[FeFe]-hydrogenases and photobiological hydrogen production

The promise of efficient, economic and renewable H-2 photoproduction from water can potentially be met by green algae. These organisms are able to functionally link photosynthetic water oxidation to the catalytic recombination of protons and electrons to generate H-2 gas through the activity of the hydrogenase enzyme. Green algal hydrogenases contain a unique metallo-catalytic H-cluster that performs the reversible H-2 oxidation /evolution reactions. The H-cluster, located in the interior of the protein structure is irreversibly inactivated by O-2, the by-product of water oxidation. We developed an Escherichia coli expression system to produce [FeFe]-hydrogenases from different biological sources and demonstrated that clostridial [FeFe]-hydrogenases have higher tolerance to O-2 inactivation compared to their algal counterparts. We have been using computational simulations of gas diffusion within the Clostridium pasteurianum CpI hydrogenase to identify the pathways through which O-2 can reach its catalytic site. Subsequently, we modify the protein structure at specific sites along the O-2 pathways (identified by the computational simulations) by site-directed mutagenesis with the goal of generating recombinant enzymes with higher O-2 tolerance. In this paper, we review the computational simulation work and report on preliminary results obtained through this strategy.