Enzymes, the catalysts for biological reactions, are able to efficiently perform very difficult chemical transformations. This project has two major long-range goals: (i) to help implement the conversion of cellulose, the major constituent of plant-derived biomass in nature, into green and sustainable biofuels; (ii) to understand how nature fertilizes the earth through the conversion of atmospheric nitrogen molecules into bioavailable nitrogen. Cellulose is potentially a renewable source of biofuels, but despite years of study, the efficient and economical degradation of cellulose remains a limiting factor in the industrial conversion of cellulose to biofuels. Polysaccharide monooxygenases are enzymes that use a copper active site to catalyze cellulose degradation, and their utilization could help to overcome the prohibitive costs of cellulose degradation. The reduction of nitrogen to yield two moles of ammonia, available as fertilizer, is catalyzed by the metalloenzyme, nitrogenase. The agronomic, economic, and social significance of nitrogenase can be appreciated by recognizing that it supplies approximately 50% of the N atoms in humans. The remainder is produced by the high-temperature/pressure Haber-Bosch industrial process, which uses H2 generated from CH4 as reductant and as a result produces vast amounts of CO2 and accounts for ~2% of the world's total energy consumption. The goal of this project is to understand the mechanism of these two important enzymes. This project will contribute to the training of graduate students and postdocs, with special attention to increasing the participation of women and underrepresented minorities in the scientific enterprise.
This project is founded on techniques developed in the laboratory of the investigator for freeze-trapping reactive intermediates, reducing them in the frozen solid by radiolytic cryoreduction, and performing EPR/ENDOR characterizations of paramagnetic states. In the case of polysaccharide monooxygenases, a primary intermediate is freeze-trapped and paramagnetic one-electron reduced forms generated by cryoreduction. A step-annealing protocol propels this along its reaction pathway in a controlled fashion for study of successive intermediates that have only fleeting existence at ambient temperatures. This work is paralleled by studies of Cu-oxy, hydroperoxy, and peroxo model complexes, both with and without substrate present. The investigator and his group have established that nitrogenase functions by sequential accumulation of 4e-/4H+ to generate an activated state that undergoes reductive elimination of H2 coupled to N2 displacement and N≡N triple bond cleavage. The subsequent delivery of 4 additional electrons/protons then produces 2 NH3 as products to complete the catalytic cycle. In this project, the characterization of the nitrogenase mechanism by trapping intermediates through freeze-quench or cryoreduction with analysis by EPR/ENDOR, and by monitoring their interconversion, is sought.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
