Novel microbial based enzymatic CO2 fixation mechanisms: Conformational control of enzymatic reactivity
The broad, long-term goal of the research is to provide insight into the mechanism of novel carboxylation and electron bifurcation reactions as paradigms to study how enzyme utilized protein conformational change to protect reactive intermediates and/or control the pathway in a catalytic cycle. catalyzed by enzymes involved in bacterial alkene, ketone, and epoxide metabolism. We are examining two paradigms in which conformational change is a key mechanistic feature of 1) protecting reaction intermediates that are sensitive to breakdown in the presence of water and 2) directing electron flow in reactions that split electron pairs along two pathways in reactions that fine tune metabolic efficiency in microbial metabolism. Both paradigms are of key importance in defining the molecular determinants of reactions relevant to CO 2 capture and fuel upcycling. In the first paradigm the compounds acetone and bicarbonate are activated by the addition of phosphate creating reaction intermediates (phosphoenolacetone and carboxyphosphate) that are very sensitive to breakdown in the presence of water. We are studying the mechanism of how the enzyme protects these reactive intermediates from water by encapsulating them upon their formation within a hydrophobic protein channel leading to an enzyme active site approximately 40 Å away. In the second paradigm, we are examining how the direction of electron transfer reactions can be modulated through protein conformational change that alters either the difference driving force or distance or both between electron transfer pathways in enzymes that catalyze electron bifurcation. We are using a combination of enzyme kinetics, mass spectrometry, spectroscopy, and structural analysis through X-ray diffraction and scattering methods to characterize the mechanism of conformational changes along the catalytic path. Within the context of the mission of the Department of Energy and the core activities of the Energy Biosciences, the results obtained in the study will reveal new insights into the fundamentals of novel carboxylation / CO 2 capture chemical transformations and electron transfer reactions that equate to biological fuel upcycling.