Hydrogenases, enzymes that catalyze the reversible conversion of protons and electrons into molecular hydrogen, offer promising avenues for sustainable energy production. My research centered on the roles of HydE and HydG, two essential maturase enzymes responsible for assembling the active site of [FeFe] hydrogenases, specifically the formation of the H-cluster, which is critical to the enzyme’s catalytic function.

We focused on investigating the mechanism of HydE, aiming to stabilize its intermediates using a combination of X-ray crystallography and EPR spectroscopy. Due to their structural similarity, a key challenge we faced was distinguishing between SAM and its inactive counterpart, SAH. Despite this obstacle, our EPR studies provided valuable insights into the redox behavior of the [Fe4S4] cluster in the presence of Complex B. Using different reducing agents such as DTH and MV•⁺. We demonstrated that Complex B plays a significant role in modulating the redox state of the cluster. Our ability to now precisely control these reaction conditions lays the groundwork for stabilizing and structurally characterizing key intermediates, facilitating their analysis through crystallography.

A key accomplishment of this work was the successful formation and structural characterization of Complex B, which was confirmed as a (κ3-cystéinato)Fe2+(CN)(CO)2 species through X-ray crystallography at a resolution of 1.3 Å. My research demonstrated that Complex B is not freely released into solution but transferred directly from HydG to HydE through transient protein-protein interactions. This direct transfer prevents the degradation of Complex B and maintains its stability during the maturation process.
This thesis advances our understanding of the assembly of the [FeFe] hydrogenase active site, providing significant insights into the enzymatic pathways that govern this process. The findings offer potential applications in optimizing biohydrogen production, contributing to the development of sustainable energy technologies.
[FeFe] hydrogenase active site, providing significant insights into the enzymatic pathways that govern this process. The findings offer potential applications in optimizing biohydrogen production, contributing to the development of sustainable energy technologies.
​
​