single-subunit cytochrome aa(3) derived from the cytochrome subcomplex, in order to facilitate fur ther studies on the terminal oxidase segment of Sulfolobus. The optical and EPR spectroscopic analyses suggest the presence of two different low-spin heme centers and one high-spin heme center in the purified cytochrome a(583)-aa(3) subcomplex, and one low-spin and one high-spin hemes in cytochrome aa(3), respectively. The Rieske-type FeS center detected in the purified cytochrome supercomplex was absent in two forms of the a-type cytochrome oxidase, indicating its association with cytochrome b(562). The crystal field parameters of the low-spin heme a(583) center indicate that its axial ligands may be similar to those of cytochromes c, rather than conventional bis-histidine ligation, In spite of the absence of any c-type cytochrome, a ferrocytochrome c oxidase activity was detected in the archaeal purified cytochrome a(583)-aa(3) subcomplex with no quinol oxidase activity, but not in the purified cytochrome oxidase supercomplex, which has been tentatively interpreted as a representative of electron transfer from the Rieske FeS center to cytochrome a(583) in vivo. Thus, our results indicate the following scheme for the intramolecular electron transfer of the terminal oxidase supercomplex from Sulfolobus sp. strain 7: [caldariellaquinol -->] b(562) --> Rieske FeS center --> a(583) aa(3) --> molecular oxygen.

DOI： 10.1074/jbc.270.52.30893

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The aerobic respiratory system of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, is unusual in that it consists of only a- and b-type cytochromes but no c-type cytochromes. In previous studies, a novel cytochrome oxidase a(583)-aa(3) subcomplex has been purified, which showed a ferrocytochrome c oxidase but no caldariellaquinol oxidase activity (Wakagi, T., Yamauchi, T., Oshima, T., Muller, M., Azzi, A., and Sone, N. (1989) Biochem. Biophys. Res. Commun, 165, 1110-1114), We show here that the cytochrome subcomplex could be copurified with a non CO-reactive cytochrome b(562) as a novel terminal oxidase ''supercomplex,'' which also contained a Rieske-type FeS cluster at g(y) = 1.89. It contained one copper and all four heme centers detectable in the archaeal membranes by the low temperature spectrophotometry and the potentiometric titration: cytochromes b(562) (+146 mV), a(583) (+270 mV), and aa(3) (+117 and +325 mV). The presence of one copper atom indicates that it contains the conventional heme a(3)-Cu-B binuclear center for reducing molecular oxygen. In conjunction with the presence of a Rieske-type FeS center, inhibitor studies suggest that the terminal oxidase segment of the respiratory chain of Sulfolobus sp, strain 7 is a functional fusion of respiratory complexes III and IV, where cytochrome b(562) and the Rieske-type FeS center probably play a central role in the oxidation of caldariellaquinol. This archaeal terminal oxidase supercomplex reconstitutes the in vitro succinate oxidase respiratory chain for the first time together with caldariellaquinone and the purified cognate succinate:caldariellaquinone oxidoreductase complex. The reconstitution system requires caldariellaquinone for the activity, and is highly sensitive to cyanide and 2-heptyl-4-hydroxy-quinoline-N-oxide, These results are also discussed in terms of the evolutionary considerations.

DOI： 10.1074/jbc.270.52.30881

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The dicluster-type ferredoxin is a key electron carrier in the cytoplasm of the aerobic and thermoacidophilic archaeon, Sulfolobus sp. strain 7, and contains 1 aspartate and 7 cysteine residues as possible ligands to two FeS clusters. The optical, electron paramagnetic resonance (EPR), and cyclic voltammetric studies suggest the presence of one each of [3Fe-4S](1+,0) (-280 mV) and [4Fe-4S](2+,1+) (-530 mV) clusters in the purified Sulfolobus ferredoxin, and the lower potential [4Fe-4S] center was scarcely reducible by excess dithionite even at pH 9. While the Sulfolobus ferredoxin has been known to function as an electron acceptor of 2-oxoacid:ferredoxin oxidoreductase (Kerscher, L., Nowitzki, S., and Oesterhelt, D. (1982) fur. J. Biochem. 128, 223-230), it is not known whether one or both of two clusters is reduced during the steady-state turnover of the enzyme. Here we show by combinations of the optical and EPR spectroscopies that only the higher potential [3Fe-4S] cluster is reduced at the physiological pH during the steady-state turnover of the purified 2-oxoacid:ferredoxin oxidoreductase at 50 degrees C. The functional significance and evolutionary implications of the [3Fe-4S] center in dicluster-type ferredoxins are discussed.

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担当区分：最終著者,　責任著者  

DOI： 10.1021/acs.biochem.8b00438

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

The hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe-2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 angstrom resolution and belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = 60.72, c = 83.31 angstrom. The asymmetric unit contains one protein molecule.

DOI： 10.1107/S1744309110019263

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担当区分：筆頭著者,　責任著者   記述言語：英語   出版者・発行元：HINDAWI LTD  

The general importance of the Fe-S cluster prosthetic groups in biology is primarily attributable to specific features of iron and sulfur chemistry, and the assembly and interplay of the Fe-S cluster core with the surrounding protein is the key to in-depth understanding of the underlying mechanisms. In the aerobic and thermoacidophilic archaea, zinc-containing ferredoxin is abundant in the cytoplasm, functioning as a key electron carrier, and many Fe-S enzymes are produced to participate in the central metabolic and energetic pathways. De novo formation of intracellular Fe-S clusters does not occur spontaneously but most likely requires the operation of a SufBCD complex of the SUF machinery, which is the only Fe-S cluster biosynthesis system conserved in these archaea. In this paper, a brief introduction to the buildup and maintenance of the intracellular Fe-S world in aerobic and hyperthermoacidophilic crenarchaeotes, mainly Sulfolobus, is given in the biochemical, genetic, and evolutionary context.

DOI： 10.1155/2010/842639

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Two-dimensional electron spin-echo envelope modulation (ESEEM) analysis of the uniformly N-15-labeled archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 has been conducted in comparison with the previously characterized high-potential protein homologs. Major differences among these proteins were found in the hyperfine sublevel correlation (HYSCORE) lineshapes and intensities of the signals in the (++) quadrant, which are contributed from weakly coupled (non-coordinated) peptide nitrogens near the reduced clusters. They are less pronounced in the HYSCORE spectra of ARF than those of the high-potential protein homologs, and may account for the tuning of Rieske-type clusters in various redox systems. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.febslet.2009.09.050

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

MitoNEET (a mammalian mitochondrial outer membrane protein) is a potential pharmacological and clinical target of the insulin-sensitizer pioglitazone. The thermophilic homologue of mitoNEET (TTHA0026) from Thermus thermophilus HB8 has been heterologously overproduced in Escherichia coli and purified as a water-soluble prototypal protein containing the mitoNEET-like [2Fe-2S] cluster. The resultant recombinant protein, named Tth-NEET0026, has been crystallized in its oxidized form by the hanging-drop vapour-diffusion method using 17%(w/v) polyethylene glycol 4000, 8.5%(v/v) 2-propanol, 15%(v/v) glycerol and 0.085 M HEPES-NaOH pH 7.2. The dark reddish crystals diffracted to 1.80 angstrom resolution and belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = 45.51, c = 84.26 angstrom. The asymmetric unit contains one protein molecule.

DOI： 10.1107/S1744309108035975

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL PUBLISHING  

The iron-sulfur (Fe-S) cluster-biosynthesis (ISC) system of the gamma-proteobacterium Pseudomonas putida JCM 20004 contains a constitutively expressed vertebrate-type [2Fe-2S] ferredoxin, FdxB, which lacks the conserved free cysteine residue near the Fe-S cluster site that has been proposed to function in the catalysis of biological Fe-S cluster assembly in other bacterial homologues. Recombinant FdxB was heterologously overproduced in Escherichia coli, purified and crystallized in its oxidized form by the hanging-drop vapour-diffusion and streak-seeding methods using 1.6 M trisodium citrate dihydrate pH 6.5. The thin needle-shaped crystals diffract to 1.90 angstrom resolution and belong to the hexagonal space group P6(1)22, with unit-cell parameters a = 87.58, c = 73.14 angstrom. The asymmetric unit contains one protein molecule.

DOI： 10.1107/S1744309107045757

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL PUBLISHING  

In place of the Rieske [2Fe-2S] cluster, an archetypal mononuclear iron site has rationally been designed into a hyperthermophilic archaeal Rieske [2Fe-2S] protein (sulredoxin) from Sulfolobus tokodaii by three residue replacements with reference to the Pyrococcus furiosus rubredoxin sequence. The resulting sulredoxin variant, SDX-triple (H44I/A45C/H64C), has been purified and crystallized by the hanging-drop vapour-diffusion method using 65%(v/v) 2-methyl-2,4-pentanediol, 0.025 M citric acid and 0.075 M sodium acetate trihydrate pH 4.3. The crystals diffract to 1.63 angstrom resolution and belong to the triclinic space group P1, with unit-cell parameters a = 43.56, b = 76.54, c = 80.28 angstrom, alpha = 88.12, beta = 78.82, gamma = 73.46 degrees. The asymmetric unit contains eight protein molecules.

DOI： 10.1107/S1744309106034476

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

The rate of quinol oxidation by cytochrome bc(1)/b(6)f complex is in part associated with the redox potential (E-m) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (S-t) and Tyr-O eta (as with the Y156W variant, which has a modified histidine N-epsilon pK(a,ox)) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-S-b/t stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S-b) and Ser-O gamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S-b/t stretching vibrations.

DOI： 10.1110/ps.052035406

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

DOI： 10.1021/ja0562393

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

DOI： 10.1021/ja045898x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Proteins of the Rieske and Rieske-type family contain a [2Fe-2S] cluster with mixed ligation by two histidines and two cysteines, and play important roles in various biological electron transfer reactions. We report here the comparative orientation-selected ESEEM and HYSCORE studies of the reduced clusters from two hyperthermophilic Rieske-type proteins; a high-potential, archaeal Rieske protein called sulredoxin (SDX) from Sulfolobus tokodaii with weak homology to the cytochrome be-associated Rieske proteins, and a low-potential, archaeal homolog of an oxygenase-associated Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus. N-14 ESEEM and HYSCORE spectra of SDX and ARF show well-defined variations, which are primarily determined by changes of quadrupole couplings (up to 50% depending on the selected orientation) of the two coordinated nitrogens. These are due to variations in coordination geometry of the histidine imidazole ligands rather than to variations of hyperfine couplings of these nitrogens, which do not exceed 8-10%. The measured quadrupole couplings and their differences in the two proteins are consistent with those calculated using the reported crystal structures of high- and low-potential Rieske proteins. These results suggest that exploration of quadrupole tensors might provide a more accurate method for characterization of the histidine coordination in different proteins and mutants than hyperfine tensors, and might have potential applications in a wider range of biological systems.

DOI： 10.1007/s00775-004-0571-y

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL MUNKSGAARD  

The hyperthermophilic archaeal sulredoxin from Sulfolobus tokodaii is a water-soluble high-potential Rieske [2Fe - 2S] protein with unique pH-dependent redox properties compared with its mesophilic homologues in cytochrome bc(1)/b(6)f complexes. The oxidized recombinant sulredoxin has been crystallized by the hanging-drop vapour-diffusion method using 30%(v/v) polyethylene glycol 400, 0.1 M cadmium chloride and 0.1 M sodium acetate pH 4.6. The crystals diffracted to beyond 2.0 Angstrom resolution and belong to the cubic space group F4(1)32, with unit-cell parameter a = 163.00 +/- 0.05 Angstrom. The asymmetric unit contains one sulredoxin molecule. Three-wavelength MAD data were collected.

DOI： 10.1107/S0907444904014295

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

The biological [2Fe-2S] clusters play important roles in electron transfer and cellular signaling for a variety of organisms from archaea, bacteria to eukarya. The two recombinant hyperthermophilic archaeal [2Fe-2S] cluster-binding proteins, SdhC and the N-terminal domain fragment of SdhB, of Sulfolobus tokodaii respiratory complex II overproduced in Escherichia coli are thermostable as isolated, but moderately sensitive to reduction with excess dithionite. We used iron K-edge X-ray absorption spectroscopy to monitor the structural changes of their Fe sites in the irreversible [2Fe-2S] cluster degradation process. Regardless of the differences in the cluster-ligating cysteine motifs and the XAS-detectable [2Fe-2S](2+) cluster environments, a complete reductive breakdown of the [2Fe-2S] clusters resulted in the appearance of a new Fourier transform (FT) peak at similar to3.3 Angstrom with a concomitant loss of the Fe-Fe interaction at ca. 2.7 Angstrom for both proteins. On the basis of the unambiguous assignment of the 3.3 Angstrom FT peak, our results suggest that a biological [2Fe-2S] cluster breakdown under reducing conditions generally releases Fell from the polypeptide chain into the aqueous solution, and the Fe2+ might then be recruited as a secondary ferrous iron source for de novo biosynthesis and/or regulation of iron-binding enzymes in the cellular system.

DOI： 10.1021/bi035078h

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COLD SPRING HARBOR LAB PRESS  

Proteins containing Rieske-type [2Fe-2S] clusters play important roles in many biological electron transfer reactions. Typically, [2Fe-2S] clusters are not directly involved in the catalytic transformation of substrate, but rather supply electrons to the active site. We report herein X-ray absorption spectroscopic (XAS) data that directly demonstrate an average increase in the iron-histidine bond length of at least 0.1 Angstrom upon reduction of two distantly related Rieske-type clusters in archaeal Rieske ferredoxin from Sulfolobus solfataricus strain P-1 and bacterial anthranilate dioxygenases from Acinetobacter sp. strain ADP1. This localized redox-dependent structural change may fine tune the protein-protein interaction (in the case of ARF) or the interdomain interaction (in AntDO) to facilitate rapid electron transfer between a lower potential Rieske-type cluster and its redox partners, thereby regulating overall oxygenase reactions in the cells.

DOI： 10.1110/ps.0222402

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The SdhC subunit of the archaeal respiratory complex 11 (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 in Escherichia 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 We-M 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. tokodaii respiratory complex 11, 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.

DOI： 10.1074/jbc.207312200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Genes encoding the NarG and NarH subunits of the molybdo-iron-sulfur enzyme, a nitrate reductase from a denitrifying halophilic euryarchaeota Haloarcula marismortui, were cloned and sequenced. An incomplete cysteine motif reminiscent of that for a [4Fe-4S] cluster binding was found in the NarG subunit, and complete cysteine arrangements for binding one [3Fe-4S] cluster and three [4Fe-4S] clusters were found in the NarH subunit. In conjunction with chemical, electron paramagnetic resonance, and subcellular localization analyses, we firmly establish that the H. marismortui enzyme is a new archaeal member of the known membrane-bound nitrate reductases whose homologs are found in the bacterial domain. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0014-5793(02)02524-3

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担当区分：最終著者,　責任著者   記述言語：英語   出版者・発行元：The Japanese Biochemical Society  

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and nitric oxide through two stepwise oxygenation reactions involving Nω-hydroxy-L-arginine, an enzyme-bound intermediate. The Nω-hydroxy-L-arginine- and arginine-bound NOS ferriheme centers show distinct, high-spin electron paramagnetic resonance signals. Iron X-ray absorption spectroscopy (XAS) has been used to examine the structure of the ferriheme site in the Nω-hydroxy-L-arginine-bound full-length neuronal NOS in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin. Iron XAS shows that the high-spin ferriheme sites in the Nω-hydroxy-L-arginine- and arginine-bound forms are strikingly similar, both being coordinated by the heme and an axial thiolate ligand, with an Fe-S distance of ca. 2.29 Å. Cu2+inhibition slightly affects the spin-state equilibrium, but causes no XAS-detectable changes in the immediate ferriheme coordination environment of neuronal NOS. The structure and ligand geometry of the high-spin ferriheme in arginine-bound neuronal NOS are essentially identical to those of the Nω-hydroxy-L-arginine-bound form and only slightly affected by the divalent cation inhibitor of consitutive NOS. © 2001 Japanese Biochemical Society.

DOI： 10.1093/oxfordjournals.jbchem.a002972

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担当区分：筆頭著者,　責任著者   記述言語：英語   出版者・発行元：ACADEMIC PRESS INC  

DOI： 10.1016/S0076-6879(01)34453-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

A membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium. Solubilization and purification were done aerobically in the presence of Triton X-100. Three chromatography steps were done for purification; Butyl-Sepharose, Mono-Q, and Superose 6, in this order. Purification was completed with 6.73% yield of total activity and with 21.4-fold increase of specific activity when compared with the values for the membrane fraction. The purified hydrogenase was shown to be a tetramer with alpha(2)beta(2) structure, with a molecular mass of 60,000 Da for the large subunit and 38,000 Da for the small subunit. The purified hydrogenase directly reduced methionaquinone with an apparent fi, of around 300 mu M and with a turnover number around 2900 (min(-1)). Metal analysis and EPR properties of the hydrogenase have shown that the enzyme is one of the [NiFe]-hydrogenases. Also, optimum pH and temperature for reaction, thermal stability, and electron acceptor specificity were reported. Finally, a model is presented for energy and central metabolism of H. thermophilus strain TK-6.

DOI： 10.1271/bbb.64.492

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The zfx gene encoding a zinc-containing ferredoxin from Thermoplasma acidophilum strain HO-62 was cloned and sequenced. It is located upstream of two genes encoding an archaeal homolog of nascent polypeptide-associated complex alpha subunit and a tRNA nucleotidyltransferase. This gene organization is not conserved in several euryarchaeoteal genomes, The multiple sequence alignments of the zfx gene product suggest significant sequence similarity of the ferredoxin core fold to that of a low potential 8Fe-containing dicluster ferredoxin without a zinc center. The tightly bound zinc site of zinc-containing ferredoxins from two phylogenetically distantly related Archaea, T. acidophilum HO-62 and Sulfolobus sp. strain 7, was further investigated by x-ray absorption spectroscopy. The zinc K-edge x-ray absorption spectra of both archaeal ferredoxins are strikingly similar, demonstrating that the same zinc site is found in T. acidophilum ferredoxin as in Sulfolobus sp, ferredoxin, which suggests the structural conservation of isolated zinc binding sites among archaeal zinc-containing ferredoxins, The sequence and spectroscopic data provide the common structural features of the archaeal zinc-containing ferredoxin family.

DOI： 10.1074/jbc.274.33.23160

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Mouse neuronal nitric-oxide synthase 2 (nNOS2) is a unique natural variant of constitutive neuronal nitric-oxide synthase (nNOS) specifically expressed in the central nervous system having a 105-amino acid deletion in the heme-binding domain as a result of in-frame mutation by specific alternative splicing. The mouse nNOS2 cDNA gene was heterologously expressed in Escherichia coli, and the resultant product was characterized spectroscopically in detail. Purified recombinant nNOS2 contained heme but showed no L-arginine- and NADPH-dependent citrulline-forming activity in the presence of Ca2+-promoted calmodulin, elicited a sharp electron paramagnetic resonance (EPR) signal at g = 6.0 indicating the presence of a high spin ferriheme as isolated and showed a peak at around 420 nm in the CO difference spectrum, instead of a 443-nm peak detected with the recombinant wild-type nNOS1 enzyme. Thus, although the heme domain of nNOS2 is capable of binding heme, the heme coordination geometry is highly abnormal in that it probably has a proximal non-cysteine thiolate ligand both in the ferric and ferrous states. Moreover, negligible spectral perturbation of the nNOS2 ferriheme was detected upon addition of either L-arginine or imidazole. These provide a possible rational explanation for the inability of nNOS2 to catalyze the cytochrome P450-type monooxygenase reaction.

DOI： 10.1074/jbc.274.25.17559

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The role of two essential residues at the N-terminal hook region of neuronal nitric-oxide synthase (nNOS) in nitric-oxide synthase activity was investigated. Full-length mouse nNOS proteins containing single-point mutations of Thr-315 and Asp-314 to alanine were produced in the Escherichia coli and baculovirus-insect cell expression systems, The molecular properties of the mutant proteins were analyzed in detail by biochemical, optical, and electron paramagnetic resonance spectroscopic techniques and compared with those of the wildtype enzyme. Replacement of Asp-314 by Ala altered the geometry around the heme site and the substrate-binding pocket of the heme domain and abrogated the ability of nNOS to form catalytically active dimers, Replacement of Thr-315 by Ala reduced the protein stability and altered the geometry around the heme site, especially in the absence of bound (6R)-5,6,7,8-tetrahydro-L-biopterin cofactor. These results suggest that Asp-314 and Thr-315 both play critical structural roles in stabilizing the heme domain and subunit interactions in mouse nNOS.

DOI： 10.1074/jbc.274.12.7705

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Japanese Biochemical Society  

Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) were found to inhibit strongly the citrulline formation activity of neuronal nitric oxide synthase (nNOS
EC 1.14.13.39). Such inhibition was not observed with any other phospholipid examined. A kinetic analysis of purified nNOS showed no significant change in apparent K(m) for L-Arg or NADPH caused by these inhibitory phospholipids. Electron paramagnetic resonance analysis revealed no significant spectral perturbation of the ferriheme or flavin semiquinone upon the addition of PIP2. On the other hand, a lower enhancement of the NADPH diaphorase activity by Ca2+-calmodulin was observed in the presence of PIP2 and PA, and the citrulline formation activity was protected from phospholipid inhibition by preincubation with Ca2+-calmodulin. Moreover, trypsin digestion analysis showed that the cleavage site within the calmodulin-binding site of nNOS was specifically protected from trypsin by the addition of PIP2 and PA. These results strongly suggest that PIP2 and PA inhibit the citrulline formation activity of nNOS by blocking the interaction of the enzyme with Ca2+-calmodulin.

DOI： 10.1093/oxfordjournals.jbchem.a022523

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担当区分：筆頭著者,　責任著者   記述言語：英語  

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記述言語：英語   出版者・発行元：Agency for Scientific Publ.  

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Irreversible conversion of the purified zinc-containing 7Fe ferredoxin from the thermoacidophilic archaeon Salfolobus sp, strain 7 was found to occur under aerobic conditions at pH 5.0 and at 4 degrees C, This process accompanied a substantial increase of the electron paramagnetic resonance signal attributed to a [3Fe-4S](1+) cluster, and the converted form containing similar to 6 Fe/Zn (mol/mol) had a net charge different from that of the native 7Fe ferredoxin, These data provide evidence for the formation of a stable intermediate product of the archaeal ferredoxin having two [3Fe-4S] clusters and a zinc center by artificial oxidative degradation, This further explains the discrepancy that a zinc center and two [3Fe-4S] clusters (but not a zinc center and one [3Fe-4S] cluster plus one [4Fe-4S] cluster) are observed in the crystal structure at pH 5.0. (C) 1997 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(97)01286-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Neuronal nitric oxide synthase (nNOS; EC 1.14.13.39) activity in supernatant of rat cerebellum homogenate was unstable and chelating reagent protected the activity from the rapid decrease, The main target ion of the chelating reagent was found to be Ca2+. Although the enzyme was very unstable after purification by the procedures including DEAE-cellulose chromatography and ammonium sulfate precipitation, the inactivation was neither accelerated by addition of Ca2+ nor protected by EGTA. Upon addition of boiled supernatant of rat cerebellum homogenate, this purified enzyme became more active and stable, but rapid inactivation occurred again by addition of Ca2+, suggesting the existence of previously unreported Ca2+-dependent stabilizer/activator in the boiled supernatant. This factor was concentrated by organic solvent and the effects on the enzyme were completely canceled by addition of Ca2+ or phospholipase C treatment. (C) 1997 Academic Press.

DOI： 10.1006/bbrc.1997.6664

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The dicluster-type ferredoxins from the thermoacidophilic archaea such as Thermoplasma acidophilum and Sulfolobus sp. are known to contain an unusually long extension of unknown function in the N-terminal region. Recent x-ray structural analysis of the Sulfolobus ferredoxin has revealed the presence of a novel zinc center, which is coordinated by three histidine ligand residues in the N-terminal region and one aspartate in the ferredoxin core domain. We report here the quantitative metal analyses together with electron paramagnetic resonance and resonance Raman spectra of T. acidophilum ferredoxin, demonstrating the presence of a novel zinc center in addition to one [3Fe-4S] and one [4Fe-4S] cluster (Fe/Zn = 6.8 mol/mol). A phylogenetic tree constructed for several archaeal monocluster and dicluster type ferredoxins suggests that the zinc containing ferredoxins of T. acidophilum and Sulfolobus sp. form an independent subgroup, which is more distantly related to the ferredoxins from the hyperthermophiles than those from the methanogenic archaea, indicating the existence of a novel group of ferredoxins, namely, a ''zinc-containing ferredoxin family'' in the thermoacidophilic archaea. Inspection of the N-terminal extension regions of the archaeal zinc-containing ferredoxins suggested strict conservation of three histidine and one aspartate residues as possible ligands to the novel zinc center.

DOI： 10.1074/jbc.272.6.3453

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropyl-malate dehydrogenases belonging in the decarboxylating dehydrogenase family.

DOI： 10.1128/jb.179.4.1174-1179.1997

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記述言語：英語   出版者・発行元：University of Calgary Press  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Portland Press Ltd  

DOI： 10.1042/bst0250783

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

''Sulredoxin'' of Sulfolobus sp. strain 7 is an archaeal soluble Rieske-type [2Fe-2S] protein and was initially characterized by several spectroscopic techniques (Iwasaki, T., Isogai, T., Iizuka, T., and Oshima, T. (1995) J. Bacteriol. 177, 2576-2582), It appears to have tightly linked ionization affecting the redox properties of the protein, which is characteristic of the Rieske FeS proteins found as part of the respiratory chain, Sulredoxin had an E(m)(low pH) value of +188 +/- 9 mV, and the slope of pH dependence of the midpoint redox potential indicated two ionization equilibria in the oxidized form with pK(a(ox1)) of 6.23 +/- 0.22 and pK(a(ox2)) of 8.57 +/- 0.20, The absorption, CD, and resonance Raman spectra of oxidized sulredoxin are consistent with the proposed (S2FeS2Fe)-Fe-t-Fe-b[N(His)](t)(2) core structure, and deprotonation of one of the two putative coordinated histidine imidazoles, having the pK(a(ox2)) of 8.57 +/- 0.20, causes a decrease in the midpoint redox potential, the change in the optical and CD spectra, and the appearance of a new Raman transition at 278 cm(-1), without major structural rearrangement of the [2Fe-2S] cluster as well as the overall protein conformation, The redox-linked ionization of sulredoxin is also contributed by local changes involving another ionizable group having the pK(a(ox1)) of 6.23 +/- 0.22, which is probably attributed to a certain positively charged amino acid residue that may not be a ligand by itself but located very close to the cluster. We suggest that sulredoxin provides a new tractable model of the membrane-bound homologue of the respiratory chain, the Rieske FeS proteins of the cytochrome bc(1)-b(6)f complexes.

DOI： 10.1074/jbc.271.44.27659

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The role of cytochrome b(562), a fragile constituent of the respiratory terminal oxidase supercomplex of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, was investigated spectroscopically in the membrane-bound state. Cytochrome b(562) did not react with CO or cyanide in the membrane-bound state, while it was irreversibly modified to a CO-reactive form (b(559)) upon solubilization in the presence of cholate and LiCl. Cyanide titration analyses with the succinate-reduced membrane suggested that cytochrome b(562) was upstream of both the 'g(y) = 1.89' Rieske FeS cluster and the a-type cytochromes. These results show that the b-type cytochrome functions as an intermediate electron transmitter in the terminal oxidase supercomplex.

DOI： 10.1016/0378-1097(96)00372-2

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

An alternative form of a 7Fe dicluster-type ferredoxin (Fd-B) with a different charge density was purified as a minor component from the aerobic and thermoacidophilic archaeon, Sulfolobus sp. strain 7. Comparison of its properties with those of native 7Fe ferredoxin (Fd-A), a major ferredoxin, was made in terms of the molecular properties, the absorption, circular dichroism and electron paramagnetic resonance spectral properties, and the reactivity coupled with the cognate 2-oxoacid:ferredoxin oxidoreductase at 50 degrees C. Our results suggest that the Sulfolobus 7Fe ferredoxin has two spectroscopically distinct forms with slightly different conformations. (C) 1995 Academic Press, Inc.

DOI： 10.1006/bbrc.1995.1080

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

A novel pink [2Fe-2S] protein has been purified from the cytosol fraction of the thermoacidophilic archaeon Sulfolobus sp. strain 7 (originally named Sulfolobus acidocaldarius 7) and called 'sulredoxin.' Its absorption, circular dichroism, and electron paramagnetic resonance spectra suggest the presence of a Rieske-type [2Fe-2S] cluster (g-factors of 2.01, 1.91, and 1.79
average g- factor [g(av)] = 1.90) which is remarkably similar to that of Thermus thermophilus respiratory Rieske FeS protein (J. A. Fee, K. L. Findling, T. Yoshida, R. Hille, G. E. Tarr, D. O. Hearshen, W. R. Dunham, E. P. Day, T. A. Kent, and E. Munck, J. Biol. Chem. 259:124-133, 1984) and distinctively different from those of the plant-type ferredoxins (g(av) = 1.96). Sulredoxin, which is the first Rieske-type [2Fe-2S] protein isolated from an archaeal species, does not function as an electron acceptor of the cognate 2-oxoacid:ferredoxin oxidoreductase. Whether sulredoxin is derived from the archaeal membrane-bound respiratory Rieske-type FeS center (g(y) = 1.91) is the subject of further investigation.

DOI： 10.1128/jb.177.9.2576-2582.1995

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

To elucidate the ferredoxin-dependent redox system of the thermoacidophilic, aerobic archaeon Sulfolobus sp. strain 7, a novel FeS flavoprotein, which can reoxidize the reduced 7Fe ferredoxin in vitro, has been purified and characterized (designated as IFP) using the cognate 7Fe ferredoxin and 2-oxoacid:ferredoxin oxidoreductase, a key enzyme of the archaeal tricarboxylic acid cycle. IFP consists of three non-identical subunits with apparent molecular masses of 87, 32, and 22 kDa, respectively, and contains at least two FMN (E(m), 6.8 = -57 mV) and two plant-ferredoxin- type [2Fe-2S]2+,1+ clusters (E(m), 6.8 = -260 mV)/α2β2γ2 structure. Both FeS and flavin centers of IFP are slowly but fully reduced by the enzymatically reduced cognate ferredoxin under anaerobic conditions at 50°C, but not by NAD(P)H. Thus, the ferredoxin-dependent redox system of Sulfolobus sp. strain 7 is tentatively proposed as follows: 2- oxoacid:ferredoxin oxidoreductase (thiamine pyrophosphate and [4Fe-4S] cluster) → ferredoxin → IFP ([2Fe-2S] cluster → FMN).

DOI： 10.1074/jbc.270.30.17878

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：WALTER DE GRUYTER  

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記述言語：日本語  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：ELSEVIER SCIENCE INC  

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担当区分：最終著者   記述言語：日本語   出版者・発行元：医歯薬出版  

CiNii Books

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出版者・発行元：Elsevier BV  

DOI： 10.1016/s0162-0134(97)80138-9

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