Electrocatalysis of the hydrogen production by [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough

Hydrogenases are enzymes which catalyze hydrogen production/consumption reactions. In this paper, the [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough is shown to catalyze the direct hydrogen production from protons, in the absence of any promoter, at a basal pyrolytic graphite electrode using cyclic voltammetry techniques. The effect of several parameters upon catalytic current is investigated: pH, hydrogenase concentration, ionic strength, competition with another protein (bovine serum albumin), cycling repetition, mode of electrode polishing. The extent and efficiency of the hydrogenase electroactivity are examined in the presence of either an artificial electron carrier (methylviologen) or a physiological partner cytochrome c₃ isolated from the same bacterial strain. Results suggest that the electron-transfer process is essentially controlled by a complex between both hydrogenase and cytochrome c₃. Electrocatalysis appears to be largely governed by the adsorption of hydrogenase on the electrode surface very likely involving hydrophobic interactions.     

Ultrafast fluorescence dynamics of tryptophan in the proteins monellin and IIAGlc

The complete time-resolved fluorescence of tryptophan in the proteins monellin and IIA(Glc) has been investigated, using both an upconversion spectrophotofluorometer with 150 fs time resolution and a time-correlated single photon counting apparatus on the 100 ps to 20 ns time scale. In monellin, the fluorescence decay displays multiexponential character with decay times of 1.2 and 16 ps, and 0.6, 2.2, and 4.2 ns. In contrast, IIA(Glc) exhibited no component between 1.2 ps and 0.1 ns. For monellin, surprisingly, the 16 ps fluorescence component was found to have positive amplitude even at longer wavelengths (e.g., 400 nm). In conjunction with quantum mechanical simulation of tryptophan in monellin, the experimental decay associated spectra (DAS) and time-resolved emission spectra (TRES) indicate that this fluorescence decay time should be ascribed to a highly quenched conformer. Recent models (Peon, J.; et al. Proc. Natl.Acad. Sci. U.S.A. 2002, 99, 10964) invoked exchange-coupled relaxation of protein water to explain the fluorescence decay of monellin.     

Comparison between ultrafast fluorescence dynamics of FMN binding protein from Desulfovibrio vulgaris, strain Miyazaki, in solution vs crystal phases

Ultrafast fluorescence dynamics of FMN binding protein (FBP) from Desulfobivrio vulgaris, strain Miyaxaki F, were compared in solution and crystal phases. Fluorescence lifetimes of FBP were 167 fs (96%) and 1.5 ps (4%) in solution (tau(av) = 220 fs), and 730 fs (60%) and longer than 10 ps (40%) in crystals (tau(av) = 4.44 ps). The quenching of the fluorescence of flavin in the protein was considered to be due to photoinduced electron transfer (ET) from Trp or Tyr to the excited isoalloxazine (Iso) nearby. The average lifetime was 20 times longer in crystal vs in solution. Averaged distances between Iso and nearby Trp-32, Tyr-35, and Trp-106 were 8.42, 7.36, and 8.15 A in solution, respectively (obtained by NMR spectroscopy), and 7.05, 7.72, and 8.49 A in crystal, respectively (obtained by X-ray crystallography). The prolonged lifetime in crystal cannot be elucidated by the change in the distances between the states. It was suggested that the longer lifetime in crystal was ascribed to the absence of water molecules around FBP with rapid motional freedom, which may be the driving force for the ET in flavoproteins.     

Simultaneous analysis of ultrafast fluorescence decays of FMN binding protein and its mutated proteins by molecular dynamic simulation and electron transfer theory

Ultrafast fluorescence decays of FMN binding proteins (FBP) from Desulfovibrio vulgaris (Miyazaki F) were analyzed with an electron transfer (ET) theory by Kakitani and Mataga (KM theory). Time-dependent distances among isoalloxazine (Iso) and Trp-32, Tyr-35, and Trp-106 in wild-type FBP (WT), among Iso and Tyr-32, Tyr-35, and Trp-106 in W32Y (Trp-32 was replaced by Tyr-32), and among Iso and Tyr-35 and Trp-106 in W32A (Trp-32 was replaced by Ala-32) were determined by molecular dynamic simulation (MD). Electrostatic energies between Iso anion and all other ionic groups, between Trp-32 cation and all other ionic groups, and between Tyr-32 cation and all other ionic groups were calculated in WT, W32Y, and W32A, from the MD coordinates. ET parameters contained in KM theory, such as frequency (nu 0), a coefficient of the ET process (beta), a critical distance of the ET process ( R 0), standard free energy related to the electron affinity of the excited Iso ( G Iso (0)), and the static dielectric constant in FBP species (epsilon 0), were determined with and without inclusion of the electrostatic energy, so as to fit the calculated fluorescence decays with the observed decays of all FBP species, by a nonlinear least-squares method according to the Marquardt algorithm. In the analyses the parameters, nu 0, beta, and R 0 were determined separately between Trp residues and Tyr residues among all FBP species. Calculated fluorescence intensities with the inclusion of the electrostatic energy fit quite well with the observed ones of all WT, W32Y, and W32A.     

Properties and binding forms of cadmium and nickel in protein extracts from bean seeds (Phaseolus vulgaris L.)

A special ultrafiltration procedure in combination with photometry and atomic absorption spectrometry is described. This technique allows not only the determination of the qualitative distribution patterns of metals but also of the quantitative binding parameters of these metals to functional groups of organic compounds. The technique was applied to protein extracts of bean seeds (Phaseolus vulgaris L.). The results show that Cd and Ni have different binding mechanisms in these seeds. Both metals differ not only in their preferred binding behaviour to certain molecular size fractions but also in their potential binding sites and the binding properties. Whereas Cd in seeds from Cd-treated plants prefers binding partners with molecular weights >30 kD and <0.5 kD, Ni is preferably bound to compounds with molecular weights <5 kD. Cd exhibits a tendency to bind more selectively to sulfhydryl groups, but Ni more to non-specific binding sites. Sulfhydryl groups account for 71% of the total capacity for Cd whereas for Ni it is only 36%.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

Excited state dynamics in recombinant water-soluble chlorophyll proteins (WSCP) from cauliflower investigated by transient fluorescence spectroscopy

The present study describes the fluorescence emission properties of recombinant water-soluble chlorophyll (Chl) protein (WSCP) complexes reconstituted with either Chl a or Chl b alone (Chl a only or Chl b only WSCP, respectively) or mixtures of both pigments at different stoichiometrical ratios. Detailed investigations were performed with time and space correlated ps fluorescence spectroscopy within the temperature range from 10 to 295 K. The following points were found: (a) The emission spectra at room temperature (295 K) are well characterized by bands with a dominating Lorentzian profile broadened due to phonon scattering and peak positions located at 677, 684 and 693 nm in the case of Chl a only WSCP and at 665, 675 and 689 nm for Chl b only WSCP. In addition, all spectra contain minor bands in the longer wavelength region. (b) The emission spectra at 10 K of samples suspended in buffer containing 50% glycerol are dominated by bands peaking at 668 nm for Chl b only WSCP and at 685 nm for Chl a only WSCP and samples reconstituted with mixtures of Chl a and Chl b. (c) At 10 K and in buffer with 50% glycerol the decay kinetics of WSCP samples with Chl a only are dominated by a component with a time constant of 6.2 (+/-0.2) ns at 685 nm while those of WSCP containing mixtures of Chl a and Chl b are characterized by a slightly shorter value of 6.0 (+/-0.2) ns. WSCP containing Chl b only exhibits a distinctly longer value of 7.0 (+/-0.3) ns at an emission wavelength of 668 nm. (d) The decay associated emission spectra at 10 K of all samples exhibit at least 3 decay components with time constants of 80-120 ps, 2-4 ns and 6-7 ns in 50% glycerol. These results are consistently described within the framework of our previously presented model (J. Phys. Chem. B 2007, 111, No. 46, 13325; J. Phys. Chem. B 2007, 111, No. 35, 10487) , for the structural motifs of chlorophyll binding to the tetrameric protein matrix of WSCP. It is shown that formation of strongly coupled open sandwich dimers does not lead to quenching of 1Chl a* or 1Chl b*.     

Ultrafast dynamics and excited state deactivation of [Ru(bpy)2Sq]+ and its derivatives

Femtosecond transient absorption spectroscopy has been employed to understand the excited state dynamics of [Ru(bpy)(2)Sq](+) (I; bpy is 2,2'-bipyridyl, and Sq is the deprotonated species of the semiquinone form of 1,2-dihydroxy benzene) and its derivatives, a widely studied near-infrared (NIR) active electrochromic dye. Apart from the well-defined dpi(Ru) --> pi(bpy)-based metal-to-ligand charge transfer (MLCT) transition bands at approximately 480 nm, this class of molecules generally shows another dpi(Ru) --> pi(Sq)(SOMO)-based intense MLCT band at around 900 nm, which is known to be redox active and bleaches reversibly upon a change in the oxidation state of the coordinated dioxolene moiety. To have better insight into the photoinduced electron transfer dynamics associated with this MLCT transition, detailed investigations have been carried out on exciting this MLCT band at 800 nm. Immediately after photoexcitation, bleach at 900 nm has been observed, whose recovery is found to follow a triexponential function with major contribution from the ultrafast component. This ultrafast component of approximately 220 fs has been ascribed to the S(1) to S(0) internal conversion process. In addition to the bleach, we have detected two transient species absorbing at 730 and 1000 nm with a formation time approximately 220 fs for both species. The excited state lifetimes for these two transient species have been measured to be 1.5 and 11 ps and have been attributed to excited singlet ((1)MLCT) and triplet ((3)MLCT) states, respectively. Transient measurements carried out on the different but analogous derivatives (II and III) have also shown similar recovery dynamics except that the rate for the internal conversion process has increased with the decrease in the S(1) to S(0) energy gap. The observed results are consistent with the energy gap law for nonradiative decay from S(1) to S(0).     

Effect of gamma irradiation on the microbiological quality and on the functional properties of proteins in dry red kidney beans (Phaseolus vulgaris)

Gamma-irradiation was found to affect the physicochemical properties of dry red kidney beans. The highest dose used (8 kGy) significantly (P0.05) modified the extent of deamidation, the number of sulfhydryl groups, as well as the solubility and the hydrophobicity of the protein. Deamidation, protein solubility and hydrophobicity all increased with the irradiation dose while the number of sulfhydryl groups was reduced by the treatment. Furthermore, irradiation also affected the outgrowth of natural filamentous fungi contaminants present on the dry beans. A dose of 1.5 kGy reduced the number of filamentous fungi by 2 log cycles immediately after treatment. However, the highest dose used (3 kGy) did not eliminate the filamentous fungi completely. Moreover, the filamentous fungi population was a lot less diversified on the irradiated samples. Species of Aspergillus sp. and Penicillium sp. were more abundant on the unirradiated beans while the beans irradiated at 3 kGy contained were predominantly infected by species of Rhizopus sp. , Cladosporium sp. and Alternaria sp.     

Ultrafast generation of thick poly(ether amine) (PEA) brush on a gold surface and its protein resistance

We demonstrate an ultrafast, convenient and universal approach to fabricate a poly(ether amine) (PEA) brush on a gold surface, which exhibited excellent performance of protein resistance with long-term stability.     

Removal of uranyl ions from UO2(NO3)2 solution by means of Chlorella vulgaris and Dunaliella salina algae

This paper deals with a study of the biosorption of UO₂ ²⁺ ions on two green algae: Chlorella vulgaris and Dunaliella salina. By investigating the retention degree versus contact time from Langmuir and Freundlich biosorption isotherms, kinetic investigations and FTIR spectra it was found that the biosorption process was greater for Chlorella vulgaris than for Dunaliella salina. A new kinetics method is proposed to establish the reaction order concerning the biosorption process of uranyl ions on these biomasses.      

Synthesis of chiral lithium carbamates from (S)-2-(N,N-dialkyl-aminomethyl)pyrrolidines and (S)-methoxymethylpyrrolidine

A convenient synthesis of chiral lithium N-alkyl carbamates 1a–4a from chiral pyrrolidines 1–4, LiH and CO₂ is described. The yields are good to excellent. A combined experimental (¹H, ⁶Li-HOESY, cryoscopy) and theoretical study (B3LYP/6-311++G(d,p)) succeeded in assigning the predominant solution state structure of 1a.     

Syntheses of (E)- and (Z)-volkendousin

The first syntheses of the antitumor agents (E)-volkendousin (1) and acetonide 3 have been accomplished by efficient routes from readily available dehydroisoandrosterone (7) using allylic oxidation with SeO₂ to introduce the 4/gb-hydroxy group and 16-ketone. This sequence should make these compounds readily available for further biological evaluation.     

Ultrafast electronic and vibrational dynamics of stabilized A state mutants of the green fluorescent protein (GFP): Snipping the proton wire

Two blue absorbing and emitting mutants (S65G/T203V/E222Q and S65T at pH 5.5) of the green fluorescent protein (GFP) have been investigated through ultrafast time resolved infra-red (TRIR) and fluorescence spectroscopy. In these mutants, in which the excited state proton transfer reaction observed in wild-type GFP has been blocked, the photophysics are dominated by the neutral A state. It was found that the A* excited state lifetime is short, indicating that it is relatively less stabilised in the protein matrix than the anionic form. However, the lifetime of the A state can be increased through modifications to the protein structure. The TRIR spectra show that a large shifts in protein vibrational modes on excitation of the A state occurs in both these GFP mutants. This is ascribed to a change in H-bonding interactions between the protein matrix and the excited state.     

Li (i=1–3) subshell fluorescence and Coster-Kronig yields for elements with 70Z92

The atomic inner-shell vacancy decay processes comprised of radiative and nonradiative transitions are characterized by the fluorescence, Auger and Coster-Kronig yields. We present a review of our recent measurements of the L_i (i=1, 2, 3) sub-shell fluorescence yields (ω_i) and the L₁–L₃ Coster-Kronig transition probabilities (f₁₃) for elements with 70Z92 following photoionization. The comparison of the measured ω_i and f₁₃ values with the theoretical ones, based on the relativistic Dirac–Hartree–Slater (RDHS) model calculations, indicated the need for more refined calculations of transition probabilities for different Coster-Kronig transitions.     

Asymmetric Synthesis of （R）- and （S）-Moprolol

A simple and effective procedure for the enantioselective synthesis of （R）- and （S）-moprolol was described. The key step was the asymmetric synthesis of enantiopure （R）- and （S）-guaifenesin, which were synthesized from enantioenriched （R）-3-chloro-l,2-propanediol and （S）-epichlorohydrin via kinetics of hydrolysis resolution of racemic epichlorohydrin by chiral Salen-Co^Ⅲ complex. The e.e. values of both the optical compounds were above 98%, and the chemical structures of the target compounds were confirmed by ^1H NMR, ^13C NMR, IR, and MS.     

Stereoselective synthesis of (1R,2S)-2-amino-1,3-diols from (R)-cyanohydrins

Vinyl substituted (1R,2S)-amino alcohols 5 were obtained by addition of vinyl magnesium bromide to the corresponding cyanohydrin O-trimethylsilyl ethers (R)-2. The O- and N-protected vinyl amino alcohols 6 were ozonized at −78°C in methanol yielding (1R,2S)-2-amino-1,3-diols7 in high enantiomeric and diastereomeric excesses. For purification, compounds 7 in some cases were acetylated to give the derivatives (1R,2S)-8. Racemic 6a was converted by oxidative ozonolysis at −78°C in methanolic NaOH solution to the corresponding methyl N-acetyl-β-hydroxy propanoate 9a. The configuration of (1R,2S)-8a was confirmed by x-ray crystallographic analysis.     

Ab Initio Study of Conformational Properties of ( Z, Z)-, ( E, Z)-, and ( E, E)-Cyclonona-1,5-dienes

Ab initio calculations at the HF/6-31G^* level of theory for geometry optimization and MP2/6-31G^*//HF/6-31G^* for a single point total energy calculation are reported for the important energy-minimum conformations and transition-state geometries of (Z,Z)-, (E,Z)-, and (E,E)-cyclonona-1,5-dienes. The C₂ symmetric chair conformation of (Z,Z)-cyclonona-1,5-diene is calculated to be the most stable form; the calculated energy barrier for ring inversion of the chair conformation via the C_s symmetric boat-chair geometry is 58.3kJmol^–1. Interconversion between chair and twist-boat-chair (C₁) conformations takes place via the twist (C₁) as intermediate. The unsymmetrical twist conformation of (E,Z)-cyclonona-1,5-diene is the most stable form. Ring inversion of this conformation takes place via the unsymmetrical chair and boat-chair geometries. The calculated strain energy for this process is 63.5kJmol^–1. The interconversion between twist and the boat-chair conformations can take place by swiveling of the trans double bond with respect to the cis double bond and requires 115.6kJmol^–1. The most stable conformation of (E,E)-cyclonona-1,5-diene is the C₂ symmetric twist-boat conformation of the crossed family, which is 5.3kJmol^–1 more stable than the C_s symmetric chair–chair geometry of the parallel family. Interconversion of the crossed and parallel families can take place by swiveling of one of the double bonds and requires 142.0kJmol^–1.     

Ab Initio Study of Conformational Properties of ( Z, Z)-, ( E, Z)-, and ( E, E)-Cyclonona-1,5-dienes

Summary. Ab initio calculations at the HF/6-31G^* level of theory for geometry optimization and MP2/6-31G^*//HF/6-31G^* for a single point total energy calculation are reported for the important energy-minimum conformations and transition-state geometries of (Z,Z)-, (E,Z)-, and (E,E)-cyclonona-1,5-dienes. The C₂ symmetric chair conformation of (Z,Z)-cyclonona-1,5-diene is calculated to be the most stable form; the calculated energy barrier for ring inversion of the chair conformation via the C_s symmetric boat-chair geometry is 58.3kJmol^–1. Interconversion between chair and twist-boat-chair (C₁) conformations takes place via the twist (C₁) as intermediate. The unsymmetrical twist conformation of (E,Z)-cyclonona-1,5-diene is the most stable form. Ring inversion of this conformation takes place via the unsymmetrical chair and boat-chair geometries. The calculated strain energy for this process is 63.5kJmol^–1. The interconversion between twist and the boat-chair conformations can take place by swiveling of the trans double bond with respect to the cis double bond and requires 115.6kJmol^–1. The most stable conformation of (E,E)-cyclonona-1,5-diene is the C₂ symmetric twist-boat conformation of the crossed family, which is 5.3kJmol^–1 more stable than the C_s symmetric chair–chair geometry of the parallel family. Interconversion of the crossed and parallel families can take place by swiveling of one of the double bonds and requires 142.0kJmol^–1.