UC Riverside

Carbon dioxide (CO2) is a potent greenhouse gas that has been building up in the Earth’s atmosphere since the beginning of the industrial revolution, resulting in anthropogenic climate change that constitutes an existential threat to human society. Adoption of renewable energies alone is likely to be insufficient to tackle this threat and current methods of capturing CO2 rely on the challenging and costly practice of burying trapped CO2 underground. In recent years, biological systems that can capture and convert CO2 to a much more practical compound have been the focus of many studies. Molybdenum-containing formate dehydrogenases are very interesting as they interconvert CO2 with the so-called feedstock chemical formate. Elucidation of the highly efficient catalytic mechanism by which enzymes catalyze this interconversion under mild conditions is expected to lead to the development of new bio-inspired catalysts, providing a means to effectively capture CO2. Moreover, such catalysts will lead to an attractive means to store energy in the form of chemical bonds. In the present work, the structure and function of the FdsDABG formate dehydrogenase from Cupriavidus necator, a cytosolic NAD+-dependent enzyme, and the FdhF formate dehydrogenase from Pectobacterium atrosepticum, an NAD+ independent formate dehydrogenase also found in the cytosol, have been investigated. Various techniques have been employed, including kinetic steady-state assays, rapid reaction kinetics, X-ray crystallography, electron paramagnetic resonance, extended X-ray absorption fine structure and electrochemical methods to investigate these formate dehydrogenases. This work has revealed that for FdsDABG, acid/base catalysis does not have a significant impact on the mechanism of formate oxidation, consistent with this enzyme specifically catalyzing a hydride transfer reaction utilizing CO2 as the substrate for reverse catalysis. In addition, inactivation of FdsDABG in air is shown to occur through a superoxide-mediated process which can be prevented by superoxide dismutase. X-ray crystal analysis of FdsBG has yielded information regarding the position and arrangement of its redox-active cofactors, including details of the NAD+/NADH binding site. Finally, UV/Visible absorption and formate reduction experiments of FdhF confirm the recombinant enzyme’s functionality and provide insights on how the electron transfer process occurs between the molybdenum center and its sole iron-sulfur cluster. The robust findings of our investigations provide compelling evidence supporting a hydride transfer mechanism for molybdenum containing formate dehydrogenases.