Minimal ATP-Independent N₂-Reducing Systems Defined by L-Cluster-Bound Nitrogenase Assembly Platforms

Area of Science:

• Biochemistry
• Bioinorganic Chemistry
• Evolutionary Biology

Background:

• Nitrogenase (NifH/NifDK) catalyzes N2 to NH3 conversion using the M-cluster metallocofactor.
• M-cluster assembly involves NifB-mediated L-cluster formation and NifEN maturation.
• The precise role of the L-cluster in nitrogenase assembly and function remains incompletely understood.

Purpose of the Study:

• To investigate the intrinsic nitrogen (N2) reducing activity of the L-cluster and its associated proteins.
• To explore the evolutionary implications of the L-cluster's reactivity for nitrogenase evolution.
• To determine the role of the L-cluster in linking biogeochemical cycles.

Main Methods:

• In vitro assays using L-cluster-bound NifB (NifBL) and NifEN (NifENL) with chemical reductants or photoexcited quantum dots.
• In vivo N2 fixation experiments in NifH-deficient E. coli strains.
• Bioinformatic analysis of L-cluster topology in ancient enzymes.

Main Results:

• L-cluster-bound NifB and NifEN exhibit intrinsic ATP-independent N2 reduction activity.
• N2 reduction capability is acquired by NifB only upon L-cluster formation.
• In vivo N2 fixation is supported by NifBL and NifENL in NifH-deficient strains, with YfhL as the electron donor.

Conclusions:

• The L-cluster possesses intrinsic N2-reducing activity, suggesting a simpler, one-component ancestral nitrogenase.
• The L-cluster may represent an evolutionary link between nitrogen, carbon, and sulfur biogeochemical cycles.
• This discovery reframes our understanding of nitrogen fixation evolution and the function of metalloenzymes.