Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN^?) ligands. Hydrogen (H₂) and oxygen (O₂) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. ⁵⁷Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fe_d in a [4Fe4S-2Fe]^3 ? complex with the net spin on Fe_d whereas Hox-CO shows an apical CN^? at Fe_d in a [4Fe4S-2Fe(CO)]^3 ? complex with net spin sharing among Fe_p and Fe_d (proximal or distal iron ions in [2Fe]). At ambient O₂ pressure, a novel H-cluster species (Hox-O₂) accumulated in C169A, assigned to a [4Fe4S-2Fe(O₂)]^3 ? complex with an apical superoxide (O₂^?) carrying the net spin bound at Fe_d. H₂ exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)]^3 ? complex with the net spin on the reduced cubane, an apical hydride at Fe_d, and a proton at a cysteine ligand. Hox-O₂ and Hhyd are stabilized by impaired O₂^– protonation or proton release after H₂ cleavage due to interruption of the proton path towards and out of the active site.     

Cascade‐Type Prelithiation Approach for Li‐Ion Capacitors

An industry‐relevant method for pre‐lithiation of lithium‐ion capacitors to balance the first charge irreversibility is demonstrated, which addresses the prime bottleneck for their market integration. Based on a composite positive electrode that integrates pyrene monomers and an insoluble lithiated base, Li₃PO₄, a “cascade‐type” process involving two consecutive irreversible reactions is proposed: i) oxidative electropolymerization of the pyrene moieties releases electrons and protons; ii) protons are captured by Li₃PO₄ and exchanged for a stoichiometric amount of Li⁺ into the electrolyte. (¹H, ¹⁹F, and ³¹P) NMR spectroscopy, operando X‐ray diffraction, and Raman spectroscopy support this mechanism. By decoupling the irreversible source of lithium ions from electrons, the cascade‐type pre‐lithiation allows the simultaneous enhancement of the capacity of the positive electrode, thanks to p‐doping of the resulting polymer. Remarkably, the proton scavenging properties of Li₃PO₄ also boost the polymerization process, which enables a 16% increase in capacity without detrimental effect on power properties and cyclability. Full cells integrating a cheap carbon black based negative electrode, show much‐improved capacity of 17 mAh g^‐1_electrodes (44 F g^‐1_electrodes, 3–4.4 V) and excellent stability over 2200 cycles at 1 A g^‐1. Thanks to its versatile chemistry and flexibility this approach in principle can be applied to any kind of ion‐battery.     

Phe(475) and Glu(446) but not Ser(445) participate in ATP-binding to the alpha-subunit of Na(+)/K(+)-ATPase

The ATP-binding site of Na(+)/K(+)-ATPase is localized on the large cytoplasmic loop of the alpha-subunit between transmembrane helices H(4) and H(5). Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser(445), Glu(446), and Phe(475) were proposed to be close to the binding pocket. Replacement of Phe(475) with Trp and Glu(446) with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser(445) with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser(445) is close to the binding site, although it does not participate in binding.     

Genetic engineering of brewing yeast to reduce the content of ethanol in beer

The GPD1 gene encoding the glycerol-3-phosphate dehydrogenase was overexpressed in an industrial lager brewing yeast (Saccharomyces cerevisiae ssp. carlsbergensis) to reduce the content of ethanol in beer. The amount of glycerol produced by the GPD1-overexpressing yeast in fermentation experiments simulating brewing conditions was increased 5.6 times and ethanol was decreased by 18% when compared to the wild-type. Overexpression of GPD1 does not affect the consumption of wort sugars. Only minor changes in the concentration of higher alcohols, esters and fatty acids could be observed in beer produced by the GPD1-overexpressing brewing yeast. However, the concentrations of several other by-products, particularly acetoin, diacetyl and acetaldehyde, were considerably increased.     

Arachidonic acid mediates dual effect of TNF-alpha on Ca2+ transients and contraction of adult rat cardiomyocytes

Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A₂ (PLA₂) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca²⁺ concentration([Ca²⁺]_i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca²⁺]_i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca²⁺]_i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca²⁺]_i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA₂.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA₂/AA pathway in mediating thecontractile effects of TNF-.

     

Effects of longitudinal stretch on VSM tone and distensibility of muscular conduit arteries

With progressing age, large arteries diminish their longitudinal stretch, which in extreme cases results in tortuosity. Increased age is also associated with loss of vessel distensibility. We measured pressure-diameter curves from muscular porcine carotid arteries ex vivo at different longitudinal stretch ratios (lambda(z) = 1.4 and 1.8) and under different vascular smooth muscle (VSM) conditions (fully relaxed, normal VSM tone, and maximally contracted). Distensibility was found to be halved by decreasing longitudinal stretch from lambda(z) = 1.8 to 1.4 at physiological pressures. This counterintuitive observation is possible because highly nonlinear elastic modulus of the artery and anisotropic properties. Furthermore, a significantly larger basal VSM contraction was observed at lambda(z) = 1.8 than 1.4, although this was clearly not related to a myogenic response during inflation. This dependence of VSM tone to longitudinal stretch may have possible implications on the functional characteristics of the arterial wall.     

Chance or design? Some specific considerations concerning synaptic boutons in cat visual cortex

To understand the rules by which axons lay down their synaptic boutons we analyzed the linear bouton distributions in 39 neurons (23 spiny, 13 smooth) and 3 thalamic axons, which were filled intracellularly with horseradish peroxidase (HRP) during in vivo experiments in cat area 17. The variation of the total number of boutons and the total axonal length was large (789–7912 boutons, 12–126 mm). The overall linear bouton density for smooth cells was higher than that of spiny cells and thalamic afferents (mean ± sd, 110 ± 21 and 78 ± 27 boutons per mm of axonal length). The distribution of boutons varied according to their location on the tree. Distal axon collaterals (first and second order segments in Horton-Strahler ordering) of smooth neurons had a 3.5 times higher, spiny cells and thalamic afferents a 2 times higher bouton density compared to the higher order (more proximal) segments. The distribution of interbouton intervals was positively skewed and similar for cells of the same type. In most cases a γ-distribution fitted well, but the distributions had a tendency to have a heavier tail. To a first approximation these bouton distributions are consistent with both diffuse and specific models of interneuronal connections. Quite simple rules can explain these distributions and the connections between the different classes of neurons.     

Geochemistry and Microbial Populations in Sediments of the Northern Baffin Bay,Arctic

The Northern Baffin Bay between Greenland and Canada is a remote Arctic area restricted in primary production by seasonal ice cover, with presumably low sedimentation rates, carbon content and microbial activities in its sediments. Our aim was to study the so far unknown subseafloor geochemistry and microbial populations driving seafloor ecosystems. Shelf sediments had the highest organic carbon content, numbers of Bacteria and Archaea, and microcosms inoculated from Shelf sediments showed highest sulfate reduction and methane production rates. Sediments in the central deep area and on the southern slope contained less organic carbon and overall lower microbial numbers. Similar 16S rRNA gene copy numbers of Archaea and Bacteria were found for the majority of the sites investigated. Sulfate in pore water correlated with dsrA copy numbers of sulfate-reducing prokaryotes and differed between sites. No methane was found as free gas in the sediments, and mcrA copy numbers of methanogenic Archaea were low. Methanogenic and sulfate-reducing cultures were enriched on a variety of substrates including hydrocarbons. In summary, the Greenlandic shelf sediments contain vital microbial communities adapted to their specific environmental conditions.     

Circadian Pattern of Wheel‐Running Activity of a South American Subterranean Rodent (Ctenomys cf knightii)

Circadian rhythms are regarded as essentially ubiquitous features of animal behavior and are thought to confer important adaptive advantages. However, although circadian systems of rodents have been among the most extensively studied, most comparative biology is restricted to a few related species. In this study, the circadian organization of locomotor activity was studied in the subterranean, solitary north Argentinean rodent, Ctenomys knightii. The genus, Ctenomys, commonly known as Tuco‐tucos, comprises more than 50 known species over a range that extends from 12°S latitude into Patagonia, and includes at least one social species. The genus, therefore, is ideal for comparative and ecological studies of circadian rhythms. Ctenomys knightii is the first of these to be studied for its circadian behavior. All animals were wild caught but adapted quickly to laboratory conditions, with clear and precise activity‐rest rhythms in a light‐dark (LD) cycle and strongly nocturnal wheel running behavior. In constant dark (DD), the rhythm expression persisted with free‐running periods always longer than 24 h. Upon reinstatement of the LD cycle, rhythms resynchronized rapidly with large phase advances in 7/8 animals. In constant light (LL), six animals had free‐running periods shorter than in DD, and 4/8 showed evidence of “splitting.” We conclude that under laboratory conditions, in wheel‐running cages, this species shows a clear nocturnal rhythmic organization controlled by an endogenous circadian oscillator that is entrained to 24 h LD cycles, predominantly by light‐induced advances, and shows the same interindividual variable responses to constant light as reported in other non‐subterranean species. These data are the first step toward understanding the chronobiology of the largest genus of subterranean rodents.