Jessica Candido Soares

• Iron-sulfur clusters (FeS) are versatile cofactors found in metalloproteins. Most FeS clusters are coordinated exclusively by cysteine residues. However, for an increasing number of proteins the presence of non-cysteinyl coordination has been noted, with a not fully understood role. At least eight other amino acids act as ligands for FeS clusters, with histidine being the first identified and possibly also the most common amino acid to substitute cysteine. Coordination by two histidine residues is found in Rieske and Apd1-like proteins, whereas MitoNEET and presumably also the IscR protein have one histidine ligand. Suggested roles of the histidine residue(s) are tuning of the redox potential, proton-coupled electron transfer (PCET) or modulation of cluster stability. In this thesis the redox properties of the [2Fe-2S]1+/2+ cluster of Escherichia coli IscR and the pKa values of the histidine ligand in both redox states were investigated to demonstrate that PCET occurs. Combining UV-Vis, CD, EPR, and Mössbauer spectroscopic techniques with redox titrations pKa values of 8.0 and ~11.1 were determined for the histidine ligand in the oxidized and reduced form of IscR, respectively. The redox potentials varied between -49 and -221 mV, at low and high pH values, respectively, with a maximal slope of approximately -55 mV/pH unit between the pK values. Additionally, the impact of these findings on the physiological function in the cellular environment is discussed. Lack of coordination by a fourth cysteine can lead to cluster instability, as seen in aconitase, which is subject to [4Fe-4S] to [3Fe-4S] cluster conversion. In the second project the YjdI protein from E. coli was characterized. YjdI exhibited g=2.01 and g=12 EPR signals characteristic of a [3Fe-4S]1+/0 cluster but converted to [4Fe-4S]1+ upon reconstitution and reduction. Studies including mutagenesis and Mössbauer spectroscopy will be needed to reveal the fourth cluster ligand of reconstituted YjdI. PCET has been extensively studied and linked to function in proton translocation for Rieske proteins. For the reduced state of three different Rieske proteins protein film voltammetry indicated that the two pK values are identical and above 12. However, despite EPR spectroscopic characterization of the alkaline (fully deprotonated) species, an intermediate monoprotonated species in the reduced state has not been reported yet. Resorting to a titration between pH 9.5 and 14 of Thermus thermophilus Rieske protein EPR spectroscopy revealed not only the fully protonated and deprotonated species, but also a monoprotonated species with g=2.03, 1.90 and 1.78. Due to the proximity of the pKa values only mixtures of species were found. Strikingly, the system exhibited cooperative behavior: the second pKa (11.8) is lower than the first pKa (12.1). Overall, these findings deepen the understanding of the redox properties of IscR, contribute to the characterization of YjdI, and demonstrate cooperativity of consecutive ligand deprotonations for the first time in the FeS field. These findings emphasize the need for further research into PCET and properties of FeS proteins.