**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** THE CURRENT POPULATION DEPENDS ON THE HABER-BOSCH PROCESS FOR THE AMMONIA-DERIVED FERTILIZERS THAT ARE RESPONSIBLE FOR INCREASED CROP YIELDS. WHILE THE SOCIETAL BENEFITS OF THE HABER-BOSCH PROCESS CANNOT BE OVERSTATED, THE PROCESS REQUIRES LARGE, ENERGY-INTENSIVE FACILITIES, AND THE RESULTANT AMMONIA MUST BE SHIPPED AROUND THE WORLD. THIS RESULTS IN A SIZEABLE CARBON FOOTPRINT. FURTHERMORE, OVER-APPLICATION OF THESE FERTILIZERS TO THE SOIL SURFACE LEADS TO NITRATE RUN-OFFS THAT CAUSE AQUEOUS DEAD-ZONES. CONSIDERING THE SUBSTANTIAL ENVIRONMENTAL IMPACT OF CURRENT FERTILIZER PRACTICES, A BENIGN SUBSTITUTE IS DESIRABLE. AS AN ALTERNATIVE TO THE HABER-BOSCH PROCESS, WE CONSIDER NATURE'S AMMONIA-GENERATING ENZYME, NITROGENASE. WHILE NITROGENASE IS ABLE TO CATALYZE THE CONVERSION OF NITROGEN TO AMMONIA, THIS PROCESS IS ALSO ENERGETICALLY COSTLY, AND THE ENZYME IS EXCEPTIONALLY INTOLERANT OF OXYGEN. IN THIS PROJECT, WE WILL TAKE LESSONS FROM NITROGENASE TO ENGINEER A ROBUST PROTEIN-BASEDANALOGUE THAT IS CAPABLE OF NITROGEN FIXATION. ULTIMATELY, WE ENVISION THAT THIS SYSTEM COULD BE GENETICALLY ENCODED INTO SOIL MICROORGANISMS AND INTRODUCED INTO CROP SOIL AS A SUBSTITUTE FOR HABER-BOSCH-DERIVED CHEMICAL FERTILIZERS. THIS WILL RESULT IN SUBSTANTIALLY REDUCING THE PRIMARY ENVIRONMENTAL IMPACTS OF ARTIFICIAL NITROGEN FIXATION.IN THE PROPOSED PROJECT, THE FOCUS WILL BE ON THE ENGINEERING AND CHARACTERIZATION OF VANADIUM PROTEIN ANALOGUES OF VANADIUM NITROGENASE. THE STARTING POINT WILL BE THE DEVELOPMENT OF MONOMETALLIC VANADIUM AZURIN (VAZ) MODELS. AZURIN WAS SELECTED BECAUSE IT IS A STABLE AND EASILY MODIFIABLE PROTEIN SCAFFOLD THAT WILL REPLICATE THE ENVIRONMENT OF THE VANADIUM ION IN VANADIUM NITROGENASE BY INCLUDING COORDINATING HISTIDINE RESIDUES AND VANADIUM-SULFUR INTERACTIONS. THE COMPLEXITY OF THE MODEL SYSTEMS WILL THEN BE INCREASED BY ENGINEERING A BIMETALLIC VANADIUM-IRON CUAAZURIN ANALOGUE (VFEAZ). THE INCLUSION OF VANADIUM-IRON INTERACTIONS AND A SECONDARYSUBSTRATE BINDING SITE IN THE VFEAZ MODEL WILL IMPROVE THE FIDELITY OF THE MODEL SYSTEMS TO VANADIUM NITROGENASE. THIS WILL ALLOW US TO BETTER UNDERSTAND WHAT IT IS ABOUT THE NITROGENASE ENZYME THAT MAKES IT ABLE TO CATALYZE THIS CHALLENGING CHEMISTRY.THE MODEL PROTEIN SCAFFOLDS AZURIN (AZ) AND CUAAZURIN (CUAAZ) WILL BE EXPRESSED AND PURIFIED IN THE NON-METALATED (APO) FORM USING ESTABLISHED MOLECULAR BIOLOGY PROTOCOLS. THE APO-PROTEIN WILL THEN BE METALATED WITH AQUEOUS SOLUTIONS OF METAL (VANADIUM AND IRON) PRECURSORS AND THE REACTIVITY OF THE RESULTANT VAZ AND VFEAZ MODELS WITH CHEMICAL REDOX AGENTS AS WELL AS KNOWN SUBSTRATES (DINITROGEN), PLAUSIBLE ACTIVATED INTERMEDIATES (DIAZENE AND HYDRAZINE), AND INHIBITORS (CARBON MONOXIDE) OF NITROGENASE WILL BE TESTED. THE ISOLABLE REDOX AND SUBSTRATE-BOUND SPECIES WILL BE CHARACTERIZED WITH A SUITE OF ESTABLISHED EXPERIMENTAL AND COMPUTATIONAL METHODOLOGIES TO DEVELOP AN UNDERSTANDING OF THEIR GEOMETRIC AND ELECTRONIC STRUCTURES. IN PARTIC,ULAR, ULTRAVIOLET-VISIBLE ABSORBANCE, RESONANCE RAMAN, CONTINUOUS-WAVE AND PULSED ELECTRON PARAMAGNETIC RESONANCE, AND METAL K-EDGE X-RAY ABSORBANCE SPECTROSCOPIES WILL PROVIDE INFORMATION ON THE GEOMETRIC AND ELECTRONIC STRUCTURES OF THE SAMPLES OF INTEREST. THE EXPERIMENTAL RESULTS OF THOSE STUDIES WILL BE COMPARED TO COMPUTATIONAL RESULTS OBTAINED FROM DENSITY FUNCTIONAL THEORY, TIME-DEPENDENT DENSITY FUNCTIONAL THEORY, AND MULTIREFERENCE CALCULATIONS TO TIE EXPERIMENTAL SPECTRA AND FUNCTION TO OPTIMIZED STRUCTURES. A COMPARISON BETWEEN THE STRUCTURES OF THE MODEL SYSTEMS AND NATURALLY OCCURRING VANADIUM-NITROGENASE WILL GUIDE FURTHER OPTIMIZATION OF THE PROTEIN SCAFFOLD. SINCE STRUCTURE AND FUNCTION ARE CLOSELY LINKED, THIS WILL ENABLE US TO RATIONALLY DESIGN FUNCTIONAL MODELS OF NITROGENASE.THE DEVELOPMENT OF A GENETICALLY ENCODABLE ARTIFICIAL NITROGENASE HAS THE POTENTIAL TO PROVIDE AN ALTERNATIVE PATHWAY FOR THE GENERATION OF AMMONIA-BASED FERTILIZERS WITH REDUCED ENVIRONMENTAL IMPACT. THE RESULTS OF THESE STUDIES WILL BE DISSEMINATED THROUGH PEER-REVIEWED JOURNAL MANUSCRIPTS AS WELL AS POSTER AND ORAL PRESENTATIONS AT CONFERENCES. WHEN MAJOR MILESTONES ARE ACHIEVED, SUCH AS ATMOSPHERIC NITROGEN BINDING AND CONVERSION, A BROADER AUDIENCE WILL BE REACHEDBY WORKING WITH THE INSTITUTION'S SCIENTIFIC COMMUNICATION OFFICES FOR PRESS RELEASES.