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Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe 7 S 9 MoC-homocitrate) as a critical N 2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E 4 (4H), which has accumulated 4[e − /H + ] as two bridging hydrides, Fe2–H–Fe6 and Fe3–H–Fe7, and protons bound to two sulfurs. E 4 (4H) is poised to bind/reduce N 2 as driven by mechanistically-coupled H 2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H 2 as the enzyme relaxes to state E 2 (2H), containing 2[e − /H + ] as a hydride and sulfur-bound proton; accumulation of E 4 (4H) in α-V70I is enhanced by HP suppression. EPR and 95 Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e − /H + ] to the E 0 state of the WT MoFe protein and to both α-V70I conformations generating E 2 (2H) that contains the Fe3–H–Fe7 bridging hydride; accumulation of another 2[e − /H + ] generates E 4 (4H) with Fe2–H–Fe6 as the second hydride. E 4 (4H) in WT enzyme and a minority α-V70I E 4 (4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2–H–Fe6 followed by slower HP of Fe3–H–Fe7, which leads to transient accumulation of E 2 (2H) containing Fe3–H–Fe7. In the dominant α-V70I E 4 (4H) conformation, HP of Fe2–H–Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3–H–Fe7 occurs first and the resulting E 2 (2H) contains Fe2–H–Fe6. It is this HP suppression in E 4 (4H) that enables α-V70I MoFe to accumulate E 4 (4H) in high occupancy. In addition, HP suppression in α-V70I E 4 (4H) kinetically unmasks hydride reductive-elimination without N 2 -binding, a process that is precluded in WT enzyme. 

Understanding how Nature accomplishes the reduction of inert nitrogen gas to form metabolically tractable ammonia at ambient temperature and pressure has challenged scientists for more than a century. Such an understanding is a key aspect toward accomplishing the transfer of the genetic determinants of biological nitrogen fixation to crop plants as well as for the development of improved synthetic catalysts based on the biological mechanism. Over the past 30 years, the free-living nitrogen-fixing bacterium Azotobacter vinelandii emerged as a preferred model organism for mechanistic, structural, genetic, and physiological studies aimed at understanding biological nitrogen fixation. This review provides a contemporary overview of these studies and places them within the context of their historical development. 

Kang et al . (Reports, 19 June 2020, p. 1381) report a structure of the nitrogenase MoFe protein that is interpreted to indicate binding of N 2 or an N 2 -derived species to the active-site FeMo cofactor. Independent refinement of the structure and consideration of biochemical evidence do not support this claim. 

Nitrogen (N) is an essential but generally limiting nutrient for biological systems. Development of the Haber-Bosch industrial process for ammonia synthesis helped to relieve N limitation of agricultural production, fueling the Green Revolution and reducing hunger. However, the massive use of industrial N fertilizer has doubled the N moving through the global N cycle with dramatic environmental consequences that threaten planetary health. Thus, there is an urgent need to reduce losses of reactive N from agriculture, while ensuring sufficient N inputs for food security. Here we review current knowledge related to N use efficiency (NUE) in agriculture and identify research opportunities in the areas of agronomy, plant breeding, biological N fixation (BNF), soil N cycling, and modeling to achieve responsible, sustainable use of N in agriculture. Amongst these opportunities, improved agricultural practices that synchronize crop N demand with soil N availability are low-hanging fruit. Crop breeding that targets root and shoot physiological processes will likely increase N uptake and utilization of soil N, while breeding for BNF effectiveness in legumes will enhance overall system NUE. Likewise, engineering of novel N-fixing symbioses in non-legumes could reduce the need for chemical fertilizers in agroecosystems but is a much longer-term goal. The use of simulation modeling to conceptualize the complex, interwoven processes that affect agroecosystem NUE, along with multi-objective optimization, will also accelerate NUE gains.