Distinct Valence States of the [4Fe4S] Cluster Revealed in the Hydrogenase CrHydA1

Harnessing biological systems for the hydrogen economy requires a thorough understanding of the responsible iron-sulfur (FeS) cluster-containing proteins. This study focuses on the HydA1 hydrogenase of Chlamydomonas reinhardtii (CrHydA1), a simplistic and well-characterized [FeFe]-hydrogenase and employs electron paramagnetic resonance (EPR) spectroscopy for studying the complex paramagnetic states of its FeS cluster. We report the existence of two distinct cluster species arising from a single [4Fe4S]-cluster within the apo-form of the enzyme. Multi-frequency and variable-temperature pulsed EPR experiments revealed the interconversion of the two dominating species, characterized by their axial and rhombic EPR responses. The species interconvert depending on redox potentials and pH levels. Molecular dynamics simulations and quantum mechanical calculations identified two secondary-sphere residues, Arg227 and Lys228, as potential causes of the binary response of the [4Fe4S] cluster. These amino acids can adopt inner and outer conformations relative to the [4Fe4S] cluster, causing valence rearrangement and thus providing a plausible explanation for the observed spectroscopic differences. The dynamic behavior of these cluster species not only suggests a central role in the catalytic mechanisms of hydrogenases but also offers valuable insights for the analysis of other FeS-containing metalloproteins with comparable characteristics.