Princess R. Cabotaje: Mining Nature’s Platinum: Discovery, Characterization, and Optimization of Hydrogenases for Green H2 Applications

Abstract

Establishing a sustainable hydrogen (H2) economy requires developing catalysts that are not reliant on rare and expensive metals like platinum. As summarized in Chapter 1, microorganisms utilize hydrogenases to harness molecular H2 for their metabolic processes. Remarkably, these ancient gas-processing enzymes rely on earth-abundant metals to reach activities on par with platinum. Investigating the biodiversity and understanding the mechanism of these enzymes offer a promising route to develop sustainable catalysts for H2-processing. 

This thesis employed a multidisciplinary approach, including bioinformatics, biochemistry, synthetic chemistry, and biophysics, to investigate two hydrogenase superfamilies, namely [FeFe] and [NiFe] hydrogenases. Key methods are outlined in Chapter 2. The main aim was to provide insights into their evolution, diversity, structure, function, and potential for green H2 applications.

Chapter 3 challenges the prevailing view of [FeFe] hydrogenase domain distribution by revealing their presence and activity in diverse archaeal lineages, including DPANN, Asgard, and Thermoplasmata, which notably host [NiFe]-[FeFe] hybrids. The study provides a new minimal size requirement for functional hydrogenase and contests the notion of evolutionary independence between the two hydrogenase superfamilies.

Chapter 4 identifies a distinct proton transfer pathway in the putatively sensory Group D [FeFe] hydrogenases, diverging from the well-characterized pathway in the catalytic Group A enzymes. The study supports the critical role of proton transfer in controlling overall enzymatic function and provides insights for engineering catalysts that integrate both proton transfer and redox chemistry.

Chapter 5 demonstrates how rational engineering approaches informed by evolutionary insights can enhance both H2 production and O2 tolerance in a Group D [FeFe] hydrogenase, TamHydS. This work reveals the synergistic impact of protein scaffold modifications on catalytic performance and highlights the potential of integrating phylogenetic information and protein engineering for developing robust and efficient biocatalysts.

Chapter 6 elucidates the mechanism by which Huc, an isolated O2-tolerant [NiFe] hydrogenase, is able to efficiently oxidize trace atmospheric H2. This exceptional capability arises from the intrinsic properties of the enzyme, particularly its high affinity for H2 and elevated overpotential, rather than being dependent on the cellular context. The insights gained from this study provide valuable guidance for the design and engineering of (a)biotic catalysts capable of operating under ambient conditions.