Metal Centers in Hydrogenases, Bimetallic Oxidases, and Model Complexes Studied by Advanced X-ray Spectroscopy Techniques and Quantum Chemistry

Abstract

Numerous metal cofactors in proteins are able to catalyze the activation of small molecules such as H2O, H2, O2, and CO2. Determination of their molecular and electronic properties as well as understanding of their catalytic reaction mechanisms attracts much research interest. In this thesis, two different biological systems based on iron and mixed iron/manganese cofactor sites were predominantly studied. [FeFe]-hydrogenases catalyze reversible hydrogen turnover whereas ligand-binding oxidases activate oxygen. An extensive X-ray absorption and emission spectroscopy and DFT study on a series of synthetic compounds mimicking the diiron site of [FeFe]-hydrogenases was performed. This revealed relations of ligand variation (substitution at terminal ligands and the bridging dithiolate group) to the electronic properties of the diiron complexes. Good agreement between experimental and calculated data was achieved. The study may provide a basis for future improvement of hydrogen forming catalysts. Successful H- cluster formation in HydA1 [FeFe]-hydrogenase (from green algae) reconstituted with synthetic diiron complexes was proven by XAS/XES studies. Comparison of XAS data of freeze-dried and native O2-sensitive [FeFe]-hydrogenases contributed to establishing lyophilization as a method for preventing O2-induced H-cluster degradation. Bimetallic cofactor sites in the ligand-binding oxidase GkR2lox (from Geobacillus kaustophilus) were investigated by a wide range of synchrotron-based X-ray techniques (XAS, XES, and nuclear resonance vibrational spectroscopy) as well as DFT. This disclosed the molecular structure and electronic configuration including the protonation state of the FeFe and MnFe active sites. The atomic level description of the cofactor structures was significantly improved in combination with XRD data. Site-selective NRVS on 57Fe labelled GkR2lox as well as assignment of vibrational bands with DFT were established. Detailed XAS/TRXF investigations on mutant variants of GkR2lox were performed and revealed the role of amino acid ligands in cofactor metalation and Fe or Mn specificity. Relations between the mechanism of O2 activation and cofactor structure were provided. Determination of the physiological role of R2lox proteins requires further studies to unravel the interplay of ether cross-linking in the protein scaffold and O2 activation. The described studies have further established advanced X-ray spectroscopy and quantum chemical methods as a viable tool for characterization of cofactors in enzymes and biomimetic molecules under functional conditions. New insight in the properties of the active sites in hydrogenases and oxidases were obtained. Various contributions to investigations of metal centers in further metalloproteins and synthetic compounds with X-ray methods were made, but are not included in these thesis to keep the focus on binuclear active sites (see own references). The presented results were compiled in several publications, making the developed methodology and scientific findings accessible to a larger community.