On reversible H ₂ loss upon N ₂ binding to FeMo-cofactor of nitrogenase

Abstract

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 FeMocofactor 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 to two ammonia (NH 3 ) molecules-is primarily catalyzed by the Mo-dependent nitrogenase. This enzyme comprises an electron-delivery Fe protein and MoFe protein, which contains the active site FeMo-cofactor ( Fig. 1A) (1, 2). The nitrogenase catalyzed reaction is generally thought to have the limiting stoichiometry shown in Eq. 1 (3),This equation conveys one of the most puzzling aspects of nitrogenase function, for it incorporates an obligatory formation of one mole of H 2 per mole of N 2 reduced, which requires the waste of two reducing equivalents and four ATP (1, 2). A kinetic framework for nitrogenase function that incorporates the stoichiometry of Eq. 1 is provided by the Lowe-Thorneley model (1, 2, 4), which describes transformations among catalytic intermediates (denoted E n ), where n is the number of electrons and protons (n = 0-8) delivered to MoFe protein. N 2 reduction requires activation of the MoFe protein to the E 4 state in which FeMo-cofactor has accumulated four electrons and four protons stored as two hydrides that bridge Fe atoms [Fe-H-Fe] and two protons presumably bound to sulfides of FeMo-cofactor (Fig. 1B) (5-8). The binding of N 2 to the E 4 state is coupled to the stoichiometric evolution of one H 2 per N 2 reduced.We recently presented a draft mechanism for N 2 reduction by nitrogenase that incorporates a mechanistic explanation for obligatory, reversible H 2 loss upon N 2 binding (5). We considered two models for this process, both built on our characterization of the key E 4 state, and tested them against the numerous constraints imposed by turnover under N 2 plus D 2 or T 2 (5, 9-16). In particular, these constraints include the key findings that during catalytic reduction of N 2 (see Scheme S1), a molecule of D 2 or T 2 will reduce two protons to form two HD or HT without D + /T + exchange with solvent, even though neither D 2 nor T 2 by themselves reacts with nitrogenase during turnover under Ar (5). One model, involving protonation of one of the hydrides to form H 2 , and its replacement by N 2 , was shown to violate ...

Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia

“…It is also possible that a decrease in commitment to catalysis results from competition at the active between N 2 and H 2 (12,13), as the latter is produced more abundantly by alternative nitrogenases (8). Additional studies on the intrinsic isotope effects of the nitrogenases and their expression at the levels of the enzyme and organism may help improve our understanding of the mechanism of N 2 reduction.…”

Mechanism of Nitrogen Fixation by Nitrogenase: The Next Stage

“…157 For example, reduction of acetylene and N 2 are mutually exclusive, with complicated inhibition kinetics between these two substrates. 217,234 Therefore, it was of interest to determine the effect of varying the N 2 partial pressure on the formation of C 2 H 3 D and C 2 H 2 D 2 at fixed C 2 H 2 and D 2 pressures.…”

Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme

“…6,7 According to the mechanism, during turnover under D 2 /N 2 , reaction of the E 4 (2N2H) intermediate with D 2 generates dideutero-E 4 with two [Fe-D-Fe] bridging deuterides which do not exchange with solvent. 14 This E 4 (4H) isotopologue, which we denote E 4 (2D − ;2H + ) ( Chart 2 ), would relax through E 2 (D − ;H + ) to E 0 with successive loss of two HD 7,10 .…”