The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a [NiFe] hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR(-). The results establish an elementary mechanistic understanding of catalysis in a [NiFe] hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.

Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.

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Background: Hydrogenases are complex metalloenzymes catalyzing the evolution of hydrogen gas but lacking an efficient system to produce recombinant forms. Results: An NADP(H)-dependent hydrogenase was overproduced by almost an order of magnitude in a hyperthermophilic microorganism. Conclusion: Homologous overproduction of an affinity-tagged hydrogenase was achieved. Significance: Native and mutant forms of hydrogenase can now be generated for in vitro biochemical analyses and bioenergy systems.

Background:The hydrogen-evolving membrane-bound hydrogenase (MBH) functions as a simple respiratory system in anaerobic microbes. Results: Affinity-tagged MBH was solubilized from membranes of a hyperthermophile as an intact 14-subunit complex. Conclusion: Solubilized MBH was catalytically active, and a structural model based on small angle x-ray scattering (SAXS) was obtained. Significance:The successful purification of a respiratory hydrogenase has enabled biochemical and structural studies.

Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O2-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining ‘minimal’ hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg−1 in H2 production using the artificial electron donor methyl viologen) and thermostable (t1/2 ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H2 in vitro without the need for an intermediate electron carrier.

Five psychrophilic bacterial strains were isolated from soil samples collected above the treeline of alpine environments. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these organisms represent four novel species of the genus Deinococcus; levels of sequence similarity to the type strains of recognized Deinococcus species were in the range 89.3-94.7 %. Strains T , T , ME-04-01-32 T and ME-04-04-52 T grew aerobically, with optimum growth at 10 6C and at pH 6-9. The major respiratory menaquinone was MK-8. At the time of writing, the genus Deinococcus comprised 25 recognized species, three of which were isolated from low-temperature environments, namely Antarctic soils or rocks (Hirsch et al., 2004). The optimal growth temperatures of species of the genus Deinococcus fall across a broad range from 9 to 50 u C. The majority of species are reported to have optimal growth temperatures in the mesophilic range. Exceptions are three slightly thermophilic species, namely Deinococcus geothermalis, D. murrayi and D. maricopensis (Ferreira et al., 1997;Rainey et al., 2005), and three psychrophilic species, namely Deinococcus frigens, D. marmoris and D. saxicola (Hirsch et al., 2004).The most studied characteristic of members of the genus Deinococcus is their ability to survive exposure to ionizingThe GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains T , T , ME-04-01-32 T and ME-04-04-52 T are EF635404, EF635405, EF635406, EF635407 and EF635408, respectively.A two-dimensional thin-layer chromatograph of total polar lipids, graphs showing the effect of gamma radiation, UV radiation and desiccation on the survival of the new isolates, and a table detailing the fatty acid compositions of the new isolates and recognized Deinococcus species are available as supplementary material with the online version of this paper.

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