Nikolay A. Zorin

Continuous monitoring of the activation and activity of [NiFe]-hydrogenases by membrane-inlet mass spectrometry

The hydrogen-deuterium (H + /D 2 ) exchange reaction catalyzed by [NiFe]-hydrogenases in the D 2 /H 2 O system has been used to study enzyme activation and activity by membrane-inlet mass spectrometry. The activation of the [NiFe]-hydrogenases from Thiocapsa roseopersicina (HynSL), Desulfovibrio fructosovorans (HynSL), Desulfomicrobium baculatum (HysSL), Rhodobacter capsulatus (HupUV), and of the bidirectional tetrameric HoxFUYH enzymes from Synechocystis PCC 6308 (Gloeocapsa alpicola) and Anabaena variabilis ATCC 29413 was determined in response to oxygen depletion and to reductant addition (molecular hydrogen, reduced methyl viologen). Natural physiological activators (NADH, NADPH) of the bidirectional [NiFe] hydrogenases could also be identified by the H + /D 2 exchange reaction. The data are discussed in the light of current models of hydrogenase catalytic mechanism.

A hydrogen biosensor made of clay, poly(butylviologen), and hydrogenase sandwiched on a glass carbon electrode

A hydrogen gas (H(2)) biosensor was developed in which hydrogenase (H(2)ase) was immobilized and sandwiched between two layers of a montmorillonite clay and poly(butylviologen) (PBV) mixture on a glass carbon electrode. The immobilized PBV efficiently enhanced the electron transfer among the electrode, H(2)ase, and methyl viologen in solution. Both PBV and methyl viologen acted as the electron carrier in the clay-PBV-H(2)ase modified electrode. The clay-PBV-H(2)ase electrode catalyzed the oxidation of H(2) to protons (H(+)) with the electrons being transferred by viologen groups to the electrode. The activation energy of this process was 38+/-2 kJ/mol at pH 7. The catalytic current of the clay-PBV-H(2)ase electrode increased linearly when exposed to increasing concentrations of H(2) gas. In contrast, this electrode showed no activity when exposed to three combustible compounds, namely, carbon monoxide, methane and methanol. The optimum pH range for the oxidation of H(2) by the clay-PBV-H(2)ase electrode was from 7 to 10. Electron transfer process in the clay-PBV-H(2)ase electrode is discussed.

Characterization of the Hydrogen-Deuterium Exchange Activities of the Energy-Transducing HupSL Hydrogenase and H 2 -Signaling HupUV Hydrogenase in Rhodobacter capsulatus

Rhodobacter capsulatus synthesizes two homologous protein complexes capable of activating molecular H(2), a membrane-bound [NiFe] hydrogenase (HupSL) linked to the respiratory chain, and an H(2) sensor encoded by the hupUV genes. The activities of hydrogen-deuterium (H-D) exchange catalyzed by the hupSL-encoded and the hupUV-encoded enzymes in the presence of D(2) and H(2)O were studied comparatively. Whereas HupSL is in the membranes, HupUV activity was localized in the soluble cytoplasmic fraction. Since the hydrogenase gene cluster of R. capsulatus contains a gene homologous to hoxH, which encodes the large subunit of NAD-linked tetrameric soluble hydrogenases, the chromosomal hoxH gene was inactivated and hoxH mutants were used to demonstrate the H-D exchange activity of the cytoplasmic HupUV protein complex. The H-D exchange reaction catalyzed by HupSL hydrogenase was maximal at pH 4. 5 and inhibited by acetylene and oxygen, whereas the H-D exchange catalyzed by the HupUV protein complex was insensitive to acetylene and oxygen and did not vary significantly between pH 4 and pH 11. Based on these properties, the product of the accessory hypD gene was shown to be necessary for the synthesis of active HupUV enzyme. The kinetics of HD and H(2) formed in exchange with D(2) by HupUV point to a restricted access of protons and gasses to the active site. Measurement of concentration changes in D(2), HD, and H(2) by mass spectrometry showed that, besides the H-D exchange reaction, HupUV oxidized H(2) with benzyl viologen, produced H(2) with reduced methyl viologen, and demonstrated true hydrogenase activity. Therefore, not only with respect to its H(2) signaling function in the cell, but also to its catalytic properties, the HupUV enzyme represents a distinct class of hydrogenases.

Reversible hydrogenase of Anabaena variabilis ATCC 29413: catalytic properties and characterization of redox centres.

The catalytic and spectroscopic properties of the reversible hydrogenase from the cyanobacterium Anabaena variabilis have been examined. The hydrogenase required reductive activation in order to elicit hydrogen‐oxidation activity. Carbon monoxide was a weak (K i = 35 μM), reversible and competitive inhibitor. A flavin with the chromatographic properties of FMN, and nickel were detected in the purified enzyme. A. variabilis hydrogenase exhibited electron paramagnetic resonance (EPR) spectra in its hydrogen‐reduced state, indicative of [2Fe‐2S] and [4Fe‐4S] clusters. Although no EPR signals due to nickel were detected, the results are consistent with the enzyme being a flavin‐containing hydrogenase of the nickel‐iron type.

Biomolecular device for photoinduced hydrogen production

In order to construct a molecular device for photoinduced hydrogen production, a model has been designed and first results in the framework of this multicomponent system are presented. This device should involve Photosystem 1, Photosystem 2 and hydrogenase in a modular configuration which allows to combine appropriate proteins from various-mainly thermophilic-organisms. Parts of this modular system can be easily exchanged and separately characterized and optimized. Here the optimization of one component of this device, the hydrogenase from Thiocapsa roseopersicina, is shown. The isolated hydrogenase was deposited as Langmuir-Blodgett (LB) film on quartz glass and ITO electrodes, respectively, and its activity was measured in dependence of counter ions, presence of oxygen and number of immobilized layers. While poly-L-lysine or poly-butyl-viologen as counter ion in the subphase stabilized the protein complex on quartz glass (up to about 30 mN/m surface pressure), Ca 2+ resulted in a dramatic activity loss at a much lower surface pressure (15 mN/m). Also, the presence of even smallest amounts of oxygen or an excess amount of protein on an ITO electrode resulted in a significant decrease of the hydrogen production as did the increase in the number of layers-as shown by electrochemical measurements.

Photoinduced hydrogen evolution by use of porphyrin, EDTA, viologens and hydrogenase in solutions and Langmuir?Blodgett films

Abstract Photoinduced hydrogen evolution was investigated by use of a zinc porphyrin, EDTA, viologens and hydrogenase (H2ase) in the solutions and Langmuir–Blodgett (LB) films. An almost linear increase of hydrogen evolution rate was observed with the increase of H2ase concentrations from 1 to 5 μg / ml . For the zinc porphyrin, EDTA and methyl viologen, when their concentrations increased to a given value, hydrogen evolution did not show obvious increase. Phospholipid-porphyrin mixed LB films were prepared and used as photosensitizer for the photoinduced hydrogen evolution. Spectroscopic studies of the deoxygenated solutions indicated a “new” absorption band (in the solutions) or sharp peaks (in the LB films) when the sample solutions were irradiated, which was ascribed to the formation of an excited complex of porphyrin–EDTA (or -EDTA breakdown products). This excited complex was unstable to air.

Langmuir-Blodgett film of hydrogenase for electrochemical hydrogen production

Abstract LB films of thermostable hydrogenase from Thiocapsa roseopersicina were prepared on electrodes. Poly- l -lysine was used as a counter ion which stabilizes the protein and improves the protein transfer onto substrates. Both hydrogen production and oxidation activities were observed in the hydrogenase monolayer on substrate. The monolayer film of hydrogenase showed a high thermal stability as in solution, i.e. denaturation did not occur up to 70°C. Electrochemical hydrogen evolution was observed by biasing the ITO electrode on which the LB film of hydrogenase was mounted in the presence of methyl viologen as a mediator.

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.

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.

Hydrogen production by model systems including hydrogenases from phototrophic bacteria

Abstract Hydrogenases of purple (Rhodobacter capsulatus, Thiocapsa roseopersicina) and green (Chlorobium limicola, Chloroflexus aurantiacus) bacteria catalyse the reactions of H 2 evolution (uptake) and isotopic exchange in the system D 2 H 2 O at a high rate (20–60 units mg −1 protein; 30°C). The enzyme active center includes a proton-accepting group, an [FeS] cluster and nickel. The data proving the direct participation of nickel in H 2 activation are obtained. The hydrogenases from phototrophic bacteria are characterized by a high stability to denaturating factors. Thiocapsa roseopersicina hydrogenase absorbed at the carbon electrode accelerates electrochemical H 2 oxidation and evolution. Biocatalytic systems containing chloroplasts, ferredoxin and hydrogenase produce H 2 at a rate up to 100 μmol (h mg Chl) −1 but exhibit low stability. Thiocapsa roseopersicina hydrogenase adsorbed on the particles of inorganic semiconductor TiO 2 carries out H 2 photoproduction. The quantum yield of this process was 30% at I light = 1 × l 0 4 erg cm −2 s −1 .