The HydS C-terminal domain of the Thiocapsa bogorovii HydSL hydrogenase is involved in membrane anchoring and electron transfer.

Thiocapsa bogorovii BBS (former name Thiocapsa roseopersicina) contains HydSL hydrogenase belonging to 1e subgroup of NiFe hydrogenases (isp-type). The operon of these hydrogenases contains gene for small subunit (hydS), gene for large subunit (hupL), and genes isp1 and isp2 between them. It is predicted that last two genes code electron transport careers for electron transfer from/to HydSL hydrogenase. However, the interaction between them is unclear. The aim of this study was to determine structural and functional role of T. bogorovii HydS C-terminal end. For this purpose, we modelled all subunits of the complex HydS-HydL-Isp1-Isp2. Hydrophobicity surface analysis of the Isp1 model revealed highly hydrophobic helices suggesting potential membrane localization, as well as the hydrophilic C-terminus, which is likely localized outside of membrane. Isp1 model was docked with models of full length and C-terminal truncated HydSL hydrogenases and results illustrate the possibility of HydSL membrane anchoring via transmembrane Isp1 with essential participation of C-terminal end of HydS in the interaction. C-terminal end of HydS subunit was deleted and our studies revealed that the truncated HydSL hydrogenase detached from cellular membranes in contrast to native hydrogenase. It is known that HydSL hydrogenase in T. bogorovii performs the reaction of elemental sulfur reduction (S0 + H2 = ≥H2S). Cells with truncated HydS produced much less H2S in the presence of H2 and S0. Thus, our data support the conclusion that C-terminal end of HydS subunit participates in interaction of HydSL hydrogenase with Isp1 protein for membrane anchoring and electron transfer.

Interaction of HydSL hydrogenase from Thiocapsa roseopersicina with cyanide leads to destruction of iron-sulfur clusters.

The effects of cyanide on enzymatic activity and absorption spectra in the visible and mid-IR (2150-1850cm-1) regions were characterized for purified HydSL hydrogenase from the purple sulfur bacterium Thiocapsa (T.) roseopersicina BBS. Prolonged incubation (over hours) of T. roseopersicina hydrogenase with exogenous cyanide was shown to result in an irreversible loss of activity of the enzyme in both the oxidized (as isolated) and H2-reduced states. The frequency position of the active site CO and CN- ligand stretching bands in the Fourier transform infrared (FTIR) spectrum of the oxidized form of hydrogenase was not influenced by cyanide treatment. The 410-nm absorption band characteristic of hydrogenase iron­sulfur clusters showed a bleaching concomitantly with cyanide inactivation. A new band at 2038cm-1 was present in the FTIR spectrum of the cyanide-inactivated preparation, which band is assignable to ferrocyanide as a possible product of a destructive interaction of hydrogenase with cyanide. The results are interpreted in terms of a slow destruction of iron­sulfur clusters of hydrogenase in the presence of cyanide accompanied by a release of iron ions in the form of ferrocyanide into the surrounding solution. Such a slow and irreversible cyanide-dependent inactivation seems to be complementary to a recently described rapid, reversible inhibitory reaction of cyanide with the active site of hydrogenases [S.V. Hexter, M.-W. Chung, K.A. Vincent, F.A. Armstrong, J. Am. Chem. Soc. 136 (2014) 10470-10477].

Preparation of Hydrogenase and Viologen-Functionalized Carbon Nanotube Composites for Electrochemical Oxidation of Hydrogen.

Bionanocomposites of hydrogenase and viologen-functionalized carbon nanotubes (H2ase/V-MWNTs) were prepared and characterized by using infrared spectra and scanning electron microscope. Cyclic voltammograms revealed two couples of redox waves corresponded to the electron transfer processes of viologens and [4Fe-4S]2+/1+ clusters of hydrogenase. The current intensity was enhanced in the H2 atmosphere, which suggested that the present bio-nanocomposites could be used as heterogeneous bio-catalyst to catalyze reversible reaction between protons and H2.

Biochemical and Structural Characterization of Enolase from Chloroflexus aurantiacus: Evidence for a Thermophilic Origin.

Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate during both glycolysis and gluconeogenesis, and is required by all three domains of life. Here, we report the purification and biochemical and structural characterization of enolase from Chloroflexus aurantiacus, a thermophilic anoxygenic phototroph affiliated with the green non-sulfur bacteria. The protein was purified as a homodimer with a subunit molecular weight of 46 kDa. The temperature optimum for enolase catalysis was 80°C, close to the measured thermal stability of the protein which was determined to be 75°C, while the pH optimum for enzyme activity was 6.5. The specific activities of purified enolase determined at 25 and 80°C were 147 and 300 U mg(-1) of protein, respectively. K m values for the 2-phosphoglycerate/phosphoenolpyruvate reaction determined at 25 and 80°C were 0.16 and 0.03 mM, respectively. The K m values for Mg(2+) binding at these temperatures were 2.5 and 1.9 mM, respectively. When compared to enolase from mesophiles, the biochemical and structural properties of enolase from C. aurantiacus are consistent with this being thermally adapted. These data are consistent with the results of our phylogenetic analysis of enolase, which reveal that enolase has a thermophilic origin.

Pd(II)-Directed Encapsulation of Hydrogenase within the Layer-by-Layer Multilayers of Carbon Nanotube Polyelectrolyte Used as a Heterogeneous Catalyst for Oxidation of Hydrogen.

A metal-directed assembling approach has been developed to encapsulate hydrogenase (H2ase) within a layer-by-layer (LBL) multilayer of carbon nanotube polyelectrolyte (MWNT-PVPMe), which showed efficient biocatalytic oxidation of H2 gas. The MWNT-PVPMe was prepared via a diazonium process and addition reactions with poly(4-vinylpyridine) (PVP) and methyl iodide (MeI). The covalently attached polymers and organic substituents in the polyelectrolyte comprised 60-70% of the total weight. The polyelectrolyte was then used as a substrate for H2ase binding to produce MWNT-PVPMe@H2ase bionanocomposites. X-ray photoelectron spectra revealed that the bionanocomposites included the elements of Br, S, C, N, O, I, Fe, and Ni, which confirmed that they were composed of MWNT-PVPMe and H2ase. Field emission transmission electron microscope images revealed that the H2ase was adsorbed on the surface of MWNT-PVPMe with the domains ranging from 20 to 40 nm. Further, with the use of the bionanocomposites as nanolinkers and Na2PdCl4 as connectors, the (Pd/MWNT-PVPMe@H2ase)n multilayers were constructed on the quartz and gold substrate surfaces by the Pd(II)-directed LBL assembling technique. Finally, the as-prepared LBL multilayers were used as heterogeneous catalysts for hydrogen oxidation with methyl viologen (MV(2+)) as an electron carrier. The dynamic processes for the reversible color change between blue-colored MV(+) and colorless MV(2+) (catalyzed by the LBL multilayers) were video recorded, which confirmed that the H2ase encapsulated within the present LBL multilayers was of much stronger stability and higher biocatalytic activity of H2 oxidation resulting in potential applications for the development of H2 biosensors and fuel cells.

Turning cellulose waste into electricity: hydrogen conversion by a hydrogenase electrode.

Hydrogen-producing thermophilic cellulolytic microorganisms were isolated from cow faeces. Rates of cellulose hydrolysis and hydrogen formation were 0.2 mM L(-1) h(-1) and 1 mM L(-1) h(-1), respectively. An enzymatic fuel cell (EFC) with a hydrogenase anode was used to oxidise hydrogen produced in a microbial bioreactor. The hydrogenase electrode was exposed for 38 days (912 h) to a thermophilic fermentation medium. The hydrogenase activity remaining after continuous operation under load was 73% of the initial value.

Photo-induced H2 production by [NiFe]-hydrogenase from T. roseopersicina covalently linked to a Ru(II) photosensitizer.

The potential of hydrogen as a clean renewable fuel source and the finite reserves of platinum metal to be utilized in hydrogen production catalysts have provided the motivation for the development of non-noble metal-based solutions for catalytic hydrogen production. There are a number of microorganisms that possess highly efficient hydrogen production catalysts termed hydrogenases that generate hydrogen under certain metabolic conditions. Although hydrogenases occur in photosynthetic microorganisms, the oxygen sensitivity of these enzymes represents a significant barrier in directly coupling hydrogen production to oxygenic photosynthesis. To overcome this barrier, there has been considerable interest in identifying or engineering oxygen tolerant hydrogenases or generating mimetic systems that do not rely on oxygen producing photocatalysts. In this work, we demonstrate photo-induced hydrogen production from a stable [NiFe]-hydrogenase coupled to a [Ru(2,2'-bipyridine)(2)(5-amino-1,10-phenanthroline)](2+) photocatalyst. When the Ru(II) complex is covalently attached to the hydrogenase, photocatalytic hydrogen production occurs more efficiently in the presence of a redox mediator than if the Ru(II) complex is simply present in solution. Furthermore, sustained hydrogen production occurs even in the presence of oxygen by presumably creating a local anoxic environment through the reduction of oxygen similar to what is proposed for oxygen tolerant hydrogenases. These results provide a strong proof of concept for engineering photocatalytic hydrogen production in the presence of oxygen using biohybrid mimetic systems.

Langmuir-Blodgett films of pyridyldithio-modified multiwalled carbon nanotubes as a support to immobilize hydrogenase.

Pyridylthio-modified multiwalled carbon nanotubes (pythio-MWNTs) have been prepared by a reaction of the oxidized MWNTs with S-(2-aminoethylthio)-2-thiopyridine hydrochloride. The obtained pythio-MWNTs nanocomposites formed stable floating monolayers at the air-water interface, which were transferred onto substrate surfaces by the Langmuir-Blodgett (LB) method. Compositions and morphologies of the LB films were characterized by absorption, Raman, X-ray photoelectron spectra as well as by scan electron microscopy and atomic force microscopy. These pythio-MWNTs LB films were then used as a support to immobilize hydrogenase (H(2)ase) to form bionanocomposite of pythio-MWNTs-H(2)ase. Cyclic voltammograms for indium tin oxide electrode covered with the pythio-MWNTs-H(2)ase films were investigated in both Ar and H(2) saturated 0.05 M KCl electrolyte solutions at pH from 4.0 to 9.0. A reversible redox couple of [4Fe-4S](2+/1+) clusters of H(2)ase was recorded when the pH value was 6.0 and 9.0, with reduction and oxidation potentials appearing at about -0.70 and -0.35 V vs Ag/AgCl, respectively. It was revealed that the H(2)ase was of high catalytic activity and strong stability in the LB films of pythio-MWNTs-H(2)ase. Hence, we suggested that the present bionanocomposites could be used as heterogeneous biocatalyst to catalyze reversible reaction between protons and H(2), resulting in potential applications in biohydrogen evolution and H(2) biofuel cells.

Hydrogen enhances nickel tolerance in the purple sulfur bacterium Thiocapsa roseopersicina.

A common microbial strategy for detoxifying metals involves redox transformation which often results in metal precipitation and/or immobilization. In the present study, the influence of ionic nickel [Ni(II)] on growth of the purple sulfur bacterium Thiocapsa roseopersicina was investigated. The results suggest that Ni(II) in the bulk medium at micromolar concentrations results in growth inhibition, specifically an increase in the lag phase of growth, a decrease in the specific growth rate, and a decrease in total protein concentration when compared to growth controls containing no added Ni(II). The inhibitory effects of Ni(II) on the growth of T. roseopersicina could be partially overcome by the addition of hydrogen (H(2)) gas. However, the inhibitory effects of Ni(II) on the growth of T. roseopersicina were not alleviated by H(2) in a strain containing deletions in all hydrogenase-encoding genes. Transmission electron micrographs of wild-type T. roseopersicina grown in the presence of Ni(II) and H(2) revealed a significantly greater number of dense nanoparticulates associated with the cells when compared to wild-type cells grown in the absence of H(2) and hydrogenase mutant strains grown in the presence of H(2). X-ray diffraction and vibrating sample magnetometry of the dense nanoparticles indicated the presence of zerovalent Ni, suggesting Ni(II) reduction. Purified T. roseopersicina hyn-encoded hydrogenase catalyzed the formation of zerovalent Ni particles in vitro, suggesting a role for this hydrogenase in Ni(II) reduction in vivo. Collectively, these results suggest a link among H(2) metabolism, Ni(II) tolerance, and Ni(II) reduction in T. roseopersicina .

Transformation of metals and metal ions by hydrogenases from phototrophic bacteria.

The ability of hydrogenases isolated from Thiocapsa roseopersicina and Lamprobacter modestohalophilus to reduce metal ions and oxidize metals has been studied. Hydrogenases from both phototrophic bacteria oxidized metallic Fe, Cd, Zn and Ni into their ionic forms with simultaneous evolution of molecular hydrogen. The metal oxidation rate decreased in the series Zn > Fe > Cd > Ni and depended on the pH. The presence of methyl viologen in the reaction system accelerated this process. T. roseopersicina and L. modestohalophilus cells and their hydrogenases reduced Ni(II), Pt(IV), Pd(II) or Ru(III) to their metallic forms under H2 atmosphere. These results suggest that metals or metal ions can serve as electron donors or acceptors for hydrogenases from phototrophic bacteria.

Immobilization of active hydrogenases by encapsulation in polymeric porous gels.

Hydrogenases encapsulated in porous polymeric silica gels retain significant levels of hydrogen production activity when compared to hydrogenases in solution using reduced methyl viologen as an electron donor. Encapsulated hydrogenases remain active after storage at room temperature for longer than four weeks and are less sensitive to proteolytic digestion. Nanoscopic confinement of active hydrogenases in solids paves the way for their potential use in hydrogen producing catalytic materials applications.

Alpha2-Macroglobulin Modulates Interactions between Lymphocytes and Fibroblasts.

The aim of the present study was to evaluate the influence of apha2-macroglobulin (alpha(2)M) on lymphocyte adhesion to fibroblasts. Peripheral blood lymphocytes from healthy donors and two fibroblast lines (human diploid embryo fibroblasts M-19 and mouse transformed fibroblasts L929) were used in the experiments. alpha(2)M treatment of fibroblast monolayer appeared to result in the enhancement of lymphocyte adhesion to fibroblasts. The number of attached lymphocytes was increased by 2-2.5 times. It should be noted that the effect of alpha(2)M didn't depend on the conformational molecule changes, since either native or methylamine or plasmin transformed alpha(2)M approximately at the same fashion increased the lymphocyte adhesion to both allogeneic and xenogeneic fibroblasts. B lymphocytes were predominant cells that were attached to fibroblast monolayer without alpha(2)M treatment. However the percentage of adherent T lymphocytes was increased substantially after the fibroblast monolayer treatment by alpha(2)M. Subpopulation analysis has shown that fibroblast pretreatment by alpha(2)M didn't result in a selective adhesion of CD4(+) or CD8(+) T lymphocytes, but increased the adhesiveness for both T lymphocyte subpopulations. The data obtained demonstrate that besides its participation in the processes of fibroblast adhesion alpha(2)M is capable to modify the contact interaction of these cells with lymphocytes that may have an influence on the functional consequences of this process.

Adhesion of Fibroblasts to Immobilized alpha(2)-Macroglobulin.

This study examines effects of alpha(2)-macroglobulin (alpha(2)M) on adhesion of fibroblasts. Native alpha(2)M and transformed form of alpha(2)M, alpha(2)M-plasmin, were bound to plastic. Adhesion of mouse L929 and human embryo M-19 fibroblasts to immobilized alpha(2)M was estimated under various conditions by counting adherent cells using videomicroscopy and computer-assisted image analysis. alpha(2)M-plasmin, bound to plastic, induced adhesion and spreading of mouse L929 and human M-19 fibroblasts. Neither native alpha(2)M nor plasmin alone did not induce fibroblast adhesion. The adhesion to alpha(2)M-plasmin was undetectable at 4 degrees C, as well as when sodium azide was added or divalent ions were removed. These findings provide novel information on alpha(2)M functions. On the basis of these observations we hypothesized that alpha(2)M, immobilized in the extracellular matrix, can participate in the regulation of microenvironment effects on the cells, and, in particular, influence on fibroblast adhesion.