Genetic engineering and purification of the hydrogenases from the hyperthermophilic archaeon Pyrococcus furiosus
McTernan, Patrick Michael
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Due to the inevitable depletion of fossil fuels on earth, a major shift in research has occurred that emphasizes the generation of a renewable energy source. More specifically, this renewable energy source should be carbon neutral and capable of delivering a high energy yield upon use. Hydrogen gas meets these criteria as it can be produced in nature without the generation of greenhouse gases and the conversion of hydrogen to water yields three times more energy than gasoline on a weight basis. Although this may be true, more research is needed in order to understand the mechanisms and restrictions associated with biological hydrogen production. In order to gain this better understanding, the hydrogenase enzyme, which is responsible for hydrogen production in nature, has been investigated. The generation and analysis of hydrogenase can greatly help in understanding how these enzymes generate hydrogen and how these enzymes could be engineered for biofuel production. The hydrogenases from Pyrococcus furiosus are the focus of this study. P. furiosus is a hyperthermophilic archaeon that optimally grows at 1000 C and ferments both peptides and carbohydrates. It contains three [NiFe]-hydrogenases, two of which are found in the cytoplasm and are termed soluble hydrogenase I (SHI) and soluble hydrogenase II (SHII). The third is a 14-subunit membrane-bound hydrogenase (MBH) that is proposed to function as a novel respiratory system within the membrane. MBH shows a strong preference to evolve hydrogen, which is rare among [NiFe]-hydrogenases, and will be the focus of this dissertation. In the literature, previous attempts to purify MBH using standard purification techniques from P. furiosus lead to the purification of only five subunits of the fourteen-subunit complex. Herein, a new protocol is described that yields the entire 14-subunit MBH complex for biochemical and structural analyses. The genetic system that was recently developed for P. furiosus has been successfully used to place the SHI operon under a stronger promoter. This lead to the over-expression of the enzyme by 10-fold, and was used to engineer an affinity tag at the N-terminus of one of the subunits, which greatly improved the yield of the hydrogenase. This genetic system was also applied to MBH to split the native operon into two different transcriptional units. From the recombinant strain, both the 14-subunit intact MBH (S-MBH) and a completely soluble MBH sub-complex (C-MBH) were purified. Both forms of MBH were characterized at the enzymatic and structural level. From this analysis, the implications of a crystal structure of C-MBH on how other respiratory systems have evolved over time are discussed.