• Laun, Konstantin

Abstract

In nature, highly selective and efficient enzymes have been evolved to catalyze a wide variety of fundamental reactions at very low overpotentials. These enzymes can work under various conditions, i.a. different catalytic activities, in the presence of oxygen, specific bias or at very high temperatures. Enzymatic solutions for CO2 conversion are of special interest given their high selectivity and mild reaction conditions. Here, metal-dependent molybdenum (Mo)-containing formate dehydrogenases are of particular interest for industrial applications since they are the only class of enzymes capable of producing a liquid in-demand product using electrons and CO2 as the only substrate. To understand how this enzyme functions in its natural environment, dynamic and structural studies have been performed to elucidate the specific molecular mechanisms. This thesis establishes vibrational spectroscopy as a tool to gain mechanistic insights into the CO2 reducing formate dehydrogenase (FDH) from Rhodobacter capsulatus and the model system TMAO reductase TorA from Escherichia coli. In the first part of this thesis, inhibitors such as azide or cyanate were employed to study substrate binding at the active site of the FDH enzyme. Infrared (IR) spectroscopy was complemented with DFT calculations to clarify the binding of azide and cyanate at the Mo ion or its second coordination sphere. The IR spectroscopic characterization of the inhibitor-bound FDH revealed two bands. One of which was found to depend on the cofactor, while the second band was assigned to a cofactor-independent species. Monitoring the response to reduction and oxidation allowed ruling out covalent binding to the Mo ion for the cofactor-dependent species which instead interacts electrostatically with the nearby metal site. The exact position of the cofactor-dependent binding sites for azide and cyanate were elucidated by two active site variants, in which His386 and Arg587 were exchanged. In the second part of this work, a minimal photocatalytic three-component system including the photosensitizer (PS), the biocatalyst FDH and sacrificial electron donor EDTA was established to study the FDH under turnover conditions. EDTA has been shown to regenerate the PS and releases the substrate CO2. The entire light-triggered process can be followed by gas chromatography-mass spectrometry, IR and UV-vis spectroscopy. Furthermore, the IR band associated to the cofactordependent azide species decreases in the presences of CO2 under catalytic turnover conditions. This observation verifies the relevance of that binding site for catalysis. The last part of this thesis is dedicated to the elucidation of the first ligation sphere of the Mo ion. For this purpose, the monomeric TMAO reductase TorA, which exclusively harbours the Mo cofactor, was studied using resonance Raman (RR) spectroscopy and complementary DFT calculations. The wildtype enzyme was compared to two active site variants, e.g. the S191C and the S191A. For the very first time, the Mo=S stretching mode was detected by RR spectroscopy. This band was found in both the active wildtype and the S191C variant, but was absent in the inactive S191A variant. Instead, the latter variant displays a RR band at 852 cm–1 due to the Mo=O stretching. Hence, it is suggested that the Mo=S ligand is required for catalysis. In addition, DFT calculations and RR spectroscopy support the view that Ser191 dissociates from the Mo ion during catalysis upon two-electron reduction.

Topics

• impedance spectroscopy
• molybdenum
• Oxygen
• density functional theory
• gas chromatography
• spectrometry
• Ultraviolet–visible spectroscopy
• vibrational spectroscopy
• gas chromatography-mass spectrometry