De novo designed metalloproteins as a new generation of artificial hydrogenases

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PROJECT SUMMARY/ABSTRACT Hydrogenases are complex, metal-containing enzymes that generate energy for certain organisms by catalyzing the reversible interconversion between H+ and H2 gas. Unraveling the intricate details about the function of these enzymes will significantly advance the H2-based, carbon-neutral alternative energy production. However, the complexity of these enzymes due to the presence of multiple metallic cofactors, low production yield, and deactivation, makes studying these enzymes challenging. Our long term goals are to design artificial biomolecular hydrogenases (ArHs) as simpler functional analogs of these metalloenzymes. De novo metalloprotein design is an appealing and well-established approach to model complex metalloproteins within minimal protein scaffolds. Although the designed systems are less complex, they serve as water-soluble functional analogs of the native metalloenzymes and provide a functional view of the chemistry. Employing this approach, we propose to pursue three Specific Aims describing the overall design principles and functional/mechanistic attributes of the ArHs inspired by the [NiFe] hydrogenases. The overall objectives of this proposal are: i) to design mononuclear (Ni), binuclear (Ni-Fe), and multinuclear (Ni3) active sites within suitable de novo scaffolds; ii) characterize the physical and catalytic properties of the ArHs; iii) determine the timescales of electron transfer; iv) outline the H+ transfer pathways; v) characterize the reaction intermediates; and vi) elucidate how metals and protein scaffold work in synergy to influence the properties/reactivity, such that a holistic mechanistic view of H-H bond formation can be attained. Our strong preliminary results presented here attest that our objectives are achievable. Collectively, the results from this proposed work will impact the fields of metalloprotein design, bioinorganic chemistry, and alternative energy research. A novel class of ArHs will emerge, which will provide functional vignettes into the working principles of H+ reduction pertaining to the native enzymes. The modular design parameters and outcomes from this study will enable us to prepare biosynthetic catalysts with novel properties and functions in the future.