Akio Urushiyama

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

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.

Redox-linked Ionization of Sulredoxin, an Archaeal Rieske-type [2Fe-2S] Protein from Sulfolobus sp. Strain 7

“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 Em(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 pKa(ox1) of 6.23 ± 0.22 and pKa(ox2) of 8.57 ± 0.20. The absorption, CD, and resonance Raman spectra of oxidized sulredoxin are consistent with the proposed St2FeSb2Fe[N(His)]t2 core structure, and deprotonation of one of the two putative coordinated histidine imidazoles, having the pKa(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 pKa(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 bc1-b6f complexes.

Novel Zinc-containing Ferredoxin Family in Thermoacidophilic Archaea

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.

Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the (2Fe-2S) center

The rate of quinol oxidation by cytochrome bc1/b6f complex is in part associated with the redox potential (Em) 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 (St) and Tyr‐Oη (as with the Y156W variant, which has a modified histidine Nε pKa,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‐Sb/t stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (Sb) and Ser‐Oγ, which is weak and/or unfavorably oriented for extensive coupling with the Fe‐Sb/t stretching vibrations.

Structures and characterization of reaction products of bis(1,1,1,5,5,5-hexafluoropentane-2,4-dionato)copper(ii) with 3-methyl- and 3,5-dimethyl-1h-pyrazoles

Abstract The reactions of [Cu(hfac)2] (Hhfac = 1,1,1,5,5,5-hexafluoropentane-2,4-dione) with 3-methyl- and 3,5-dimethyl-1 H-pyrazoles in light petroleum have been studied. These reactions give the 1 :2 adducts of [Cu(hfac)2(5-Hmpz)2] and [Cu(hfac)2(3,5-Hdmpz)2], respectively. The former adduct changes into complexes [Cu4(tfa)2(μ4-ttpt)2(5-Hmpz)4] (1a) and [Cu(hfac)(tfa)(5-Hmpz)2] (1b) (Htfa = trifluoroacetic acid; H3ttpt = 2,4,6-tris(trifluoromethyl) tetrahydropyran-2,4,6-triol; 5-Hmpz = 5-methyl-1H-pyrazole), while the latter does not change to give similar complexes. The crystal structures of { bd1a} and 1b have been determined. Complex 1a is tetranuclear, and four copper atoms linked together by two tridentate ttpt ligands are arranged in a parallelogram. The geometry about atoms Cu(1) and Cu(2) is distorted square-pyramidal, the coordination type being CuO5 and CuN2O3, respectively. Complex 1b is mononuclear and the geometry about the copper atom is a distorted tetragonal bipyramid. The axial positions are occupied by two oxygen atoms, one from a bidentate hfac ligand and the other from a monodentate tfa ligand. The two axial Cu-O distances are considerably different [2.294(7) and 2.522(6) , respectively] and the O(2)-Cu-O(3) axial bond angle is 162.0(2)°. The structures of 1:2 adducts are also discussed on the basis of IR and UV spectroscopy. Copyright

Multilevel based analysis of the thermoluminescence of CaSO4:RE (RE=Tm, Dy, Tb, and Sm)

Multitrapping levels feature of thermoluminescent (TL) phosphors of CaSO4:RE (RE=Dy, Tb, and Sm) were analyzed using a variable heating rate method (including extremely slow heating rates of 0.001°Cs−1), the postannealing method, and the analysis of TL decay curves using x-ray induced luminescence (XIL) spectroscopy after x-ray excitation was cut off at temperatures between 100 and 240 °C. All the data confirmed the multilevel feature of the TL levels. The ranges of activation energies E of these components in the main glow were determined to be (a) 1.64-2.03 eV for CaSO4:Dy, (b) 1.66-2.01 eV for CaSO4:Tb, and (c) 1.66-2.00 eV for CaSO4:Sm. The frequency factor s was determined to be 1019s−1 for all components of the phosphors. The previously reported TL properties of the CaSO4:Tm phosphor were reinvestigated using the XIL decay analysis. Assuming that the most populated level is located at the center of the main glow, the four phosphors have similar E values of ∼1.8eV. The present results imply that the ...

A novel 6Fe (2 × [3Fe-4S]) ferredoxin from Mycobacterium smegmatis

A novel ferredoxin was purified from Mycobacterium smegmatis by a series of hydrophobic chromatographies in the presence of high concentrations of ammonium sulfate and sodium chloride. The ferredoxin exhibited the same peptide map and N‐terminal amino acid sequence as the known 7Fe ferredoxin from the same bacterium. On the other hand, this ferredoxin was found to contain ∼6 Fe/mol ferredoxin and was also shown to contain only [3Fe‐4S] clusters by resonance Raman spectroscopy, indicating that it is a novel 6Fe ferredoxin which contains two [3Fe‐4S] clusters.

The vibrational fine structure of the 1A2g ← 1A1g polarized absorption band of trans-[Co(NH3)4(CN)2]+. The excited state geometry

Abstract The low temperature polarized absorption, far-infrared, and Raman spectra of trans-[Co(NH 3 ) 4 (CN) 2 ]Cl (H) and of the deuterated (D) compound

Elucidation of the annealing process required in the preparation of the thermoluminescence phosphor of CaSO4:Tm

The thermoluminescence phosphor of CaSO4:Tm was subjected to physicochemical studies. Careful thermo mass spectrometry and thermogravimetry/differential thermal analysis studies for a solid sample prepared by 200°C evaporation of a sulfuric acid medium without annealing by an extremely slow speed of temperature elevation were carried out. The data revealed, along with an initial weight loss corresponding to a small, nonstoichiometric amount of crystal water at 200°C, evidence for reaction with H2O vapor in air between 300 and 500°C and a 550°C and a subsequent release of SOx at higher temperatures until the beginning of bursting decomposition of CaSO4 bulk above 700°C. H1 NMR study revealed a broad signal at 9.9ppm, which was easily assigned to the crystal water involved. This initial solid has no thermostimulated luminescence (TL) efficiency. The first signal at 9.9ppm disappeared at 200°C. A new signal appeared at 13.1ppm by annealing from 300°C and increased significantly in intensity during annealing ...

Cyanidioschyzon merolae ferredoxin: a high resolution crystal structure analysis and its thermal stability.

Cyanidioschyzon merolae (Cm) is a single‐cell red alga that grows under moderately thermophilic (40–50 °C), acidic (pH 1–3) conditions. We purified a Cm ferredoxin (Fd) that was characterized as a plant‐type [2Fe–2S] Fd by physicochemical techniques. X‐ray crystallography revealed that the overall three‐dimensional structure of CmFd was highly similar to, but slightly different from, the [2Fe–2S] Fd from Spinacia oleracea, whose growth temperature is 15–20 °C. Therefore, slight structural differences, including non‐covalent‐bond number and amino acid sequence, may underlie the differential thermostabilities of the plant‐type Fds.