Cytoplasmic and membrane-bound hydrogenases from Pyrococcus furiosus

Hydrogenases catalyze the simplest of chemical reactions, the reversible interconversion of protons, electrons, and hydrogen gas. These enzymes have potential to be utilized for several biotechnological applications, such as in vitro hydrogen production from renewable materials and in enzyme-based fuel cells for electricity generation. Based on the metal content of their catalytic sites, hydrogenases are classified as either [NiFe], [FeFe], or mononuclear Fe enzymes, and [NiFe] hydrogenases are further categorized into five groups based on the sequences of the catalytic subunits. This chapter describes recombinant engineering strategies, purification procedures, and catalytic properties of two distinct types of [NiFe] hydrogenase from Pyrococcus furiosus, a microorganism with an optimal growth temperature of 100°C. These enzymes are termed soluble hydrogenase I (SHI, group 3) and membrane-bound hydrogenase (MBH, group 4). The two hydrogenases were affinity-tagged to facilitate their purification and the purified enzymes have been used for biochemical, mechanistic, and structural analyses. The results have provided us with new insights into how catalysis by SHI is achieved, which could also lead to the development of catalysts for economic hydrogen production, and knowledge of how MBH couples hydrogen gas production to conservation of energy in the form of an ion gradient. The methods described in this chapter provide the basis for these studies.