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Metal–ligand cooperativity is an essential feature of bioinorganic catalysis. The design principles of such cooperativity in metalloenzymes are underexplored, but are critical to understand for developing efficient catalysts designed with earth abundant metals for small molecule activation. The simple substrate requirements of reversible proton reduction by the [NiFe]-hydrogenases make them a model bioinorganic system. A highly conserved arginine residue (R355) directly above the exogenous ligand binding position of the [NiFe]-catalytic core is known to be essential for optimal function because mutation to a lysine results in lower catalytic rates. To expand on our studies of soluble hydrogenase-1 from Pyrococcus furiosus (Pf SH1), we investigated the role of R355 by site-directed-mutagenesis to a lysine (R355K) using infrared and electron paramagnetic resonance spectroscopic probes sensitive to active site redox and protonation events. It was found the mutation resulted in an altered ligand binding environment at the [NiFe] centre. A key observation was destabilization of the Nia3+–C state, which contains a bridging hydride. Instead, the tautomeric Nia+–L states were observed. Overall, the results provided insight into complex metal–ligand cooperativity between the active site and protein scaffold that modulates the bridging hydride stability and the proton inventory, which should prove valuable to design principles for efficient bioinspired catalysts.

The genome of the archaeon Pyrobaculum aerophilum (Topt ~ 100 °C) contains an operon (PAE2859–2861) encoding a putative pyranopterin-containing oxidoreductase of unknown function and metal content. These genes (with one gene modified to encode a His-affinity tag) were inserted into the fermentative anaerobic archaeon, Pyrococcus furiosus (Topt ~ 100 °C). Dye-linked assays of cytoplasmic extracts from recombinant P. furiosus show that the P. aerophilum enzyme is a thiosulfate reductase (Tsr) and reduces thiosulfate but not polysulfide. The enzyme (Tsr–Mo) from molybdenum-grown cells contains Mo (Mo:W = 9:1) while the enzyme (Tsr–W) from tungsten-grown cells contains mainly W (Mo:W = 1:6). Purified Tsr–Mo has over ten times the activity (Vmax = 1580 vs. 141 µmol min−1 mg−1) and twice the affinity for thiosulfate (Km = ~ 100 vs. ~ 200 μM) than Tsr–W and is reduced at a lower potential (Epeak = − 255 vs − 402 mV). Tsr–Mo and Tsr–W proteins are heterodimers lacking the membrane anchor subunit (PAE2861). Recombinant P. furiosus expressing P. aerophilum Tsr could not use thiosulfate as a terminal electron acceptor. P. furiosus contains five pyranopterin-containing enzymes, all of which utilize W. P. aerophilum Tsr–Mo is the first example of an active Mo-containing enzyme produced in P. furiosus.

Metal–ligand cooperativity is an essential feature of bioinorganic catalysis. The design principles of such cooperativity in metalloenzymes are underexplored, but are critical to understand for developing efficient catalysts designed with earth abundant metals for small molecule activation. The simple substrate requirements of reversible proton reduction by the [NiFe]-hydrogenases make them a model bioinorganic system. A highly conserved arginine residue (R355) directly above the exogenous ligand binding position of the [NiFe]-catalytic core is known to be essential for optimal function because mutation to a lysine results in lower catalytic rates. To expand on our studies of soluble hydrogenase-1 from Pyrococcus furiosus (Pf SH1), we investigated the role of R355 by site-directed-mutagenesis to a lysine (R355K) using infrared and electron paramagnetic resonance spectroscopic probes sensitive to active site redox and protonation events. It was found the mutation resulted in an altered ligand binding environment at the [NiFe] centre. A key observation was destabilization of the Nia3+–C state, which contains a bridging hydride. Instead, the tautomeric Nia+–L states were observed. Overall, the results provided insight into complex metal–ligand cooperativity between the active site and protein scaffold that modulates the bridging hydride stability and the proton inventory, which should prove valuable to design principles for efficient bioinspired catalysts., Metal–ligand cooperativity is an essential feature of bioinorganic catalysis.

BackgroundThe outbreak of novel 2019 coronavirus disease (COVID-19) has imposed an enormous physical and psychological pressure on people across the world. This study focused on evaluating the prevalence and influencing factors of anxiety and depression symptoms in surgical nurses during the epidemic in China. MethodA cross-sectional, multicenter quantitative study was conducted in Anhui province (China) from March 3, 2020 to March 19, 2020, with a questionnaire package which consisted of general information questionnaire，Zung's self-rating anxiety scale (SAS), Zung's self-rating Depression Scale (SDS) and social support rating scale (SSRS). A total of 3600 surgical nurses participated in the survey by Wechat and QQ. Data were analysed using multiple linear regression models. ResultsA total of 3492 surgical nurses from 12tertiary hospitals and 12 secondary hospitals in one province of mainland China completed the survey. The prevalence rates of anxiety symptoms and depressive symptoms were 24.83% and 22.39%, respectively. The average level of anxiety and depression of surgical nurses were higher than that of the Chinese norm (P< 0.05).Levels of social support for surgical nurses were significantly negatively associated with the degree of anxiety (r = -0.630, P < 0.001) and depression (r = -0.578, P < 0.001). Fertility status (β = 1.469, P = 0.003), hospital (β = -0.611, P < 0.001), participation in care for COVID-19 patients (β = 2.229, P < 0.001), likelihood of being infected with COVID-19 (β = 1.146, P < 0.001), social support (β = -0.623, P < 0.001) were significantly influencing surgical nurses’ anxiety degree. Similarly, these characteristics were significantly associated with the odds of experiencing depression symptoms in surgical nurses. Divorce and widowed surgical nurses (β = -2.654, P < 0.001) were significantly more likely to experience depressive symptoms than single nurses. ConclusionIn this survey, we found that the surgical nurses had high anxiety and depression symptoms during the COVID-19 outbreak in China. The findings suggest that targeted psychological interventions to promote the mental health of surgical nurses with psychological problems need to be immediately implemented.

Arsenate is a notorious toxicant that is known to disrupt multiple biochemical pathways. Many microorganisms have developed mechanisms to detoxify arsenate using the ArsC-type arsenate reductase, and some even use arsenate as a terminal electron acceptor for respiration involving arsenate respiratory reductase (Arr). ArsC-type reductases have been studied extensively, but the phylogenetically unrelated Arr system is less investigated and has not been characterized from Archaea. Here, we heterologously expressed the genes encoding Arr from the crenarchaeon Pyrobaculum aerophilum in the euryarchaeon Pyrococcus furiosus, both of which grow optimally near 100°C. Recombinant P. furiosus was grown on molybdenum (Mo)- or tungsten (W)-containing medium, and two types of recombinant Arr enzymes were purified, one containing Mo (Arr-Mo) and one containing W (Arr-W). Purified Arr-Mo had a 140-fold higher specific activity in arsenate [As(V)] reduction than Arr-W, and Arr-Mo also reduced arsenite [As(III)]. The P. furiosus strain expressing Arr-Mo (the Arr strain) was able to use arsenate as a terminal electron acceptor during growth on peptides. In addition, the Arr strain had increased tolerance compared to that of the parent strain to arsenate and also, surprisingly, to arsenite. Compared to the parent, the Arr strain accumulated intracellularly almost an order of magnitude more arsenic when cells were grown in the presence of arsenite. X-ray absorption spectroscopy (XAS) results suggest that the Arr strain of P. furiosus improves its tolerance to arsenite by increasing production of less-toxic arsenate and nontoxic methylated arsenicals compared to that by the parent. IMPORTANCE Arsenate respiratory reductases (Arr) are much less characterized than the detoxifying arsenate reductase system. The heterologous expression and characterization of an Arr from Pyrobaculum aerophilum in Pyrococcus furiosus provides new insights into the function of this enzyme. From in vivo studies, production of Arr not only enabled P. furiosus to use arsenate [As(V)] as a terminal electron acceptor, it also provided the organism with a higher resistance to arsenate and also, surprisingly, to arsenite [As(III)]. In contrast to the tungsten-containing oxidoreductase enzymes natively produced by P. furiosus, recombinant P. aerophilum Arr was much more active with molybdenum than with tungsten. It is also, to our knowledge, the only characterized Arr to be active with both molybdenum and tungsten in the active site.

A series of viologen related redox mediators of varying reduction potential has been characterized and their utility as electron shuttles between CdSe quantum dots and hydrogenase enzyme has been demonstrated. Tuning the mediator LUMO energy optimizes the performance of this hybrid photocatalytic system by balancing electron transfer rates of the shuttle.

The NADPH-dependent cytoplasmic [NiFe]-hydrogenase (SHI) from the hyperthermophile Pyrococcus furiosus, which grows optimally near 100°C, is extremely thermostable and has many in vitro applications, including cofactor generation and hydrogen production. In particular, SHI is used in a cell-free synthetic pathway that contains more than a dozen other enzymes and produces three times more hydrogen (12 H2/glucose) from sugars compared to cellular fermentations (4 H2/glucose). We previously reported homologous over-expression and rapid purification of an affinity-tagged (9x-His) version of SHI, which is a heterotetrameric enzyme. However, about 30% of the enzyme that was purified contained an inactive trimeric form of SHI lacking the catalytic [NiFe]-containing subunit. Herein, we constructed a strain of P. furiosus that contained a second set of the eight genes involved in the maturation of the catalytic subunit and insertion of the [NiFe]-site, along with a second set of the four genes encoding the SHI structural subunits. This resulted in a 40% higher yield of the purified affinity-tagged enzyme and the content of the inactive trimeric form decreased to 5% of the total protein. These results bode well for the future production of active SHI for both basic and applied purposes.

A series of viologen related redox mediators of varying reduction potential has been characterized and their utility as electron shuttles between CdSe quantum dots and hydrogenase enzyme has been demonstrated. Tuning the mediator LUMO energy optimizes the performance of this hybrid photocatalytic system by balancing electron transfer rates of the shuttle.

We describe a hybrid photocatalytic system for hydrogen production consisting of nanocrystalline CdSe/CdS dot-in-rod (DIR) structures coupled to [NiFe] soluble hydrogenase I (SHI) from Pyrococcus furiosus. Electrons are shuttled to the catalyst by a redox mediator, either methyl viologen (MV2+, E0 = −446 mV vs. NHE) or propyl-bridged 2-2′-bipyridinium (PDQ2+, E0 = −550 mV vs. NHE). We demonstrate nearly equal photoreduction efficiencies for the two mediators, despite extracting ∼100 mV of additional driving force for proton reduction by PDQ2+. Femtosecond to microsecond transient absorption reveals that while electron transfer (ET) from the DIR to PDQ2+ is slower than for MV2+, in both cases the ET process is complete by 1 ns and thus it efficiently outcompetes radiative decay. Long-lived charge separation is observed for both mediators, resulting in similar net efficiencies of photoreduction. Whereas both mediators are readily photoreduced, only PDQ2+ yields measurable H2 production, demonstrating the importance of optimizing the electron shuttling pathway to take advantage of the available reducing power of the DIR excited state. H2 production in the PDQ2+ system is highly efficient, with an internal quantum efficiency (IQE) as high as 77% and a TONSHI of 1.1 × 106 under mild (RT, pH = 7.35) conditions.

Background To provide support for combined usage of phosphoinositide 3-kinase (PI3K) inhibitors or mitogen-activated protein kinase pathway inhibitors together with sorafenib in treatment of sorafenib-resistant hepatocellular carcinoma. Materials and methods The sorafenib-resistant cell lines were established to evaluate the effects of MK-2206 2HCL, a dual PI3K/mammalian target of rapamycin (mTOR) inhibitor, and PD0325901, an rat sarcoma (RAS) and/or extracellular signal-regulated kinase (ERK) inhibitor, on cell proliferation and apoptosis, as both single and combined treatments with sorafenib. In addition, multidrug resistance 1 gene expression, mutation status of key members in PI3K/mTOR, and RAS/ERK pathways and pathway activation were analyzed to identify predictors of drug response. Results Molecular studies reveal that combining MK-2206 2HCL or PD0325901 with sorafenib not only has a synergistic effect, in suppressing PI3K/protein kinase B/mTOR and RAS/MEK/ERK signaling more effectively than either treatment alone, but also prevents the cross activation of the other pathway that occurs with single treatments in both sorafenib sensitive and resistant lines. PD0325901 exhibited a stronger synergic effect with sorafenib than MK-2206 2HCL. Sorafenib-resistant cell lines were characterized by activation of both of the two pathways, as indicated by multidrug resistance 1 gene expression profiles and pathway activity analysis. Conclusions Our studies have showed that both inhibitors of PI3K/mTOR and RAS/ERK signaling are potentially effective antihepatocellular carcinoma drugs especially in treating sorafenib-resistant hepatocellular carcinoma.

Hydrogen production by water splitting energized by biomass sugars is one of the most promising technologies for distributed green H2 production. Direct H2 generation from NADPH, catalysed by an NADPH-dependent, soluble [NiFe]-hydrogenase (SH1) is thermodynamically unfavourable, resulting in slow volumetric productivity. We designed the biomimetic electron transport chain from NADPH to H2 by the introduction of an oxygen-insensitive electron mediator benzyl viologen (BV) and an enzyme (NADPH rubredoxin oxidoreductase, NROR), catalysing electron transport between NADPH and BV. The H2 generation rates using this biomimetic chain increased by approximately five-fold compared to those catalysed only by SH1. The peak volumetric H2 productivity via the in vitro enzymatic pathway comprised of hyperthermophilic glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase, NROR, and SH1 was 310 mmol H2/L h−1, the highest rate yet reported. The concept of biomimetic electron transport chains could be applied to both in vitro and in vivo H2 production biosystems and artificial photosynthesis.

Green hydrogen production through water splitting at low temperatures is highly desired for hydrogen economy. Herein, we demonstrate an in vitro non-natural enzymatic pathway to utilize the chemical energy stored in xylooligosaccharides from biomass to split water to produce a nearly theoretical yield of H-2 (i.e., approximate to 9.5 H-2 per xylose plus water). This pathway was constructed on the basis of the novel activities of phosphopentomutase catalyzing the conversion of D-xylose 1-phosphate into d-xylose 5-phosphate and of ribose 5-phosphate isomerase catalyzing the conversions of d-xylose 5-phosphate and D-xylulose 5-phosphate. This study suggests that the discovery of novel promiscuous enzyme activities is important to implement complicated biotransformations catalyzed by synthetic enzymatic pathways.

Hyperthermophiles are microorganisms that thrive in extremely hot environment from 80 oC and upwards. They are the most primitive organisms evolved to survive in the early unhostile earth. The simplest and versatile respiratory electron transport chain of these hyperthermophiles made them suitable candidates for microbial fuel cells (MFC) anode catalysts to generate power at high temperatures. We explored the extracellular electron transfer ability of a hyperthermophilic archaeon called Pyrococccus furiosus (PF). PF is an aquatic anaerobe that grows optimally at 100 oC using a wide range of substrates such as carbohydrates and peptides1. The respiratory chain of PF is simple and unique that it does not require any terminal electron acceptor, rather reduces proton to H2 through membrane bound enzyme complex called hydrogenase2. Their ability to reduce the extracellular oxidants such as insoluble Fe(III) oxide and soluble Fe(III) citrate has been investigated. PF reduced Fe(III) oxide during its growth at 90 oC and the PF culture re-suspended in MOPS buffer (pH 7.5) was found to reduce Fe(III) citrate significantly using H2 as an electron donor. Further, low scan-rate cyclic voltammetry at 90 oC using potassium ferricyanide as a mediator showed greater mediated electron transfer by PF compared to the control without PF. The proof of concept study reported here reveals the potential of PF to generate electricity in MFCs at high temperature locations such as in solfataric fields or near hydrothermal vents. Moreover, such studies also help to advance our understanding in the field of biogeochemistry of the early hot environment and exobiology. References: S. D. Hamilton-Brehm, G. J. Schut and M. W. W. Adams (2005) J Bacteriol., 187(21): 7492–7499 S Mukund and M. W. W. Adams (1991) J. Biol. Chem., 266(22), 14208-14216. Figure 1

Complex I or NADH quinone oxidoreductase (NUO) is an integral component of modern day respiratory chains and has a close evolutionary relationship with energy-conserving [NiFe]-hydrogenases of anaerobic microorganisms. Specifically, in all of biology, the quinone-binding subunit of Complex I, NuoD, is most closely related to the proton-reducing, H2-evolving [NiFe]-containing catalytic subunit, MbhL, of membrane-bound hydrogenase (MBH), to the methanophenzine-reducing subunit of a methanogenic respiratory complex (FPO) and to the catalytic subunit of an archaeal respiratory complex (MBX) involved in reducing elemental sulfur (S°). These complexes also pump ions and have at least 10 homologous subunits in common. As electron donors, MBH and MBX use ferredoxin (Fd), FPO uses either Fd or cofactor F420, and NUO uses either Fd or NADH. In this review, we examine the evolutionary trajectory of these oxidoreductases from a proton-reducing ancestral respiratory complex (ARC). We hypothesize that the diversification of ARC to MBH, MBX, FPO and eventually NUO was driven by the larger energy yields associated with coupling Fd oxidation to the reduction of oxidants with increasing electrochemical potential, including protons, S° and membrane soluble organic compounds such as phenazines and quinone derivatives. Importantly, throughout Earth's history, the availability of these oxidants increased as the redox state of the atmosphere and oceans became progressively more oxidized as a result of the origin and ecological expansion of oxygenic photosynthesis. ARC-derived complexes are therefore remarkably stable respiratory systems with little diversity in core structure but whose general function appears to have co-evolved with the redox state of the biosphere. This article is part of a Special Issue entitled Respiratory Complex I, edited by Volker Zickermann and Ulrich Brandt.

Hydrogenases catalyze the simplest of chemical reactions, the reversible interconversion of protons, electrons, and hydrogen gas. These enzymes have potential to be utilized for several biotechnological applications, such as in vitro hydrogen production from renewable materials and in enzyme-based fuel cells for electricity generation. Based on the metal content of their catalytic sites, hydrogenases are classified as either [NiFe], [FeFe], or mononuclear Fe enzymes, and [NiFe] hydrogenases are further categorized into five groups based on the sequences of the catalytic subunits. This chapter describes recombinant engineering strategies, purification procedures, and catalytic properties of two distinct types of [NiFe] hydrogenase from Pyrococcus furiosus, a microorganism with an optimal growth temperature of 100°C. These enzymes are termed soluble hydrogenase I (SHI, group 3) and membrane-bound hydrogenase (MBH, group 4). The two hydrogenases were affinity-tagged to facilitate their purification and the purified enzymes have been used for biochemical, mechanistic, and structural analyses. The results have provided us with new insights into how catalysis by SHI is achieved, which could also lead to the development of catalysts for economic hydrogen production, and knowledge of how MBH couples hydrogen gas production to conservation of energy in the form of an ion gradient. The methods described in this chapter provide the basis for these studies.

Hyperthermophilic archaea contain a hydrogen gas–evolving,respiratory membrane–bound NiFe-hydrogenase (MBH) that is very closely related to the aerobic respiratory complex I. During growth on elemental sulfur (S°), these microorganisms also produce a homologous membrane-bound complex (MBX), which generates H(2)S. MBX evolutionarily links MBH to complex I, but its catalytic function is unknown. Herein, we show that MBX reduces the sulfane sulfur of polysulfides by using ferredoxin (Fd) as the electron donor, and we rename it membrane-bound sulfane reductase (MBS). Two forms of affinity-tagged MBS were purified from genetically engineered Pyrococcus furiosus (a hyperthermophilic archaea species): the 13-subunit holoenzyme (S-MBS) and a cytoplasmic 4-subunit catalytic subcomplex (C-MBS). S-MBS and C-MBS reduced dimethyl trisulfide (DMTS) with comparable K(m) (∼490 μm) and V(max) values (12 μmol/min/mg). The MBS catalytic subunit (MbsL), but not that of complex I (NuoD), retains two of four NiFe-coordinating cysteine residues of MBH. However, these cysteine residues were not involved in MBS catalysis because a mutant P. furiosus strain (MbsLC85A/C385A) grew normally with S°. The products of the DMTS reduction and properties of polysulfides indicated that in the physiological reaction, MBS uses Fd (E(o)′ = −480 mV) to reduce sulfane sulfur (E(o)′ −260 mV) and cleave organic (RS(n)R, n ≥ 3) and anionic polysulfides (S(n)(2−), n ≥ 4) but that it does not produce H(2)S. Based on homology to MBH, MBS also creates an ion gradient for ATP synthesis. This work establishes the electrochemical reaction catalyzed by MBS that is intermediate in the evolution from proton- to quinone-reducing respiratory complexes.

Although the extremely thermophilic archaea (T(opt) ≥ 70°C) may be the most primitive extant forms of life, they have been studied to a limited extent relative to mesophilic microorganisms. Many of these organisms have unique biochemical and physiological characteristics with important biotechnological implications. These include methanogens that generate methane, fermentative anaerobes that produce hydrogen gas with high efficiency, and acidophiles that can mobilize base, precious and strategic metals from mineral ores. Extremely thermophilic archaea have also been a valuable source of thermoactive, thermostable biocatalysts, but their use as cellular systems has been limited because of the general lack of facile genetics tools. This situation has changed recently, however, thereby providing an important avenue for understanding their metabolic and physiological details and also opening up opportunities for metabolic engineering efforts. Along these lines, extremely thermophilic archaea have recently been engineered to produce a variety of alcohols and industrial chemicals, in some cases incorporating CO(2) into the final product. There are barriers and challenges to these organisms reaching their full potential as industrial microorganisms but, if these can be overcome, a new dimension for biotechnology will be forthcoming that strategically exploits biology at high temperatures.

Hydrogen gas-evolving membrane-bound hydrogenase (MBH) and quinone-reducing Complex I are homologous respiratory complexes with a common ancestor but a structural basis for their evolutionary relationship is lacking. Herein we report the cryo-EM structure of a 14-subunit MBH from the hyperthermophilie Pyrococcus furiosus. MBH contains a membrane-anchored hydrogenase module that is highly similar structurally to the quinone-binding Q-module of Complex I while its membrane-embedded ion-translocation module can be divided into a H(+)- and a Na(+)-translocating unit. The H(+)-translocating unit is rotated 180° in-membrane with respect to its counterpart in Complex I, leading to distinctive architectures for the two respiratory systems despite their largely conserved proton-pumping mechanisms. The Na(+)-translocating unit, absent in Complex I, resembles that found in the Mrp H(+)/Na(+) antiporter and enables hydrogen gas evolution by MBH to establish a Na(+) gradient for ATP synthesis near 100 °C. MBH also provides insights into Mrp structure and evolution of MBH-based respiratory enzymes.

Increasing studies have indicated the association between adipokines and multiple autoimmune diseases. This study aimed to evaluate the mRNA expression levels of vaspin, adiponectin and adrenomedullin in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE), as well as their clinical associations.A total of 46 SLE patients and 51 normal controls were recruited. The three adipokines expression levels in PBMCs from SLE patients were measured by qRT-PCR, and their associations with major clinical and laboratory parameters of SLE patients were also analysed.Compared with normal controls, vaspin expression level in PBMCs was significantly decreased (p0.001), whereas adiponectin expression level was significantly higher in SLE patients (p0.001). There was no significant difference in adrenomedullin expression level between SLE patients and normal controls. Vaspin and adrenomedullin expression levels in more active SLE were significantly lower than those in less active SLE (p=0.012, p=0.046, respectively). No significant difference in these adipokine expression levels was observed between SLE patients with and without lupus nephritis (LN). There was also no significant association between mRNA levels of these adipokines and major clinical and laboratory parameters.Altered vaspin, adiponectin expression levels, and the associations between vaspin, adrenomedullin levels and disease activity in SLE patients suggested that these adipokines might play a role in SLE.

Hydrogenases catalyze the simplest of chemical reactions, the reversible interconversion of protons, electrons, and hydrogen gas. These enzymes have potential to be utilized for several biotechnological applications, such as in vitro hydrogen production from renewable materials and in enzyme-based fuel cells for electricity generation. Based on the metal content of their catalytic sites, hydrogenases are classified as either [NiFe], [FeFe], or mononuclear Fe enzymes, and [NiFe] hydrogenases are further categorized into five groups based on the sequences of the catalytic subunits. This chapter describes recombinant engineering strategies, purification procedures, and catalytic properties of two distinct types of [NiFe] hydrogenase from Pyrococcus furiosus, a microorganism with an optimal growth temperature of 100°C. These enzymes are termed soluble hydrogenase I (SHI, group 3) and membrane-bound hydrogenase (MBH, group 4). The two hydrogenases were affinity-tagged to facilitate their purification and the purified enzymes have been used for biochemical, mechanistic, and structural analyses. The results have provided us with new insights into how catalysis by SHI is achieved, which could also lead to the development of catalysts for economic hydrogen production, and knowledge of how MBH couples hydrogen gas production to conservation of energy in the form of an ion gradient. The methods described in this chapter provide the basis for these studies.

Starch is a natural energy storage compound and is hypothesized to be a high-energy density chemical compound or solar fuel. In contrast to industrial hydrolysis of starch to glucose, an alternative ATP-free phosphorylation of starch was designed to generate cost-effective glucose 6-phosphate by using five thermophilic enzymes (i.e., isoamylase, alpha-glucan phosphorylase, 4-α-glucanotransferase, phosphoglucomutase, and polyphosphate glucokinase). This enzymatic phosphorolysis is energetically advantageous because the energy of α-1,4-glycosidic bonds among anhydroglucose units is conserved in the form of phosphorylated glucose. Furthermore, we demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch, regardless of branched and linear contents, with water to hydrogen at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. The use of a biomimetic electron transport chain enabled to achieve a maximum volumetric productivity of 90.2mmol of H2/L/h at 20g/L starch. The complete oxidation of starch to hydrogen by this in vitro synthetic (enzymatic) biosystem suggests that starch as a natural solar fuel becomes a high-density hydrogen storage compound with a gravimetric density of more than 14% H2-based mass and an electricity density of more than 3000Wh/kg of starch.

The archaeon Pyrococcus furiosus grows optimally at 100°C by converting carbohydrates to acetate, carbon dioxide and hydrogen gas (H(2)), obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H(2) production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is classified as a Group 4 hydrogenase and is encoded by a 14-gene operon that contains hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged 4-subunit cytoplasmic subcomplex of MBH (C-MBH) was engineered and expressed in P. furiosus by differential transcription of the MBH operon. It was purified under anaerobic conditions by affinity chromatography without detergent. Purified C-MBH had a Fe : Ni ratio of 14 : 1, similar to the predicted value of 13 : 1. The O(2) sensitivities of C-MBH and the 14-subunit membrane-bound version were similar (half-lives of ∼15 h in air), but C-MBH was more thermolabile (half-lives at 90°C of 8 and 25 h, respectively). C-MBH evolved H(2) with the physiological electron donor, reduced ferredoxin, optimally at 60°C. This is the first report of the engineering and characterization of a soluble catalytically active subcomplex of a membrane-bound respiratory hydrogenase.

The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions.

A hyperthermophilic archaeon called Pyrococcus furiosus (Pf), is being explored as potential microbial catalyst for use microbial fuel cells (MFCs) [Sekar et al. (2017)]. The two main enzymes membrane bound hydrogenase (MBH) and cytoplasmic soluble hydrogenases (SH) are involved in its oxidative fermentative pathways. In P. furiosus the membrane bound hydrogenase (MBH) uses reducing equivalents from the oxidative fermentative pathway to produce hydrogen, which is believed to be used by soluble hydrogenases (SHI and SHII) for nicotinamide cofactor generation. However, in anaerobic respiratory systems, the oxidative fermentative pathways and the activity of MBH and SH enzyme complexes are not fully understood. In this study, we hypothesize that genetically manipulating the expressions of SH and MBH could influence extracellular electron transfer, which could be characterized by an enhancement in organism’s ability to reduce a metal ion (e.g. soluble ferric citrate or and insoluble ferric oxide). Herein, genetic traceable Pf strain was genetically engineered to produce strains with over-expressed SHI (OESHI), over-expressed MbhJ-N (OEMbhJ-N), knocked-out SHI/SHII (ΔSHI/II) or knocked-out MbhL (ΔMbhL). Soluble ferric citrate and insoluble ferric oxide were used to study the ability of engineered Pf strains to reduce the extracellular oxidants. Experiments revealed that the ΔMbhL strain, produced the highest exo-electrogenic ability among these Pf strains. To the best of our knowledge this is the first report that compares the exoelectrogenic ability of different strains of genetically engineered Pf.

Laparoscopic common bile duct exploration (LCBDE) had become one of the main options for management of choledocholithiasis. This retrospective comparative study aimed to evaluate on the feasibility and advantages of primary closure versus conventional T-tube drainage of the common bile duct (CBD) after laparoscopic choledochotomy. In this retrospective analysis, 100 patients (47 men and 53 women) with choledocholithiasis who underwent primary closure of the CBD (without T-tube drainage) after LCBDE (Group A) were compared with 92 patients who underwent LCBDE with T-tube drainage (Group B). Both groups were evaluated with regard to biliary complications, hospital stay, and recurrence of stones. The mean operation time was 104.12 minutes for Group A and 108.92 minutes for Group B ( P = 0.069). The hospital stay was significantly shorter in Group A than that in Group B (6.95 days and 12.05 days, respectively; P < 0.001). In Group A, bile leakage occurred in two patients on postoperative Day 2 and Day 3, respectively. In Group B, bile leakage noted in one patient after removal of the T-tube on Day 14 after operation ( P = 1.000). With a median follow-up time of 40 months for both groups, stone recurrence was noted in two patients in Group A and three patients in Group B ( P = 0.672). Primary closure of the CBD is safe and feasible in selected patients after laparoscopic choledochotomy. It results in shorter duration of hospital stay without the need for carrying/care of a T-tube in the postoperative period and similar stone recurrence as that of the conventional method.

This paper discusses the grinding performance of ZnS. Its the base for ultra-precision grinding to master the mechanical properties and removal mechanism of ZnS which is an infrared material. This study determined the hardnessfracture toughness and critical grinding depth by micro-indentation test; Based on the result of nanoindentation test, constitutive model of ZnS was established and simulation of indentation and scratch were completed. Grinding removal mechanism and effect of process parameters on scratch result were explored by scratch test. It was found that test was consistent with simulation.

Iron–sulfur clusters are ubiquitous in biology and function in electron transfer and catalysis. They are assembled from iron and cysteine sulfur on protein scaffolds. Iron is typically stored as iron oxyhydroxide, ferrihydrite, encapsulated in 12 nm shells of ferritin, which buffers cellular iron availability. Here we have characterized IssA, a protein that stores iron and sulfur as thioferrate, an inorganic anionic polymer previously unknown in biology. IssA forms nanoparticles reaching 300 nm in diameter and is the largest natural metalloprotein complex known. It is a member of a widely distributed protein family that includes nitrogenase maturation factors, NifB and NifX. IssA nanoparticles are visible by electron microscopy as electron-dense bodies in the cytoplasm. Purified nanoparticles appear to be generated from 20 nm units containing ∼6,400 Fe atoms and ∼170 IssA monomers. In support of roles in both iron–sulfur storage and cluster biosynthesis, IssA reconstitutes the [4Fe-4S] cluster in ferredoxin in vitro., The biosynthesis of iron-sulfur clusters in anaerobic organisms has not been extensively investigated. Here, the authors identify and characterize a multi-subunit protein that stores iron and sulfur in thioferrate for the assembly of the clusters in Pyrococcus furiosus.

Candida albicans is an opportunistic human fungal pathogen. The ability to switch among multiple cellular forms is key to its pathogenesis. The Dbf4-dependent protein kinase gene CDC7 is conserved due to its role in initiating DNA replication. Because a C. albicans Cdc7 (Cacdc7) homozygous null was not viable, we generated a C. albicans strain with a deleted C. albicans CDC7 (CaCDC7) allele and an expression-repressible allele. Surprisingly, cells of the strain grew as hyphae under the repressed conditions. The in vitro kinase assays confirmed that CaCdc7 (K232) and CaCdc7 (T437) are critical for catalytic and phosphoacceptor of activation activity, respectively. C. albicans cells formed hyphae when expressing either the catalytically inactive CaCdc7 (K232R) or the phosphoacceptor-deficient CaCdc7 (T437A). While CaCdc7 interacted with CaDbf4, cells of the strain in which CaCDC7 was repressed were not rescued by constitutively expressing C. albicans DBF4 or vice versa. We conclude that CaDBF4-dependent CaCDC7 is an essential gene suppressing the hyphal development.

Hyperthermophiles are microorganisms that thrive in extremely hot environments with temperatures near and even above 100°C. They are the most deeply rooted microorganisms on phylogenetic trees suggesting they may have evolved to survive in the early hostile earth. The simple respiratory systems of some of these hyperthermophiles make them potential candidates to develop microbial fuel cells (MFC) that can generate power at temperatures approaching the boiling point. We explored extracellular electron transfer in the hyperthermophilic archaeon Pyrococcus furiosus (Pf) by studying its ability to generate electricity in a two-chamber MFC. Pf growing in defined medium functioned as an anolyte in a MFC operated at 90°C, generating a maximum current density of 2 A m−2 and a peak power density of 225 mW m−2 without the addition of any external redox mediator. Electron microscopy and electrochemical impedance spectroscopy of the anode with the attached Pf biofilm demonstrated bio-electrochemical behavior that led to electricity generation in the MFC via direct electron transfer. This proof of concept study reveals for the first time that a hyperthermophile such as Pf can generate electricity in MFC at extreme temperatures. Biotechnol. Bioeng. 2017;114: 1419–1427. © 2017 Wiley Periodicals, Inc.

[NiFe] hydrogenases are metalloenzymes that catalyze the reversible oxidation of H2. While electron transfer to and from the active site is understood to occur through iron–sulfur clusters, the mechanism of proton transfer is still debated. Two mechanisms for proton exchange with the active site have been proposed that involve distinct and conserved ionizable amino acid residues, one a glutamate, and the other an arginine. To examine the potential role of the conserved glutamate on active site acid–base chemistry, we mutated the putative proton donor E17 to Q in the soluble hydrogenase I from Pyrococcus furiosus using site directed mutagenesis. FTIR spectroscopy, sensitive to the CO and CN ligands of the active site, reveals catalytically active species generated upon reduction with H2, including absorption features consistent with the Nia-C intermediate. Time-resolved IR spectroscopy, which probes active site dynamics after hydride photolysis from Nia-C, indicates the E17Q mutation does not interfere wit...

Hydrogenases (H2ases) represent one of the most striking examples of biological proton-coupled electron transfer (PCET) chemistry, functioning in facile proton reduction and H2 oxidation involving long-range proton and electron transport. Spectroscopic and electrochemical studies of the [NiFe] H2ases have identified several catalytic intermediates, but the details of their interconversion are still a matter of debate. Here we use steady state and time-resolved infrared spectroscopy, sensitive to the CO ligand of the active site iron, as a probe of the proton inventory as well as electron and proton transfer dynamics in the soluble hydrogenase I from Pyrococcus furiosus. Subtle shifts in infrared signatures associated with the Nia-C and Nia-S states as a function of pH revealed an acid-base equilibrium associated with an ionizable amino acid near the active site. Protonation of this residue was found to correlate with the photoproduct distribution that results from hydride photolysis of the Nia-C state, in which one of the two photoproduct states becomes inaccessible at low pH. Additionally, the ability to generate Nia-S via PCET from Nia-C was weakened at low pH, suggesting prior protonation of the proton acceptor. Kinetic and thermodynamic analysis of electron and proton transfer with respect to the various proton inventories was utilized to develop a chemical model for reversible hydride oxidation involving two intermediates differing in their hydrogen bonding character.

Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity’s growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophilePyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used inin vitrosystems for hydrogen production and NADPH generation and these systems are also discussed.

The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a [NiFe] hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR(-). The results establish an elementary mechanistic understanding of catalysis in a [NiFe] hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.

We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi-organ damage including the kidneys. In particular, excessive deposition of C5 and C5b-9-the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE-sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up-regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE-induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro-inflammatory cytokines IL-2, TNF-α and IFN-γ in the kidney tissue (P 0.05); this was accompanied by increased expression of P-p38, P-ERK and P-JNK proteins (P 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P-p38, P-ERK and P-JNK proteins (P 0.05) and also consistently reduced the TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ (P 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE or other environmental chemicals.

Based on mitochondrial cytochrome b gene sequence analysis, the history of true sheep (Ovis) began approximately 3.12 million years ago (MYA). The evolution of Ovis resulted in three generally accepted genetic groups: Argaliforms, Moufloniforms, and Pachyceriforms. The Pachyceriforms of the subgenus Pachyceros comprise the thin-horn sheep Ovis nivicola (snow sheep), Ovis dalli (Dall and Stone sheep), and Ovis canadensis (Rocky Mountain and desert bighorn). North America wild sheep (O. canadensis and O. dalli) evolved separately from Eurasian wild sheep and diverged from each other about 1.41 MYA. Ancestral stock that gave rise to snow sheep, Moufloniforms, and Argaliforms occurred 2.3 MYA, which then gave rise to two different extant lines of snow sheep that diverged from each other about 1.96 MYA. The more recent nivicola line is genetically closer to the North American wild sheep and may represent a close association during the refugium when Alaska and Siberia were connected by the Bering land bridge. The earlier period of evolution of the Pachyceriforms suggests they may have first evolved in Eurasia, the oldest ancestor then giving rise to North American wild sheep, and that a canadensis-like ancestor most likely gave rise to nivicola. Cytogenetic analysis further validates that the standard diploid number for modern nivicola is 52.