Probing the Hydrogen Bonding Structure in the Rieske Protein (Englisch)

The use of the far‐infrared spectral range presents a novel approach for analysis of the hydrogen bonding in proteins. Here it is presented for the analysis of FeS vibrations (500–200 cm⁻¹) and of the intra‐ and intermolecular hydrogen bonding signature (300–50 cm⁻¹) in the Rieske protein from Thermus thermophilus as a function of temperature and pH. Three pH values were adequately chosen in order to study all the possible protonation states of the coordinating histidines. The FeS vibrations showed pH‐dependent shifts in the FIR spectra in line with the change of protonation state of the histidines coordinating the [2Fe2S] cluster. Measurements of the low‐frequency signals between 300 and 30 K demonstrated the presence of a distinct overall hydrogen bonding network and a more rigid structure for a pH higher than 10. To further support the analysis, the redox‐dependent shifts of the secondary structure were investigated by means of an electrochemically induced FTIR difference spectroscopic approach in the mid infrared. The results confirmed a clear pH dependency and an influence of the immediate environment of the cluster on the secondary structure. The results support the hypothesis that structure‐mediated changes in the environment of ironsulfur centers play a critical role in regulating enzymatic catalysis. The data point towards the role of the overall internal hydrogen bonding organization for the geometry and the electronic properties of the cluster.