The global population has recently exceeded 7 billion people and the demand for energy continues to expand as the number of industrialized countries grows. Currently, the energy economy is dominated by the utilization of polluting and non-renewable fossil fuels. Both the collection and use of petroleum-based fuels is destructive to the environment and is not sustainable over a long time-scale, which justifies the investigation into the development of renewable, alternative fuels. Of the various fuels that have been proposed, molecular hydrogen (H2) in particular holds great promise as a clean-burning fuel capable of supplementing the current energy economy, especially as the combustion of H2 generates only water vapor as by-product. H2 can be generated via a number of chemical processes, but current H2 technologies either require fossil fuels as inputs or are energy-inefficient. The biological production of H2, however, has garnered a great deal of interest because microorganisms are able to drive H2 synthesis using energy derived from both light and dark fermentative metabolisms. This manner of production does not depend on mining non-renewable resources and microbes can be cultured at the industrial scale without competing with arable land needed for agriculture. H2 evolution in these microorganisms is dependent on nitrogenases and/or hydrogenases, enzymes which utilize unique metal centers for catalysis. Hydrogenases have been of particular interest for industrial-scale H2 production because these enzymes are found in a diverse array of organisms and require only protons and electrons as substrates. In particular, [FeFe]-hydrogenases have very high turnover numbers and catalysis can be coupled to photosynthesis. Unfortunately, these enzymes are inactivated by molecular oxygen (O2), and a number of studies have therefore attempted to engineer O2-tolerant hydrogenases. However, engineering enzymes to introduce optimal qualities has been impeded by an incomplete understanding of the overall reaction mechanism. Substrate (protons, electrons, and 2) transport is essential to hydrogenase activity, yet relatively little information is available regarding the intraprotein transport of substrate in [FeFe]-hydrogenase. I focused my investigation on identifying and testing pathways important for substrate transport between the enzyme surface and the active site in the Clostridium pasteurianum [FeFe]-hydrogenase. I have elucidated a key pathway for proton transport and confirmed that two iron-sulfur clusters are essential in an electron transfer relay, contributing to the overall characterization of [FeFe]-hydrogenase activity. Green algae utilize [FeFe]-hydrogenases to catalyze H2 production using reducing equivalents derived from photosynthesis and these enzymes are an integral component of anaerobic metabolism in these microalgae. I explored the H2 production capabilities of a multicellular green alga, Volvox carteri, and characterized two hydrogenases likely responsible for this activity. In addition, a unique hydrogenase gene cluster discovered within the Volvox carteri genome provided interesting hints into the origin of [FeFe]-hydrogenase in green algae.
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