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

Iron-sulfur clusters play a crucial role in electron transfer for many essential enzymes, including [FeFe]-hydrogenases. This study focuses on the [4Fe4S] cluster ([4Fe]_H) of the minimal [FeFe]-hydrogenase from Chlamydomonas reinhardtii (CrHydA1) and employs advanced spectroscopy, site-directed mutagenesis, molecular dynamics simulations, and QM/MM calculations. We provide insights into the complex electronic structure of [4Fe]_H and its role in the catalytic reaction of CrHydA1, serving as paradigm for understanding [FeFe]-hydrogenases. We identified at least two distinct species within the apo-form of CrHydA1, designated 4Fe-R and 4Fe-A, with unique redox potentials and pH sensitivities. Our findings revealed that these species arise from a complex interplay of structural heterogeneity and valence isomer rearrangements, influenced by second-sphere residues. We propose that the interconversion between 4Fe-R and 4Fe-A could provide control over electron transfer in the absence of accessory FeS clusters typically found in other [FeFe]-hydrogenases. The insights gained from this study not only enhance our understanding of [FeFe]-hydrogenases but also provide a crucial foundation for future investigations into analysis of other FeS clusters across diverse biological systems.