Comment on “Structural evidence for a dynamic metallocofactor during N2 reduction by Mo-nitrogenase”

Kang et al. (Reports, 19 June 2020, p. 1381) report a structure of the nitrogenase MoFe protein that is interpreted to indicate binding of N2 or an N2-derived species to the active-site FeMo cofactor. Independent refinement of the structure and consideration of biochemical evidence do not support this claim.

Assignment of protonated R-homocitrate in extracted FeMo-cofactor of nitrogenase via vibrational circular dichroism spectroscopy

Protonation of FeMo-cofactor (FeMo-co) is important for the process of substrate hydrogenation. Its structure has been clarified as Δ-Mo*Fe7S9C(R-homocit*)(cys)(Hhis) after the efforts of nearly 30 years, but it remains controversial whether FeMo-co is protonated or deprotonated with chelated ≡C − O(H) homocitrate. We have used protonated molybdenum(V) lactate 1 and its enantiomer as model compounds for R-homocitrate in FeMo-co of nitrogenase. Vibrational circular dichroism (VCD) spectrum of 1 at 1051 cm−1 is attributed to ≡C − OH vibration, and molybdenum(VI) R-lactate at 1086 cm−1 is assigned as ≡C − Oα-alkoxy vibration. These vibrations set up labels for the protonation state of coordinated α-hydroxycarboxylates. The characteristic VCD band of NMF-extracted FeMo-co is assigned to ν(C − OH), which is based on the comparison of molybdenum(VI) R-homocitrate. Density functional theory calculations are consistent with these assignments. To the best of our knowledge, this is the first time that protonated R-homocitrate in FeMo-co is confirmed by VCD spectra.

H2 Formation Holds the Key to Opening the Fe Coordination Sites of Nitrogenase FeMo-cofactor for Dinitrogen Activation

The present quantum-mechanical and molecular-mechanics study reveals the crucial roles of H₂ formation, of H₂S shift and of N₂ bond expansion in the nitrogenase process of the reduction of N₂ to <a href="https://en.wikipedia.org/wiki/Ammonia">NH₃</a>. Proton and electron transfers to the Fe(C@Fe₆S₉)Mo unit of the FeMo-co complex weaken the Fe-S and Fe-H bonds and expose the <b>Fe</b> coordination sites, coupled with energy release due to H₂ generation. Thereby the two sites <b>Fe2</b> and <b>Fe6</b> become prepared for stronger N₂ adsorption, expanding and attenuating the ǀN≡Nǀ bond. After subsequent detachment of H₂S from its Fe binding site into a holding site of the rearranged protein residue, the <b>Fe6</b> site becomes completely unfolded, and the N₂ triple bond becomes completely activated to an ‑<u>N</u>=<u>N</u>- double bond for easy subsequent hydrogenation to NH₃. We explain in particular, why the obligatory H₂ formation is an essential step in N₂ adsorption and activation

H2 Formation Holds the Key to Opening the Fe Coordination Sites of Nitrogenase FeMo-cofactor for Dinitrogen Activation

The present quantum-mechanical and molecular-mechanics study reveals the crucial roles of H₂ formation, of H₂S shift and of N₂ bond expansion in the nitrogenase process of the reduction of N₂ to <a href="https://en.wikipedia.org/wiki/Ammonia">NH₃</a>. Proton and electron transfers to the Fe(C@Fe₆S₉)Mo unit of the FeMo-co complex weaken the Fe-S and Fe-H bonds and expose the <b>Fe</b> coordination sites, coupled with energy release due to H₂ generation. Thereby the two sites <b>Fe2</b> and <b>Fe6</b> become prepared for stronger N₂ adsorption, expanding and attenuating the ǀN≡Nǀ bond. After subsequent detachment of H₂S from its Fe binding site into a holding site of the rearranged protein residue, the <b>Fe6</b> site becomes completely unfolded, and the N₂ triple bond becomes completely activated to an ‑<u>N</u>=<u>N</u>- double bond for easy subsequent hydrogenation to NH₃. We explain in particular, why the obligatory H₂ formation is an essential step in N₂ adsorption and activation

Reconstructing the Evolutionary History of Nitrogenases: Evidence for Ancestral Molybdenum-Cofactor Utilization

ABSTRACTThe nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is one of life’s key biogeochemical innovations. The three isozymes of nitrogenase differ in their metal dependence, each binding either a FeMo-, FeV-, or FeFe-cofactor where the reduction of dinitrogen takes place. The history of nitrogenase metal dependence has been of particular interest due to the possible implication that ancient marine metal availabilities have significantly constrained nitrogenase evolution over geologic time. Here, we reconstructed the evolutionary history of nitrogenases, and combined phylogenetic reconstruction, ancestral sequence inference, and structural homology modeling to evaluate the potential metal dependence of ancient nitrogenases. We find that active-site sequence features can reliably distinguish extant Mo-nitrogenases from V- and Fe-nitrogenases, and that inferred ancestral sequences at the deepest nodes of the phylogeny suggest these ancient proteins most resemble modern Mo-nitrogenases. Taxa representing early-branching nitrogenase lineages lack one or more biosynthetic nifE and nifN genes that both contribute to the assembly of the FeMo-cofactor in studied organisms, suggesting that early Mo-nitrogenases may have utilized an alternate and/or simplified pathway for cofactor biosynthesis. Our results underscore the profound impacts that protein-level innovations likely had on shaping global biogeochemical cycles throughout the Precambrian, in contrast to organism-level innovations that characterize the Phanerozoic Eon.