Structural evolution of nitrogenase states under alkaline turnover.

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Biological nitrogen fixation is crucial for global nitrogen cycling, supplying essential bioavailable nitrogen.
• The enzyme nitrogenase, specifically the MoFe-protein, catalyzes this process.
• Understanding the structural dynamics of nitrogenase intermediates is key to elucidating its mechanism.

Purpose of the Study:

• To resolve the ambiguous structural nature of nitrogenase intermediates.
• To visualize the structural changes of the MoFe-protein during the catalytic cycle.
• To investigate the role of the nitrogenase associated factor T protein.

Main Methods:

• Time-resolved cryo-electron microscopy (cryo-EM) was used to capture multiple structures.
• Alkaline reaction mixtures of the MoFe-protein were analyzed under an acetylene atmosphere.
• In vitro binding assays were performed to assess the interaction with factor T protein.

Main Results:

• Four high-resolution cryo-EM structures of the MoFe-protein were obtained, showing sequential changes.
• Observed changes include FeMo-cofactor inorganic framework perturbations, homocitrate depletion, and S2B sulfur density diminution.
• Asymmetric displacement of the FeMo-cofactor and side chain rearrangements were identified.
• Factor T protein was shown to bind inactivated MoFe-protein in vitro.

Conclusions:

• The time-resolved structures provide experimental evidence for FeMo-cofactor distortions and S2B displacement at specific intermediates (E0-E3).
• These findings support a model of cluster rearrangements preceding nitrogen binding in the substrate reduction mechanism.
• The results offer insights into structural dynamics relevant to both biological and synthetic nitrogen fixation systems.