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Enzymatic reactions involving pyruvate: ferredoxin oxidoreductase and 2-oxoglutarate: ferredoxin oxidoreductase from a thermophilic, aerobic, chemolithoautotrophic, and hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, were investigated as the CO2 exchange reaction and CO2 fixation reaction using ferredoxin isolated from the same organism as a reductant.The reduced ferredoxin was required in the pyruvate synthetic reaction and the 2-oxoglutarate synthetic reaction by a cell extract that had been treated with a PD-10 column to remove the low molecular weight substances. [14C]Pyruvate and [14C]2-oxoglutarate were detected as products of pyruvate synthetic and 2-oxoglutarate synthetic reactions, respectively. Further evidence for the operation of pyruvate: ferredoxin oxidoreductase and 2-oxoglutarate: ferredoxin oxidoreductase was obtained from experiments on CO2 exchange reactions using the purified enzymes.

A new cyanobacterium of strain O-77 was isolated from a hot spring at Aso-Kuju National Park, Kumamoto, Japan. According to the phylogenetic analysis determined by 16S rRNA gene sequence, the strain O-77 belongs to the genus Leptolyngbya, classifying into filamentous non-heterocystous cyanobacteria. The strain O-77 showed the thermophilic behavior with optimal growth temperature of 55°C. Moreover, we have purified and characterized the oxygen-evolving photosystem II (PSII) from the strain O-77. The O2-evolving activity of the purified PSII from strain O-77 (PSIIO77) was 1275 ± 255 μmol O2 (mg Chl a)(-1) h(-1). Based on the results of MALDI-TOF mass spectrometry and urea-SDS-PAGE analysis, the purified PSIIO77 was composite of the typical PSII components of CP47, CP43, PsbO, D2, D1, PsbV, PsbQ, PsbU, and several low molecular mass subunits. Visible absorption and 77 K fluorescence spectra of the purified PSIIO77 were almost identical to those of other purified PSIIs from cyanobacteria. This report provides the successful example for the purification and characterization of an active PSII from thermophilic, filamentous non-heterocystous cyanobacteria.

NADH:ferredoxin reductase (EC 1.18.1.3) and NAD-reducing hydrogenase (EC 1.12.1.2) activities were detected in the cytoplasm of Hydrogenobacter thermophilus TK-6. NADH:ferredoxin reductase activity was detected using metronidazole, an artificial electron acceptor, which reacts specifically with reduced ferredoxin. Soluble NAD-reducing hydrogenase activity was detected after extended preincubation. The lag disappeared when cell-free extract was incubated anaerobically for more than 30 min. The electron transport system of this chemolithoautotrophic bacterium is discussed.

Thermal stratification is a common phenomenon in the surge line of Pressurized Water Reactors (PWR). The stratification temperature difference (ΔT) and cyclic behavior severities are the most prevalent during heat-up and cool-down operations of a PWR, when temperature difference between the pressurizer and the hot leg in the Reactor Coolant System (RCS) becomes the largest and inventory fluctuations in RCS are the highest. These behaviors cause cyclic damages and deformations of the pipe. In addition, USNRC recommended integrity evaluations of the piping system submitted to thermal stratified flow in USNRC Bulletin 88–11. In this reason, the importance of monitoring the fatigue induced by the thermal stratified flow in thick-wall pipes rises more and more. To raise the accuracy of solutions for the fatigue damage induced by thermal stratification, the exact global temperature profile is needed to be derived. Many studies have been performed to obtain these profiles. The purpose of this paper is to predict the global temperature profiles in the piping systems submitted to thermal-stratified flow from measured temperatures on the outside wall. It presents both the inverse transfer function method and the numerical-analysis method.Copyright © 2012 by ASME

Membrane-bound respiratory [NiFe] hydrogenase is an H2-uptake enzyme found in the periplasmic space of bacteria that plays a crucial role in energy-conservation processes. The heterodimeric unit of the enzyme from Hydrogenovibrio marinus was purified to homogeneity using chromatographic procedures. Crystals were grown using the sitting-drop vapour-diffusion method at room temperature. Preliminary crystallographic analysis revealed that the crystals belonged to space group P2(1), with unit-cell parameters a=75.72, b=116.59, c=113.40 A, β=91.3°, indicating that two heterodimers were present in the asymmetric unit.

Hydrogenases are metalloprotein enzymes that catalyse the reversible oxidation of dihydrogen to protons and electrons, a critical pathway in anaerobic metabolism. This reaction is of particular interest for hydrogen-based applications, in fuel cells for instance, but many applications are hindered by the high oxygen sensitivity that is an intrinsic feature of most hydrogenases. Two groups report the structures of oxygen-tolerant hydrogenases, one from the soil bacterium Ralstonia eutropha and the other from the marine bacterium Hydrogenovibrio marinus. The structures shed light on how redox-sensitive active-site intermediates are protected from destruction. Both enzymes feature a novel iron-sulphur centre at the active site, coordinated by a group of cysteine residues. Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H2-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H2 → 2H+ + 2e− (ref. 1). In contrast to the well-studied O2-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O2-tolerant H2 oxidation activity2,3,4; however, the mechanism of O2 tolerance is unclear5. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32 A. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues—in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes—and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O2-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O2 tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O2 in preventing the formation of an unready, inactive state of the enzyme.

For plant life extension, it is the regulatory requirement to assess reactor coolant environmental impacts on critical components of the nuclear power plant including at least those mentioned in NUREG/CR-6260[2]. The pressurizer surge line is the most easy-to-fail component in view of LWR (Light Water Reactor) environments when it comes to meeting the current ASME code limit of the fatigue evaluation. Cumulative Usage Factor (CUF) value could be increased to a maximum of 15.35 times due to the environmental effects, which makes it easy to exceed the allowable fatigue limit (1.0). This paper discusses the process of the environmental correction factor calculation described in NUREG/CR-5704[4], and five proposed schemes for reducing the environmental CUF value to the ASME code limit or below. This paper concludes that the proposed schemes are effective in lowering the environmental CUF value of the pressurizer surge line.Copyright © 2011 by ASME

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100+/-10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H(2) oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H(2) oxidation/H(2) evolution) was 1.61 x 10(2) at pH 7.0. The optimal temperature was 75 degrees C. The enzyme was quite stable under air atmosphere (the half-life of activity was c. 48 h at 4 degrees C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 degrees C. The activity increased up to 2.5-fold during incubation at 50 degrees C under H(2). Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V(max)=336 U mg(-1), k(cat)=560 s(-1), and k(cat)/K(m)=2.24 x 10(7) M(-1) s(-1). The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe-4S](+) and [4Fe-4S](+) clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H(2)-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga.

J-Resistance properties of automatic narrow gap gas tungsten arc welds (NGGTAW) for ferritic primary loop piping were investigated. The piping diameter is 1066 mm and its thickness is 76 mm. To do this, tensile, micro-hardness and J-R tests were performed and the microstructures of the welds were examined. Tensile and J-R tests were conducted at 316°C and 177°C for temperature effect and at 1 mm/min and 1000 mm/min for strain rate effect. The results were compared with the weld cases which are fabricated by the conventional SMAW process. NGGTAW welds investigated in this paper have lower J-R curves than the SMAW welds do. Fracture surfaces of the NGGTAW welds have an unusual appearance with deep ditches and holes due to the inclusions and pores. It is believed that the high density of the inclusion and the pore which exists in the NGGTAW welds is responsible for this low toughness and the cracked surface. Therefore, for an improvement of the NGGTAW weld toughness, it is recommended that sulfur content should be limited to a considerably low level and the pore should be eliminated by modifying the welding procedure specification.Copyright © 2005 by ASME

A hydrogen-oxidizing bacterium strain AH-24 was isolated, which was classified in the genus Hydrogenophaga, based on the 16S rRNA gene sequence. The isolate possessed a typical yellow pigment of Hydrogenophaga species. Its closest relative was Hydrogenophaga pseudoflava, but the assimilation profile of sugar compounds resembled that of no species of Hydrogenophaga. The optimum temperature and pH for autotrophic growth were, respectively, 33-35 degrees C and 7.0. Most hydrogenase activity (benzyl viologen reducing activity) was localized in the membrane fraction (MF), but NAD(P)-reducing hydrogenase activity was detected in neither the membrane nor the soluble fractions. Cytochromes b561 and c551 were present in MF; both were reduced when hydrogen was supplied to the oxidized MF, suggesting involvement in respiratory H2 oxidation as electron carriers. Cytochrome b561 was inferred to function as the redox partner of the membrane-bound hydrogenase.

When the flow velocity exceeds a critical value, fluid-elastic instability is believed to be a factor of the large-amplitude vibration and results in the rapid wear of heat exchanger tubes. For sub-critical flow velocities, however, the random turbulence excitation is considered the main mechanism to cause the long-term wear on steam generator tubes. Since flow-induced interactions between the tube and tube supports in the sub-critical flow velocity can cause a localized tube wear, tube movement in the clearance between the tube and tube support, as well as the normal contact force on the tubes by fluid should be maintained as low as possible. An analytical method is proposed for predicting fretting-wear damage of the double 90° U-bend tubes. This approach is based on Archard’s equation for adhesive wear with both the straight single-span tube analytical model proposed by Connors and the linear structural dynamic theory of ASME Section III, Appendix N-1300. The results from the presented method show good trend compared with the field data. It is expected that this method can be utilized to predict the fretting-wear of the double 90° U-bend tubes in steam generators.Copyright © 2006 by ASME

J-Resistance properties of automatic narrow gap gas tungsten arc welds (NGGTAW) for ferritic primary loop piping were investigated. The piping diameter is 1066 mm and its thickness is 76 mm. To do this, tensile, micro-hardness and J-R tests were performed and the microstructures of the welds were examined. Tensile and J-R tests were conducted at 316°C and 177°C for temperature effect and at 1 mm/min and 1000 mm/min for strain rate effect. The results were compared with the weld cases which are fabricated by the conventional SMAW process. NGGTAW welds investigated in this paper have lower J-R curves than the SMAW welds do. Fracture surfaces of the NGGTAW welds have an unusual appearance with deep ditches and holes due to the inclusions and pores. It is believed that the high density of the inclusion and the pore which exists in the NGGTAW welds is responsible for this low toughness and the cracked surface. Therefore, for an improvement of the NGGTAW weld toughness, it is recommended that sulfur content should be limited to a considerably low level and the pore should be eliminated by modifying the welding procedure specification.

Until now, antenna ratio was considered one of the most important parameters for plasma induced damage, and tolerable antenna ratio is very important for circuit designers to guarantee high yield circuits. In this paper, the dependence of plasma induced damage on the gate area is characterized using novel test patterns. The test chip is fabricated using 0.15 /spl mu/m CMOS technology with low-k IMD material. The variation of gate current is used to detect the plasma induced damage. It is shown that the failure rates of transistor increase as the gate area in the transistor increases although the antenna ratio maintains constant. Therefore, to guarantee the reliability of the gate oxide and transistors, we propose that the dependence of plasma induced damage on gate area should be considered when defining the maximum tolerable antenna ratio.

Two distinct ferredoxins, Fd I and Fd II, were isolated and purified to homogeneity from photoautotrophically grown Chlorobium tepidum, a moderately thermophilic green sulfur bacterium that assimilates carbon dioxide by the reductive tricarboxylic acid cycle. Both ferredoxins serve a crucial role as electron donors for reductive carboxylation, catalyzed by a key enzyme of this pathway, pyruvate synthase/pyruvate ferredoxin oxidoreductase. The reduction potentials of Fd I and Fd II were determined by cyclic voltammetry to be -514 and -584 mV, respectively, which are more electronegative than any previously studied Fds in which two [4Fe-4S] clusters display a single transition. Further spectroscopic studies indicated that the CD spectrum of oxidized Fd I closely resembled that of Fd II; however, both spectra appeared to be unique relative to ferredoxins studied previously. Double integration of the EPR signal of the two Fds yielded approximately approximately 2.0 spins per molecule, compatible with the idea that C. tepidum Fd I and Fd II accept 2 electrons upon reduction. These results suggest that the C. tepidum Fd I and Fd II polypeptides each contain two bound [4Fe-4S] clusters. C. tepidum Fd I and Fd II are novel 2[4Fe-4S] Fds, which were shown previously to function as biological electron donors or acceptors for C. tepidum pyruvate synthase/pyruvate ferredoxin oxidoreductase (Yoon, K.-S., Hille, R., Hemann, C. F., and Tabita, F. R. (1999) J. Biol. Chem. 274, 29772-29778). Kinetic measurements indicated that Fd I had approximately 2.3-fold higher affinity than Fd II. The results of amino acid sequence alignments, molecular modeling, oxidation-reduction potentials, and spectral properties strongly indicate that the C. tepidum Fds are chimeras of both clostridial-type and chromatium-type Fds, suggesting that the two Fds are likely intermediates in the evolutional development of 2[4Fe-4S] clusters compared with the well described clostridial and chromatium types.

Publisher Summary Hydrogenobacter thermophilus strain TK-6 is an aerobic thermophilic hydrogen-oxidizing bacterium isolated from hot spring in Izu, Japan. As for CO 2 fixation pathway of the strain, 14 CO 2 labeling experiment, in vitro enzyme assays, and purification of ATP:citrate lyase in the group supported the idea that the reductive TCA cycle is operative in strain TK-6. However, identification of a strong reductant that is needed for the pyruvate synthase and 2-oxoglutarate synthase and clarification of an electron transport system were definitely needed. H. thermophilus was shown to belong to a very early branching order, the Aquificales . Also, strain TK-6 was isolated from hot spring, which implies that the strain may have its original ecological niche underground where little or no oxygen exists. Taking these things into consideration, it is of great interest to examine if the strain has its ability to grow anaerobically.

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80° C. The pH optimum was 7.6–7.8. The apparent K m values for pyruvate and coenzyme A at 70° C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t 50%) at 70° C was approximately 8 h.

A membrane-bound hydrogenase was purified aerobically by one step using a hydroxyapatite column after solubilization by acetone treatment from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1. The enzyme consists of two polypeptides of 63 and 31 kDa, respectively. The amino-terminal amino acid sequences of both subunits were homologous to membrane-bound type [Ni-Fe] hydrogenases from other origins. The thermostability under a hydrogen gas atmosphere is highly stable at 50 degrees C, which is the optimum temperature for the cell growth.

This communication reports the successful merging of the chemical properties of a natural [NiFe]hydrogenase (Desulfovibrio vulgaris Miyazaki F) and our previously reported [NiRu] hydrogenase-mimic. The catalytic activity of both the natural enzyme and the mimic is almost identical, with the exception of working pH ranges, and this allows us to use them simultaneously in the same reaction flask. In such a manner, isotope exchange between D(2) and H(2)O could be conducted over an extended pH range (about 2-10) in one pot under mild conditions at ambient temperature and pressure.

In order to generate renewable and clean fuels, increasing efforts are focused on the exploitation of photosynthetic microorganisms for the production of molecular hydrogen from water and light. In this study we engineered a 'hard-wired' protein complex consisting of a hydrogenase and photosystem I (hydrogenase-PSI complex) as a direct light-to-hydrogen conversion system. The key component was an artificial fusion protein composed of the membrane-bound [NiFe] hydrogenase from the beta-proteobacterium Ralstonia eutropha H16 and the peripheral PSI subunit PsaE of the cyanobacterium Thermosynechococcus elongatus. The resulting hydrogenase-PsaE fusion protein associated with PsaE-free PSI spontaneously, thereby forming a hydrogenase-PSI complex as confirmed by sucrose-gradient ultracentrifuge and immunoblot analysis. The hydrogenase-PSI complex displayed light-driven hydrogen production at a rate of 0.58 mumol H(2).mg chlorophyll(-1).h(-1). The complex maintained its accessibility to the native electron acceptor ferredoxin. This study provides the first example of a light-driven enzymatic reaction by an artificial complex between a redox enzyme and photosystem I and represents an important step on the way to design a photosynthetic organism that efficiently converts solar energy and water into hydrogen.