The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni±Fe hydrogenase. Ten ORFs (mbhA±I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni±Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe±4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe±4S] protein. With P. furiosus soluble [4Fe±4S] ferredoxin as the electron donor the membranes produce H 2 , and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H 2 evolution to H 2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with E m < 20.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni±Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H 2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H 2 ) connected by devices for transduction, transfer, recovery and safety-valving of energy.

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Tlte effect of riboflavin and riboflavin binding proteins on the light-induced formation of reactive oxygen species and sunstruck off-flavour was studied in model beer solutions. Under model beer conditions (pH 4.0, 1 ppm riboflavin, 5% ethanol and traces of O2) hydroxyl and hydroxyethyl radicals were formed upon illumination. Radical formation was measured with the spin traps N-t-butyl-a-phenylnitrone (PBN) and 5,5-dimethyl-l-pyrroline-N-oxide (DMPO). DMPO appeared to be a better spin trap than PBN for sttidying the effect of light exposure, since PBN is photochemically active by itself. Addition of isohumulones to the model beer reduced the amount of riboflavin-induced radicals. Two different riboflavin binding proteins were tested both for their ability to scavenge riboflavin and how in turn this influenced free radical formation. The apoform of egg white riboflavin binding protein (RfBP) was more efficient in reducing radical formation than an apo-flavodoxin protein isolated from Azotobacter vinelandii. Organoleptic assessment clearly indicated that the addition of apoRfBP to model beer solutions, containing stiochiometric amounts of riboflavin as well as isohumulones and cysteine, reduced sunstruck off-flavour formation. The dual role of riboflavin and ethanol as radical propagators in oxidiatve flavour change is discussed. From these anaerobic stock solutions, samples were prepared in an anaerobic glove box (20% H2 and 80% N2).All samples were freshly prepared and well protected against light prior to illumination.Samples for flavour evaluation were prepared as described above, except that 12 ml GC sealable vials were The EPR experiments were performed immediately after illumination. EPR analysisAll EPR experiments were performed at room temperature on a Bruker ER 200D EPR spectrometer.The following parameters were used: field (

Among the most successful systems for homogeneous catalysis, hydrogenation catalysts capable of activating molecular hydrogen, take outstanding roles in research laboratories and in industry. To open up the field of continuous catalytic hydrogenations a novel membrane reactor concept was developed and successfully applied for hydrogenations with dihydrogen both for chemical and for enzymatic catalysis. The hydrogenase I of the archaeon Pyrococcus furiosus was utilized for the continuous hydrogenation of NADP to NADPH with recycling of the enzyme by means of ultrafiltration. The well known PyrPhos-Rh system was used for the enantioselective synthesis of an amino acid derivative by hydrogenation.Keywords: asymmetric catalysis; catalyst immobilization; cofactors; enzyme catalysis; homogeneous catalysis; hydrogenation; membranes; reduction Here we report our most recent efforts to extend the range of feasible reactions in the membrane reactor [1] to hydrogenation with dihydrogen as reducing agent. The direct usage of hydrogen has several advantages over the usage of hydrogen transfer agents like 2-propanol [2] since it constitutes a cheaper and more powerful means of reduction that can be used in large excess and be easily removed, thus not hampering downstream processing.For this approach a novel reactor concept was developed for the continuous dosage of gaseous reactants via a dense polymer membrane. The feasibility of the application of volume-aeration to hydrogenation was investigated for chemical and enzymatic catalysis. We chose the homogeneous hydrogenation catalyst PyrPhos [3] as the chemical representative, whereas the hydrogenase I from the hyperthermophilic archeon Pyrococcus furiosus (PfH) illustrates the enzymatic approach.[4] Both catalysts activate hydrogen; the PyrPhos system for the enantioselective reduction of activated double bonds, whereas the PfH is capable of heterolytic cleavage of hydrogen and regioselective 1,4-hydride addition to the oxidized form of the phosphorylated nicotinamide cofactor: NADP (Scheme 1). In the case of the enzymatic approach the macromolecular catalyst was recycled by means of ultrafiltration.Reactor Set-Up: For continuous dosage of dihydrogen in a continuously operated membrane reactor a new setup was developed. The general scheme is shown in Figure 1. The delivery of gaseous reactants can be achieved by pressure-enhanced diffusion through dense polymer membranes, which has been shown in fluidized bed for animal cell culture.[5] We chose polytetrafluoroScheme 1. Hydrogenation of a) 2-N-acetylamidocinnamic acid (AAZ) with PyrPhos and b) NADP with the hydrogenase from Pyrococcus furiosus (PfH) (for NADP and NADPH only the reduced nicotinamide moiety is shown).