Oriented immobilization of a membrane-bound hydrogenase onto an electrode for direct electron transfer

The interaction of redox enzymes with electrodes is of great interest for studying the catalytic mechanisms of redox enzymes and for bioelectronic applications. Efficient electron transport between the biocatalysts and the electrodes has achieved more success with soluble enzymes than with membrane enzymes because of the higher structural complexity and instability of the latter proteins. In this work, we report a strategy for immobilizing a membrane-bound enzyme onto gold electrodes with a controlled orientation in its fully active conformation. The immobilized redox enzyme is the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough, which catalyzes H(2)-oxidation reversibly and is associated with the cytoplasmic membrane by a lipidic tail. Gold surfaces modified with this enzyme and phospholipids have been studied by atomic force microscopy (AFM) and electrochemical methods. The combined study indicates that by a two-step immobilization procedure the hydrogenase can be inserted via its lipidic tail onto a phospholipidic bilayer formed over the gold surface, allowing only mediated electron transfer between the enzyme and electrode. However, a one-step immobilization procedure favors the formation of a hydrogenase monolayer over the gold surface with its lipidic tail inserted into a phospholipid bilayer formed on top of the hydrogenase molecules. This latter method has allowed for the first time efficient electron transfer between a membrane-bound enzyme in its native conformation and an electrode.     

Density functional calculations for modeling the active site of nickel-iron hydrogenases. 2. Predictions for the unready and ready States and the corresponding activation processes

ZORA relativistic DFT calculations are presented which aim to model the geometric and electronic structure of the active site of NiFe hydrogenases in its EPR-active oxidized states Ni-A (unready state) and Ni-B (ready state). Starting coordinates are taken from the X-ray structure of a mutant of Desulfovibrio fructosovorans hydrogenase refined at 1.81 A resolution. Nine possible candidates for Ni-A and Ni-B are analyzed in terms of their geometric and electronic structure. Comparison of calculated geometric and magnetic resonance parameters with available experimental data indicates that both oxidized states have a micro-hydroxo bridge between the two metal centers. The different electronic structures of both forms can be explained by a modification of a terminal cysteine in Ni-B, best modeled by protonation of the sulfur atom. A possible mechanism for the activation of both oxidized forms is presented.     

Isolation and synthesis of N-acyladenine and adenosine alkaloids from a southern Australian marine sponge, Phoriospongia sp.

Chemical fractionation of the southern Australian marine sponge Phoriospongia sp. (CMB-03107) yielded phorioadenine A (1) as a nematocidal agent and the first reported example of a 6-N-acyladenine natural product. The structure of 1 was confirmed by spectroscopic analysis and the chemical synthesis of racemic (1a) and enantiomeric (1b) analogues. HPLC–ESIMS analysis of the crude sponge extract with comparisons to the synthetic 6-N-acyladenosine 2a provided evidence that the biosynthetically related adenosine, phorioadenosine A (2), was present as a trace co-metabolite. The rare starfish metabolite asterubine (3) was also isolated as a co-metabolite, and its structure confirmed by spectroscopic analysis and chemical synthesis. Biological investigations confirmed that natural products 1–3 and synthetic analogues 1a–e and 2a were not cytotoxic to multiple mammalian cancer cell lines, or Gram-positive or -negative bacteria. Nematocidal activity (inhibition of larval development of Haemonchus contortus) detected in the Phoriospongia sp. extract was attributed to 1 (LD₉₉ 31 μg/mL), with preliminary structure–activity relationship investigations confirming the importance of the N-acyl side chain.     

The elimination of metal(I) hydride in the mass spectra of some metal(II) compounds

Metal(I) hydrides are eliminated as neutral species in the electron impact ionization mass spectra of copper(II) and palladium(II) complexes of ethylene-N,N′-3-benzoylprop-2-en-2-amine. Deuterium labelling shows that the hydrogen atom of the metal(I) hydride is derived predominantly from the ethylene bridge both for ion source reactions and for metastable ion transitions. Evidence supporting the proposed rationalization for elimination of metal(I) hydride is provided by the observation of an analogous reaction in the mass spectrum of (ethylene-N,N′-salicylaldiminato)copper(II). The mass spectrum of ethylene-d₄-N,N′-3-benzoylprop-2-en-2-amine shows an unusual rearrangement to give [C₇H₅D₂]⁺ ions involving a formal phenyl-to-methylene transfer.     

Characterization by three-dimensional representation of sequential transitions of metastable ions in a double focusing mass spectrometer

The ion currents due to products of selected ion reactions of toluene and 3-chlorophenol, occurring in the source or flight tube of a double focusing mass spectrometer for which the electric sector precedes the magnetic sector, have been represented by plotting them against two variables that reflect independently the effects of the sectors. The daughter ion produced from the molecular ion of 3-chlorophenol that decays by sequential losses of CO in the field free region preceding the electric sector and Cl˙ in the field free region preceding the magnetic sector gives rise to a peak with a distinctive shape on the resultant three-dimensional surface. A peak due to the daughter ion arising from the corresponding sequential losses of H˙ and C₂H₂ from the molecular ion of toluene could not be detected. A portion of the ion kinetic energy spectrum which would be recorded by a detector at the energy resolving slit is derived from the surface for 3-chlorophenol and is in satisfactory agreement with a published spectrum.     

In-situ X-ray imaging of III-V strained-layer relaxation processes

The important value of the X-ray topography (XRT) technique for the investigation of III-V strained-layer relaxation processes is described. In addition to post-growth ex-situ XRT studies, a unique combined XRT/MBE growth facility has been constructed which allows the generation, motion and interaction of misfit dislocations to be monitored in-situ during epilayer growth, for the first time. The in-situ data already obtained for (100) InGaAs strained-layer growth on both Czochralski- and vertical-gradient freeze-grown GaAs substrates indicates technologically important differences in the initial relaxation process, including, in the latter case, the observation of a previously unreported secondary relaxation phase. Initial results relating to the influence of both post-growth annealing and the subsequent cool-down process are also described.     

Dipolar relaxations in the glass transition region and in the liquid crystalline phase of two side-chain liquid crystalline polysiloxanes

The molecular relaxation mechanisms in the glass transition region and in the liquid crystalline phase exhibited by two side-chain liquid crystalline polysiloxanes have been studied by Thermally Stimulated Discharge Currents. These results were compared with those previously obtained by dielectric relaxation spectroscopy. It was observed that two relaxation mechanisms were present in the liquid crystalline phase, and we suggest that these might correspond to the motions of the mesogenic moieties in the liquid crystalline phase. © 1996 John Wiley & Sons, Inc.     

The use of photoinitiated free-radical cyclopolymerization in the preparation of novel side-chain liquid-crystalline polydienes

The technique of photoinitiated free-radical cyclopolymerization has been used to produce a variety of novel side-chain liquid-crystalline (SCLC) polymers in which an alicyclic ring structure is incorporated into a polymer backbone. Most of the SCLC polymers gave glass transition temperatures (T_g) around room temperature and clearing points around 60–70°C. However, the poly(1,5-hexadiene) was the exception to this, exhibiting a T_g value of 14.5°C and a clearing point of 132.5°C. Confirmation of the structure of the alicyclic ring along the polymer backbone is extremely difficult to obtain, but evidence is given in the paper to support the ring structure.