Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron-Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H₂ activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentials for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (∼ < -450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fd_ox/Fd_red ratio at which CpI can operate, consistent with the role of CpI in recycling Fd_red that accumulates during fermentation. Subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.