No description available.

No description available.

Nitrogen is a critical nutrient for growth and reproduction in living organisms. Although the Earth’s atmosphere is composed of ~80% nitrogen gas (N2), it is inaccessible to most living organisms in that form. Biological nitrogen fixation, however, can be performed by microbes that harbor the enzyme nitrogenase. This enzyme converts N2 into bioavailable ammonia (NH3) and accounts for at least half of the “fixed”nitrogen on the planet. The other major contributor to ammonia production is the industrial Haber-Bosch process. While the Haber-Bosch process has made significant advances in sustaining the global food supply through the generation of fertilizer, it requires high temperature and pressure and fossil fuels. This makes nitrogenase an ideal system for study, as it is capable of performing this challenging chemistry under ambient conditions and without fossil fuels. Nitrogenase requires energy and electrons to convert N2into NH3. The work presented here examined how the enzyme receives electrons to perform the reaction. It was discovered that some microbes employ a novel mechanism that adjusts the energy state of the electrons so that nitrogenase can accept them. Further, the slowest step that takes place in nitrogenase once the electrons are taken up was identified. Finally, by capitalizing on fundamental knowledge, a biohybrid system was designed to grow nitrogen-fixing bacteria in association with electrodes for light-driven production of fixed nitrogen that has potential to be used as a fertilizer for plant growth. Gaining an in-depth understanding of nitrogenase provides insight into one of the most challenging biological reactions, and the newfound knowledge may be a catalyst in developing more efficient systems for sustainable ammonia production.

Cytochromes P450 are diverse group of heme enzymes found in most species on Earth. In humans they are involved in metabolism of foreign compounds or steroids, bacteria employ cytochromes P450 for utilization of various hydrophobic substrates. General reaction catalyzed by cytochromes P450 is monooxygenation, when one atom of oxygen molecule is introduced into the substrate, while the other is reduced producing water. NADPH:cytochrome P450 oxidoreductase or cytochrome b5 usually serves as an electron donor providing electrons needed for activation of oxygen in eukaryotic organisms, in bacteria small FeS proteins or flavoproteins are these electron donors. It was shown earlier that bacterial electron donor flavodoxin could also interact with human cytochromes P450 in vitro. This thesis employs molecular modeling techniques to support a hypothesis that flavodoxin is responsible for reduction of human (1A2, 2A6, 2A13, 2C9, 2C19, 3A4) and bacterial (101A1 a 176A1) cytochromes P450 heterologously expressed in Escherichia coli. An initial guess of possible mutual orientations of cytochrome P450 and flavodoxin was predicted using information-driven protein-protein docking. The stability of these complexes was examined by directed dissociation method. The most stable orientation for each cytochrome P450 was further...