Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism To Achieve N₂ Reduction

Abstract

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2 reduction to 2NH3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe7). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N2 binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E4(4H) (“Janus”) state. N2 binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H2, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N2/D2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H2 formation by hydride protonolysis (k HP) but significant differences in the rate constant for H2 re with N2 binding/reduction (k re ). We now report that V-nitrogenase also exhibits HD formation during N2/D2 turnover (and H2 inhibition of N2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N2 binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant (k HP) for H2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (k re ) for the re of H2 coupled to N2 binding/reduction, which decreases in the order Mo > V > Fe.

Carbon substrate re‐orders relative growth of a bacterium using Mo‐, V‐, or Fe‐nitrogenase for nitrogen fixation

“…Previous in vitro studies have shown that electron availability, which can be manipulated by varying the dinitrogenase reductase versus dinitrogenase protein ratio, can influence the efficiency of Nase, including the specific N 2 reduction rate and the H 2 :N 2 ratio (Eady, ; Harris et al ., ). For R .…”

“…Our direct measurements of N 2 reduction and H 2 production indicate that the V‐Nase H 2 :N 2 ratio can be as low as ~1.5, half of the canonical 3:1 ratio (Table ). In fact, recent evidence has confirmed that all three forms of Nase use an analogous catalytic mechanism with the same minimum 1 H 2 : 1 N 2 stoichiometry for N 2 binding to the active site (Lukoyanov et al ., , ; Harris et al ., ; Harris et al ., ). Many of the measurements that led to the canonical 3:1 ratio for V‐Nase were conducted at 30°C (Bishop et al ., ; Dilworth et al ., ) but there are hints that the H 2 :N 2 ratio may be temperature dependent and that it is lower at 19°C, the temperature where our growth experiments were performed (Table S2; Miller and Eady, ).…”

“…Diazotrophic growth on succinate and acetate are differentiated by electron availability Previous in vitro studies have shown that electron availability, which can be manipulated by varying the dinitrogenase reductase versus dinitrogenase protein ratio, can influence the efficiency of Nase, including the specific N 2 reduction rate and the H 2 :N 2 ratio (Eady, 1996;Harris et al, 2019). For R. palustris, the key determinants of whether it is limited by electron acceptors or donors are (i) the oxidation state of the substrate relative to the biomass (Table 1) Fig.…”

Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

“…Instead, it is possible that, lacking nifEN , an alternative and/or simplified pathway for FeMo‐ or similar cofactor synthesis may have acted as a transition state between such an Mo‐independent stage (i.e., binding a cluster resembling the NifB‐cofactor; Boyd & Peters, ; Mus et al, ) and the development of the canonical FeMo‐cofactor biosynthetic pathway. It is thus reasonable to speculate that this transition to Mo‐usage may be exhibited by AncC–D ancestors. Mo‐nitrogenases are far more efficient at reducing nitrogen than V‐ or Fe‐nitrogenases (Eady, ; Harris et al, ; Harris, Yang, et al, ), and the majority of all extant nitrogenases are Mo‐dependent across both anoxic and oxic environments (Boyd et al, ; Mus et al, ; Raymond et al, ). Even those organisms that have additional V‐ or Fe‐nitrogenases still retain and preferentially express Mo‐nitrogenases (Boyd, Anbar, et al, ; Boyd, Hamilton, et al, ; Dos Santos et al, ; Hamilton et al, ; Raymond et al, ).…”

Large Hydrogen Isotope Fractionation Distinguishes Nitrogenase-Derived Methane from Other Methane Sources

“…Molecular hydrogen (H2) is an obligatory product of nitrogen fixation and, in our experiments, is generated simultaneously with the production of methane from carbon dioxide (53,54). We explored whether the buildup and isotopic composition of H2 influence methane isotope fractionation by nitrogenase, as has been observed for mcr-based methanogenesis (2,43,48,(55)(56)(57)(58)(59)(60)(61)(62)(63)(64).…”