On reversible H₂ loss upon N₂ binding to FeMo-cofactor of nitrogenase

Nitrogenase is activated for N ₂ reduction by the accumulation of four electrons/protons on its active site FeMo-cofactor, yielding a state, designated as E ₄, which contains two iron-bridging hydrides [Fe–H–Fe]. A central puzzle of nitrogenase function is an apparently obligatory formation of one H ₂ per N ₂ reduced, which would “waste” two reducing equivalents and four ATP. We recently presented a draft mechanism for nitrogenase that provides an explanation for obligatory H ₂ production. In this model, H ₂ is produced by reductive elimination of the two bridging hydrides of E ₄ during N ₂ binding. This process releases H ₂, yielding N ₂ bound to FeMo-cofactor that is doubly reduced relative to the resting redox level, and thereby is activated to promptly generate bound diazene (HN=NH). This mechanism predicts that during turnover under D ₂/N ₂, the reverse reaction of D ₂ with the N ₂-bound product of reductive elimination would generate dideutero-E ₄ [E ₄(2D)], which can relax with loss of HD to the state designated E ₂, with a single deuteride bridge [E ₂(D)]. Neither of these deuterated intermediate states could otherwise form in H ₂O buffer. The predicted E ₂(D) and E ₄(2D) states are here established by intercepting them with the nonphysiological substrate acetylene (C ₂H ₂) to generate deuterated ethylenes (C ₂H ₃D and C ₂H ₂D ₂). The demonstration that gaseous H ₂/D ₂ can reduce a substrate other than H ⁺ with N ₂ as a cocatalyst confirms the essential mechanistic role for H ₂ formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, and provides direct experimental support for the reductive elimination mechanism.