Biological nitrogen fixation � the reduction of N2 to two NH3 molecules � is catalyzed by the enzyme nitrogenase. An understanding of the nitrogenase catalytic mechanism requires, at minimum, identification and characterization of each intermediate along the reaction pathway, plus an understanding of the kinetics/dynamics through which these intermediates form. The kinetics are described by the Lowe-Thorneley (LT) model, which provides rate constants for transformations among intermediates, denoted En, as indexed by the number of electrons (and protons), n = 0-8, accumulated within the MoFe protein. One of the most puzzling aspects of nitrogenase function embodied in the LT scheme is an obligatory generation of H2 upon N2 binding, so that the limiting stoichiometry for 'enzymological' nitrogen fixation requires eight electrons/protons to reduce each N2. As the delivery of each electron requires the hydrolysis of two MgATP, the formation of H2 thus apparently 'wastes' 25% of the total energy supplied by the hydrolysis of ATP. We have trapped and characterized three of the five N2 electron-reduction stages, the key 'Janus intermediate', E4, the state at which N2 hydrogenation begins, and intermediate states that contain semi-reduced N2. This allowed us to integrate kinetics and pathway, thereby generating a first-draft mechanism for biological nitrogen fixation. The mechanism was completed with the keystone proposal that H2 release upon N2 binding involves reductive elimination of two hydrides, to yield N2 bound to doubly-reduced FeMo-co highly activated to achieve the most difficult step of nitrogen fixation, the initial hydrogenation of N2. The result is the first mechanistic proposal for every transformation of nitrogen fixation based on the properties of trapped intermediates. This mechanism has been tested against the vast body of data on which it builds, and has passed a stringent experimental test it inspired.

On the mechanism of nitrogen fixation by nitrogenase

247th National Meeting (2014)

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