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

Abstract

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.

Fig. 1.. Assessment of nitrogen-to-sulfur replacement using original structure factors.

(A) Stereo view of a 2F₀ − F_c map (blue mesh contoured at 1σ) and F₀ − F_c maps (positive green mesh and negative red mesh contoured at 3.5 σ) calculated around FeMo-co in chain A after refinement with S replacing the previously modeled N₂ molecules. The S atoms are labeled S3A and S2B to reflect the naming for the native structure of the Azotobacter vinelandii MoFe protein structure. FeMo-co is presented in ball and sticks. Coloring scheme: carbon, green and gray; oxygen, red; nitrogen, blue; iron, gray; sulfur, yellow; molybdenum, dark red. (B and C) Spherical integration of the anomalous maps showing the spread of the S positions in the FeMo cofactor in chain A (B) and in chain C (C). Files related to re-refinement are available as a Zenodo archive (6). To assess the anomalous signal at the S positions of the two FeMo cofactors, we inspected an anomalous difference electron density map “6UG0_Fig_2a_2d_Data_collected_at_7100eV”) deposited by the authors (9). The sulfur positions of the refined structure N2ase_Rib_refine_sulfur.pdb that contained μ₂-sulfides in all bridging positions were used, and electron density within the anomalous map was integrated over a sphere with r = 0.7 Å at these positions using the program MAPMAN (10). Re-refined coordinates for the MoFe nitrogenase structure were generated by refinement of 6UG0 structure with nitrogen atoms substituted for the sulfur atoms at the sulfur belt positions of FeMo-co using structure factors submitted with 6UG0. Refinement was performed using phenix.refine (11) (PHENIX-dev-3965 software suite) including reciprocal space, real-space, individual occupancy, and individual B-factor routines. Restraints published with the 6UG0 structure were used during the refinement: ICE and ICZ (FeMo-co), 1CL (P-cluster), HCA (homocitrate).