[FeFe]-hydrogenase active site assembly: tracking intermediates in the radical SAM maturase HydE – IRMA

Understanding the chemical processes involved in enzymes is a challenge that requires, in most cases, the use of complementary experimental techniques. This knowledge is often crucial for the development of biotechnological applications. The aim of the IRMA application is to perform a precise structural and temporal characterization, associated with a clear spectroscopic signature of the different radical intermediates involved in the protein HydE. The latter belongs to the so-called radical SAM enzyme superfamily and is a key player in the FeFe hydrogenase active site assembly machinery. To achieve this, we aim at coupling electron paramagnetic resonance and time-resolved serial-crystallography combined with theoretical QM/MM calculations to both establish the chemical mechanism of the enzyme and characterize the structural motions at play. In addition to a fundamental contribution to the understanding of key steps in the reactivity of this enzyme, notably the role of the protein matrix in the control of these processes, the associated methodological developments will open the way to the dynamic structural study of many oxygen-sensitive metalloenzymes. Such implementation of dynamics in structural studies is a major challenge to better understand how these enzymes really work with many potent applications.