[Project supported by FrenchBIC] The catalytic cycles of reversible and irreversible FeFe hydrogenases
During my Ph.D. I studied different homologous FeFe-hydrogenases, enzymes that catalyze the oxidation and evolution of hydrogen. Despite their structural similarity and the identical active site, different hydrogenases exhibit different catalytic properties. I am interested in the understanding of the molecular determinants of these properties. In this project I focused on “catalytic reversibility”, which is defined in relation to the thermodynamic driving force (overpotential in electrochemistry experiments) that has to be applied for the enzyme to work at a significant rate. Prototypical, or “standard”, hydrogenases (such as HydA1 from Chlamydomonas reinhardtii, Cr HydA1) are reversible catalysts: a small difference between the electrode potential and the equilibrium potential of the H+/H2 couple leads to a significant hydrogen oxidation or proton reduction current. The recently identified sensory hydrogenase (HydS) from Thermoanaerobacter mathranii (Tam HydS) shows an irreversible catalytic response, as it catalyzes the reaction in either direction only if a large overpotential is applied.
The aim of this work is to understand what differ in the catalytic cycles of these two enzymes that determines their different catalytic responses. To do so we performed a kinetic analysis of the catalytic response of the two enzymes in Protein Film Electrochemistry (PFE), and we compared it with the information that are obtained by Fourier Transformed Infrared Spectroscopy (FITR). The latter technique, indeed, informs about the different states of the active site of the hydrogenase under different conditions (like pH and redox potential).
I spent, therefore, one week at the Laboratory of Energy converting enzymes, in James Birrell group, which has a great expertise in FTIR spectroscopy, specifically applied to hydrogenases.
There, I have performed at equilibrium redox and pH titration on the enzyme HydA1 from Cr. This data allowed us to discriminate between different kinetic models when analyzing the PFE experiments and helped us to further understand the catalytic cycle of the FeFe hydrogenases, and which parameters determines the reversibility of the catalytic response.
I would like to acknowledge the FrenchBIC for the grant and James Birrell for the opportunity to work in his lab. It has been a great week of experiments and scientific discussions that taught a lot.
Publication: “Kinetic modeling of the reversible or irreversible electrochemical responses of FeFe-hydrogenases”, J. Am. Chem. Soc. (2024). doi: 10.1021/jacs.3c10693