Kasanmascheff, MügeHeghmanns, Melanie2024-04-252024-04-252023http://hdl.handle.net/2003/42451http://dx.doi.org/10.17877/DE290R-24287Iron-sulfur (FeS) clusters are essential cofactors found in all living organisms. Acting as versatile electron carriers they are indispensable for life-sustaining processes, contributing to respiration, nitrogen fixation, and hydrogen production. Several proteins containing single and multiple FeS clusters are involved in the pathway to hydrogen evolution. Electron paramagnetic resonance (EPR) spectroscopy was used to characterize their FeS clusters providing valuable information about their magnetic properties, structural features, redox states, and biological function. Understanding and tuning the complex interplay of these proteins and their clusters is required for efficient biotechnological H2 production to sustainably meet the requirements of the world's increasing energy demand. We established pulsed EPR monitored redox potentiometry, performed at higher frequencies than usual, for determining the midpoint potentials of ferredoxins (Fdxs) and variants. Exchanging a single amino acid residue in CrFdx1 fine-tuned the midpoint potential of its [2Fe2S] cluster. Moreover, the characterization of fully maturated and more complex [FeFe]-hydrogenases harboring multiple distinct FeS clusters is addressed. One of the main drawbacks of efficient hydrogen production is the oxygen sensitivity of the H-cluster. A newly discovered oxygen-protection mechanism in the [FeFe]-hydrogenase from C. beijerinckii led to a comprehensive EPR spectroscopic study, characterizing not only the H-cluster states but also discovering a new radical R•ox. Advanced spectroscopic methods explored its origin and function. Eventually, the spectral features of the complex [FeFe]-hydrogenase CpI from C. pasteurianum were revisited and revealed exchange interactions between the H-cluster and the neighbored [4Fe4S] cluster. Variants showed a change in the strength of the exchange coupling and were explored to investigate its effect on the biological function. The studies presented in this thesis, including the fine-tuning of the midpoint potential of Fdxs, the discovery and characterization of an unusual radical signal, and the investigation of exchange coupling interactions in [FeFe]-hydrogenases, shed light on the magnetic properties and functional roles of FeS clusters in essential electron transfer processes and hydrogen evolution pathways.enEPR spectroscopicIron sulfur clustersHydrogenasesHydrogen production540TextElektronenspinresonanzspektroskopieSchwefelEisenClusterHydrogenasen