Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase

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

Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production

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

A Stress Induced Source of Phonon Bursts and Quasiparticle Poisoning

The performance of superconducting qubits is degraded by a poorly characterized set of energy sources breaking the Cooper pairs responsible for superconductivity, creating a condition often called "quasiparticle poisoning." Recently, a superconductor with one of the lowest average quasiparticle densities ever measured exhibited quasiparticles primarily produced in bursts which decreased in rate with time after cooldown. Similarly, several cryogenic calorimeters used to search for dark matter have also observed an unknown source of low-energy phonon bursts that decrease in rate with time after cooldown. Here, we show that a silicon crystal glued to its holder exhibits a rate of low-energy phonon events that is more than two orders of magnitude larger than in a functionally identical crystal suspended from its holder in a low-stress state. The excess phonon event rate in the glued crystal decreases with time since cooldown, consistent with a source of phonon bursts which contributes to quasiparticle poisoning in quantum circuits and the low-energy events observed in cryogenic calorimeters. We argue that relaxation of thermally induced stress between the glue and crystal is the source of these events, and conclude that stress relaxation contributes to quasiparticle poisoning in superconducting qubits and the athermal phonon background in a broad class of rare-event searches.Comment: 13 pages, 6 figures. W. A. Page and R. K. Romani contributed equally to this work. Correspondence should be addressed to R. K. Roman

Analysis of the <i>P. furiosus</i> maturation proteins required to produce active rSHI in <i>E. coli</i>.

<p>The specific activities are shown for rSHI in cell extracts of <i>E. coli</i> resulting from the co-expression of different <i>Pf</i> processing genes in <i>E. coli</i> MW1001. Bars indicate MV-linked specific activity (µmol H₂ evolved min⁻¹ mg⁻¹). All cell extracts were heated for 30 min at 80°C prior to assay.</p

Plasmid construction for rSHI production in <i>E. coli</i>.

<p>P_hya was cloned into a set of patented plasmids constructed by inserting Invitrogen Gateway cassettes into Novagen Duet plasmids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010526#pone.0010526-Horanyi1" target="_blank">[25]</a>. (<b>a</b>) Sequences of <i>E. coli</i> anaerobic promoter P_hya, attB1 and TEV protease recognition site sequence and their encoded peptide sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010526#pone.0010526-Dougherty1" target="_blank">[31]</a>. (<b>b</b>) Elements constructed in the final expression plasmids: P_hya promoter, N-terminus of the first protein in the operon derived from the Gateway recombination cloning site (attB1) including a TEV protease site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010526#pone.0010526-Dougherty1" target="_blank">[31]</a> and <i>P. furiosus</i> genes. (<b>c</b>) Artificial Shine-Dalgarno sequences inserted between <i>P. furiosus</i> fused ORFs in plasmids pC11A2-CDABI, pC3AR2-SdFa and pRA2-EF.</p

Properties of recombinant <i>P. furiosus</i> SHI.

<p>(<b>a</b>) Gel electrophoresis (SDS-PAGE) analysis of native and recombinant SHI. Bands representing the four subunits of the SHI are indicated, all of which were confirmed by MALDI-TOF analysis (data not shown). The high molecular weight band in the native protein, not seen in the recombinant version, represents undenatured tetrameric protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010526#pone.0010526-Bryant1" target="_blank">[11]</a>. Recombinant PF0891 is approximately 2 kDa larger than the native protein due to the N-terminal extension (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010526#pone-0010526-g003" target="_blank">Fig. 3</a>). Molecular weight markers (kDa) are indicated. (<b>b</b>) Physical and catalytic properties of rSHI and the native hydrogenase purified from <i>P. furiosus</i> biomass. Hydrogen evolution was measured using either methyl viologen (MV) or NADPH as the electron donor at 80°C.</p

Purification of recombinant <i>P. furiosus</i> SHI from <i>E. coli</i>.

<p>Purification of recombinant <i>P. furiosus</i> SHI from <i>E. coli</i>.</p