Thema der Dissertation:
Mechanistic Insights into the Formate Dehydrogenase - EPR Spectroscopy and Computational Methods
Mechanistic Insights into the Formate Dehydrogenase - EPR Spectroscopy and Computational Methods
Abstract: The formate dehydrogenase (FDH) represents an enzyme at the interface of the biosphere and atmosphere as it reversibly converts formate into atmospheric carbon dioxide (CO2). Due to this property, this enzyme has the potential to be leveraged in geoengineering to effectively reduce the atmospheric CO2 concentration, which has significantly increased since the start of the industrial revolution. The enzymatic oxidation of formate to CO2 occurs at the molybdenum cofactor (MoCo) within FDH, involving the abstraction of the formate proton and two electrons, which are transferred via an electron transfer chain consisting of iron-sulfur (FeS) clusters and a flavin mononucleotide (FMN) to an electron acceptor. Despite analyses of related molybdoenzymes, the precise mechanism of the reaction and the electron transfer remains elusive. Electron paramagnetic resonance (EPR) spectroscopy, a method for probing transition metal ions in their paramagnetic states, was utilized in this work to investigate the immediate environment of the MoCo in its azide-inhibited paramagnetic Mo(V) state. Hyperfine spectroscopic investigations revealed the absence of CO2 in the Mo(V) state, as a direct consequence of rapid molecular conversion and following CO2 release, while the formate proton remains bound in close proximity to the MoCo. Although CO2 is already released in the azide-inhibited Mo(V) state, insights into its binding site can be gained by analyzing azide as a competitive inhibitor of FDH, adopting a similar orientation to that of the substrate. A combined approach of density functional theory (DFT) and EPR spectroscopy enabled the characterization of the precise binding site of the formate proton and revealed its interaction with azide close to the MoCo The electron transfer mechanism was investigated by EPR-monitored redox titration experiments resulting in the determination of the redox potentials of the FeS clusters and the electron transfer pathway through the enzyme. These investigations led to the proposal of MoCo acting as electron transfer transducer, converting a two-electron transfer into a sequential one-electron transfer. This effect is reversed by another transducer within the FdsGB subunit of FDH before the electron acceptor is reduced by two electrons. All conclusions were obtained by an extensive analysis of EPR spectra under various biochemical conditions and preparations. To minimize the preparatory effort for similarly complex research subjects, deep learning-based methods were developed in order to separate EPR signals according to their lifetimes. The performance and limitations of this method were extensively evaluated on a defined test set.
Time & Location
May 28, 2025 | 04:30 PM
Hörsaal A (1.3.14)
(Fachbereich Physik, Arnimallee 14, 14195 Berlin)