Reduced forms of molecular nitrogen (dinitrogen) are essential for the production of fertilizers and countless industrial chemicals, as well as the biosynthesis of amino and nucleic acids. The reduction of dinitrogen to ammonia (nitrogen fixation) by the Haber-Bosch process requires high temperatures and pressures, consuming over 100 billion watts of power every year. Biological dinitrogen fixation does not require such extreme conditions; indeed, the enzyme nitrogenase catalyzes ammonia synthesis under ambient conditions. After decades of intense research efforts, however, it is still not known how nitrogenase activates dinitrogen. Nitrogenase catalysis is distinct from other multielectron/multiproton catalytic reactions in its requirement of 16 ATP molecules per turnover reaction despite a favorable thermodynamic driving force. This project aims to elucidate why and how ATP hydrolysis is required in biological nitrogen fixation. Recent structural studies show that the two constituents of nitrogenase, the Fe-protein (electron donor/ATPase) and the MoFe-protein (catalytic component), can assume at least three docking geometries, depending on the ATP-hydrolysis state. By developing and utilizing several powerful chemical and biophysical tools, this project will probe whether multiple Fe-protein:MoFe-protein docking modes are functionally important, and if they are involved in timing the successive electron and proton transfers into the catalytic metal cluster (FeMoco). In parallel, photochemical methods will be utilized to investigate the possibility of driving substrate reduction at FeMoco without requiring ATP hydrolysis. The demonstration of photoactivation of nitrogenase will open new avenues for studying its mechanism that in turn could lead to the development of biocatalytic systems for ammonia and hydrogen production.

Broader Impacts: The complexity of biological nitrogen fixation requires a multidisciplinary plan of attack. This project combines a multitude of experimental approaches that will provide an expansive training ground for graduate and undergraduate students. Biological nitrogen fixation sustains a large fraction (40%) of the world's population, and the industrial Haber-Bosch process is responsible for considerable amounts of energy consumption and greenhouse gas emissions. A thorough understanding of nitrogenase mechanism could lead to the design of clean and efficient biocatalysts for ammonia production, which would have an immense economic and environmental impact. Nitrogen fixation also provides a conduit into the education goals of this project, which is to raise the awareness of students about the global energy problem, and to train scientists in energy biosciences. These goals will be addressed on several fronts, including a) the interdisciplinary training of graduate and undergraduate students in the laboratory, b) restructuring of an advanced course in Bioinorganic Chemistry to focus on redox-catalytic processes involved in global carbon, nitrogen, oxygen, and sulfur cycles, as well as the design of a multidisciplinary course on Global Energy Problem and Alternative Energy Research, and c) outreach to low-income students from underrepresented groups attending a local charter school, and their recruitment through seminars.

Project Report

Reduced forms of dinitrogen are essential for the biosynthesis of amino and nucleic acids, as well as the production of fertilizers and countless industrial chemicals. The reduction of chemically inert dinitrogen to ammonia (nitrogen fixation) by the Haber-Bosch process requires extreme conditions (>450 ?C, 270 atm N2 and H2) and accounts for ~ 1-2% of all human energy consumption. The bacterial enzyme nitrogenase, in contrast, accomplishes the same feat at ambient conditions: N2 + 8 H+ + 8 e- + 16 ATP → 2 NH3 + H2 + 16 ADP + 16 Pi Understanding how nitrogenase orchestrates the transfer of 8 electrons and 8 protons to effect the reduction of dinitrogen has been a tremendous challenge. Mo-nitrogenase is a two-protein complex, formed by the Fe-protein (FeP) and the MoFe-protein (MoFeP) (Figure 1). FeP functions as an electron shuttle to MoFeP, where substrate activation takes place at the so-called FeMo-cofactor (FeMoco; a [7Fe:1Mo:9S:1C] cluster). Despite extensive research, two critical question on nitrogenase catalysis remain unanswered, namely: "How does ATP hydrolysis regulate the interactions between FeP and MoFeP that control the delivery of multiple electrons/protons to FeMoco and the substrate?" and "What is the mechanism of substrate reduction on FeMoco?" This NSF CAREER-supported project aimed to address these questions by 1) developing light-activated electron injection strategies for the activation of FeMoco-mediated catalysis independently of ATP hydrolysis, and 2) probing the functional importance of the multiple, nucleotide-dependent docking modes between FeP and MoFeP (Figure 2). Under Aim 1, it was found that by careful placement of ruthenium-polypyridine-based photosensitizers on the MoFeP-surface, 2- and 6-electron catalytic reductions of, respectively, protons and hydrogen cyanide by FeMoco could be activated by light. On one hand, these experiments broke the long-standing dogma that ATP-hydrolysis and FeP were crucial for nitrogenase activity. On the other hand, light-activated nitrogenase catalysis was determined to be significantly less efficient than that driven by ATP hydrolysis, which supported the hypothesis that electron and proton transfer reactions within MoFeP must be conformationally gated in an ATP-dependent manner. Under Aim 2, new strategies were developed for the site-directed mutategenesis of MoFeP in the native, nitrogen-fixing organism, Azotobacter vinelandii. These strategies allowed multiple mutations to be made in regions on the MoFeP surface which enable FeP docking in different nucleotide-bound states (Figure 2). It was found that mutations even in the surface regions that appear to be "off-pathway" in the nitrogenase catalytic cycle (such as Zone 1) had considerable detrimental effects on nitrogen-fixation efficiency. These findings confirmed our hypothesis that multiple FeP-MoFeP docking modes are not a crystallographic artifact and that they have a functional significance. Under this CAREER project, two PhD students (both women) and nearly ten undergraduate and high school researchers received a broad training in bioinorganic chemistry. The PI was able to initiate and sustain several science outreach efforts both on campus to members of underrepresented minorities and outside of campus to high school students from across and outside the country. The results of the project have been disseminated in the form of many journal publications, book chapters and national/international public seminars.

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Kamal Shukla
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University of California San Diego
La Jolla
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