Nitrogenase: A draft mechanism

Biological nitrogen fixation, the reduction of N₂ to two NH ₃ molecules, supports more than half the human population. The predominant form of the enzyme nitrogenase, which catalyzes this reaction, comprises an electron-delivery Fe protein and a catalytic MoFe protein. Although nitrogenase has been studied extensively, the catalytic mechanism has remained unknown. At a minimum, a mechanism must identify and characterize each intermediate formed during catalysis and embed these intermediates within a kinetic framework that explains their dynamic interconversion. The Lowe-Thorneley (LT) model describes nitrogenase kinetics and provides rate constants for transformations among intermediates (denoted E_n, where n is the number of electrons (and protons), that have accumulated within the MoFe protein). Until recently, however, research on purified nitrogenase had not characterized any E_n state beyond E₀.In this Account, we summarize the recent characterization of three freeze-trapped intermediate states formed during nitrogenase catalysis and place them within the LT kinetic scheme. First we discuss the key E₄ state, which is primed for N ₂ binding and reduction and which we refer to as the "Janus intermediate" because it lies halfway through the reaction cycle. This state has accumulated four reducing equivalents stored as two [Fe-H-Fe] bridging hydrides bound to the active-site iron-molybdenum cofactor ([7Fe-9S-Mo-C- homocitrate]; FeMo-co) at its resting oxidation level. The other two trapped intermediates contain reduced forms of N₂. One, intermediate, designated I, has S = 1/2 FeMo-co. Electron nuclear double resonance/hyperfine sublevel correlation (ENDOR/HYSCORE) measurements indicate that I is the final catalytic state, E₈, with NH₃ product bound to FeMo-co at its resting redox level. The other characterized intermediate, designated H, has integer-spin FeMo-co (non-Kramers; S ≥ 2). Electron spin echo envelope modulation (ESEEM) measurements indicate that H contains the [-NH₂] fragment bound to FeMo-co and therefore corresponds to E₇.These assignments in the context of previous studies imply a pathway in which (i) N₂ binds at E₄ with liberation of H₂, (ii) N₂ is promptly reduced to N₂H₂, (iii) the two N's are reduced in two steps to form hydrazine-bound FeMo-co, and (iv) two NH₃ are liberated in two further steps of reduction. This proposal identifies nitrogenase as following a "prompt-alternating (P-A)" reaction pathway and unifies the catalytic pathway with the LT kinetic framework. However, the proposal does not incorporate one of the most puzzling aspects of nitrogenase catalysis: obligatory generation of H₂ upon N₂ binding that apparently "wastes" two reducing equivalents and thus 25% of the total energy supplied by the hydrolysis of ATP. Because E₄ stores its four accumulated reducing equivalents as two bridging hydrides, we propose an answer to this puzzle based on the organometallic chemistry of hydrides and dihydrogen. We propose that H ₂ release upon N₂ binding involves reductive elimination of two hydrides to yield N₂ bound to doubly reduced FeMo-co. Delivery of the two available electrons and two activating protons yields cofactor-bound diazene, in agreement with the P-A scheme. This keystone completes a draft mechanism for nitrogenase that both organizes the vast body of data on which it is founded and serves as a basis for future experiments.