Mechanism of N₂ Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H₂

Abstract

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N reduction relative to H evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe-H-Fe hydrides on the active site FeMo-cofactor to yield H is a key feature in the N reduction mechanism. The N reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N reduction. Inductively coupled plasma optical emission spectroscopy and mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N reduction (1 atm) of 181 ± 5 nmol NH min mg FeFe protein, for proton reduction (in the absence of N) of 1085 ± 41 nmol H min mg FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol CH min mg FeFe protein. Under turnover conditions, N reduction is inhibited by H and the enzyme catalyzes the formation of HD when presented with N and D. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N reduction occurring by reductive elimination of H.

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

“…Compared with the Mo-Nase, less is known about the cellular costs of alternative V-and Fe-Nase-based diazotrophy. All Nase isoforms produce H 2 as an obligatory byproduct of N 2 reduction (Simpson and Burris, 1984;Harris et al, 2018a;Harris et al, 2018b). Early in vitro data indicated that the alternative V-and Fe-Nases produce more H 2 than the Mo-Nase, resulting in greater energy and reducing power requirements (Eady, 1996).…”

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

“…Further, an ancient Mo‐independent nitrogenase may have been capable of—albeit inefficiently—reducing nitrogen by a cofactor resembling the Fe‐S‐C cluster assembled by NifB, which constitutes the biosynthetic precursor to the FeMo‐cofactor (Boyd & Peters, ; Mus et al, ; Soboh et al, ). Though our sequence analyses cannot fully assess ancestral nitrogenase dependence for a NifB‐cofactor, it is likely that the NifB‐cofactor resembles the structure and composition of the FeFe‐cofactor, excepting homocitrate (Corbett et al, ; Guo et al, ; Harris, Lukoyanov, et al, ). However, the greater similarity of AncC–D active sites to those of Mo‐nitrogenases than Fe‐nitrogenases likely suggests ancestral dependence on a cofactor incorporating Mo rather than only Fe, as would be the case for the NifB‐cofactor.…”

“…During catalysis, the electron delivery component transiently associates with and delivers electrons to the catalytic component (Hageman & Burris, ). Electrons accumulate at the active site for N 2 binding and reduction (Hoffman, Lukoyanov, Yang, Dean, & Seefeldt, ), which houses a homocitrate‐metallocluster cofactor unique to each nitrogenase form: the FeMo‐cofactor in Nif (Figure b), FeV‐cofactor in Vnf (Figure c), and FeFe‐cofactor in Anf (Figure d) (Eady, ; Harris, Lukoyanov, et al, ; Krahn et al, ; Mus et al, ; Sippel & Einsle, ; Spatzal et al, ). Available spectral evidence suggests that these cofactors are structurally similar, with the main difference being the substitution of a Mo, V, or additional Fe atom (FeV‐cofactor is also proposed to incorporate a carbonate ligand in place of one sulfur atom; Sippel & Einsle, ) (Eady, ; Krahn et al, ; Spatzal et al, ).…”

“…Available spectral evidence suggests that these cofactors are structurally similar, with the main difference being the substitution of a Mo, V, or additional Fe atom (FeV‐cofactor is also proposed to incorporate a carbonate ligand in place of one sulfur atom; Sippel & Einsle, ) (Eady, ; Krahn et al, ; Spatzal et al, ). Nevertheless, biochemical studies demonstrate variable catalytic properties among the three nitrogenase forms, including differential abilities to reduce alternative substrates (Harris, Lukoyanov, et al, ; Harris, Yang, Dean, Seefeldt, & Hoffman, ; Hu et al, ; Hu, Lee, & Ribbe, ; Zheng et al, ). These catalytic variations likely arise due to a combination of the aforementioned cofactor compositional differences and differences in the surrounding protein environment (Fixen et al, ; Harris, Yang, et al, ; Lee et al, ; Rebelein, Lee, Newcomb, Hu, & Ribbe, ; Zheng et al, ).…”

“…The active‐site FeMo‐cofactor of Mo‐nitrogenase and FeV‐cofactor of V‐nitrogenase are circled. (b–d) Catalytic genes and cofactor structures of A. vinelandii Mo‐nitrogenase (b; PDB 3U7Q; (Spatzal et al, ), V‐nitrogenase (c; PDB 5N6Y; Sippel & Einsle, ), and Fe‐nitrogenase (d; proposed structure; Harris, Lukoyanov, et al, ). Residue numbering from aligned A. vinelandii NifD.…”