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

Significance Biological reduction of dinitrogen (N2) to two ammonia (NH3) is catalyzed by the metalloenzyme, nitrogenase. A central aspect of nitrogenase function is an apparently obligatory formation of one H2 per N2 reduced, with a resulting “waste” of the energy required to deliver two electrons/protons, obtained from hydrolysis of four ATP. We here report experiments that confirm the essential mechanistic role for H2 formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, not six as in the equation N2 + 3H2 → 2NH3. These experiments were devised to test our recently proposed “reductive elimination” mechanism for H2 formation and the activation of nitrogenase for ammonia production. Our findings provide direct experimental support for that mechanism. Nitrogenase is activated for N2 reduction by the accumulation of four electrons/protons on its active site FeMo-cofactor, yielding a state, designated as E4, which contains two iron-bridging hydrides [Fe–H–Fe]. A central puzzle of nitrogenase function is an apparently obligatory formation of one H2 per N2 reduced, which would “waste” two reducing equivalents and four ATP. We recently presented a draft mechanism for nitrogenase that provides an explanation for obligatory H2 production. In this model, H2 is produced by reductive elimination of the two bridging hydrides of E4 during N2 binding. This process releases H2, yielding N2 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 D2/N2, the reverse reaction of D2 with the N2-bound product of reductive elimination would generate dideutero-E4 [E4(2D)], which can relax with loss of HD to the state designated E2, with a single deuteride bridge [E2(D)]. Neither of these deuterated intermediate states could otherwise form in H2O buffer. The predicted E2(D) and E4(2D) states are here established by intercepting them with the nonphysiological substrate acetylene (C2H2) to generate deuterated ethylenes (C2H3D and C2H2D2). The demonstration that gaseous H2/D2 can reduce a substrate other than H+ with N2 as a cocatalyst confirms the essential mechanistic role for H2 formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, and provides direct experimental support for the reductive elimination mechanism.

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