Author Correction: The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single molecule sequencing of the M13 virus genome without amplification.

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.

The efficient hemostatic effect of Antarctic krill chitosan is related to its hydration property.

Antarctic krill chitosan (A-Chitosan) was first evaluated in its hemostatic effect in this study. The prepared A-Chitosan powder showed low level of crystallinity and significantly high water binding capacity as 1293% (w/w). By mice tail amputation model and blood coagulation timing experiment, it is showed that this chitosan accelerated the tail hemostasis by 55% and shortened the blood clotting time by 38%. This efficacy was better than two other commercial chitosans investigated and was corresponding to their water binding capacities. Through examining the effect of chitosan on blood components, it could be found that platelets adhesion was mainly affected by the water binding capacity, and red blood cells aggregation was dependent on their deacetylation degree. The physicochemical properties resulted in better hydration property of chitosan would improve its hemostatic effect. These results suggested that Antarctic krill chitosan is a good candidate for hemostatic application.

The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster.

Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation.

Covalent attachment of FeFe hydrogenases to carbon electrodes for direct electron transfer.

Direct electron transfer between enzymes and electrodes is now commonly achieved, but obtaining protein films that are very stable may be challenging. This is particularly crucial in the case of hydrogenases, the enzymes that catalyze the biological conversion between dihydrogen and protons, because the instability of the hydrogenase films may prevent the use of these enzymes as electrocatalysts of H(2) oxidation and production in biofuel cells and photoelectrochemical cells. Here we show that two different FeFe hydrogenases (from Chamydomonas reinhardtii and Clostridium acetobutylicum) can be covalently attached to functionalized pyrolytic graphite electrodes using peptidic coupling. In both cases, a surface patch of lysine residues makes it possible to favor an orientation that is efficient for fast, direct electron transfer. High hydrogen-oxidation current densities are maintained for up to one week, the only limitation being the intrinsic stability of the enzyme. We also show that covalent attachment has no effect on the catalytic properties of the enzyme, which means that this strategy can also used be for electrochemical studies of the catalytic mechanism.

The quest for a functional substrate access tunnel in FeFe hydrogenase.

We investigated di-hydrogen transport between the solvent and the active site of FeFe hydrogenases. Substrate channels supposedly exist and serve various functions in certain redox enzymes which use or produce O2, H2, NO, CO, or N2, but the preferred paths have not always been unambiguously identified, and whether a continuous, permanent channel is an absolute requirement for transporting diatomic molecules is unknown. Here, we review the literature on gas channels in proteins and enzymes and we report on the use of site-directed mutagenesis and various kinetic methods, which proved useful for characterizing substrate access to the active site of NiFe hydrogenase to test the putative "static" H2 channel of FeFe hydrogenases. We designed 8 mutations in attempts to interfere with intramolecular diffusion by remodeling this putative route in Clostridium acetobutylicum FeFe hydrogenase, and we observed that none of them has a strong effect on any of the enzyme's kinetic properties. We suggest that H2 may diffuse either via transient cavities, or along a conserved water-filled channel. Nitrogenase sets a precedent for the involvement of a hydrophilic channel to conduct hydrophobic molecules.

CO disrupts the reduced H-cluster of FeFe hydrogenase. A combined DFT and protein film voltammetry study.

Carbon monoxide is often described as a competitive inhibitor of FeFe hydrogenases, and it is used for probing H(2) binding to synthetic or in silico models of the active site H-cluster. Yet it does not always behave as a simple inhibitor. Using an original approach which combines accurate electrochemical measurements and theoretical calculations, we elucidate the mechanism by which, under certain conditions, CO binding can cause permanent damage to the H-cluster. Like in the case of oxygen inhibition, the reaction with CO engages the entire H-cluster, rather than only the Fe(2) subsite.

Asymmetrical stress generated by the erythrocyte lipid flippase triggers multiple bud formation on the surface of spherical giant liposomes.

Proteo-giant liposomes were electroformed from a mixture of lecithin vesicles and inside-out vesicles from erythrocytes. After addition of Mg-ATP in the vicinity of the proteo-giant liposomes, small buds appeared on the liposome surfaces, which--via an increase in lipids in the outer monolayer--demonstrated the active transport of lipids from the inner to the outer monolayer, indicating flippase activity.