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

The Mo-nitrogenase, which consists of a reductase component (NifH) and a catalytic component (NifDK), catalyzes ATP-dependent reduction of N₂ to NH₃ at its active-site M-cluster ([(R-homocitrate)MoFe₇S₉C]). A complex metallocofactor, the M-cluster is assembled through NifB-mediated formation of the intermediate L-cluster ([Fe₈S₉C]), followed by L-to-M cluster maturation on NifEN. Here, we show that the L-cluster intrinsically endows the assembly proteins NifB and NifEN with N₂-reducing activity. Such a function is strictly dependent on the L-cluster, as NifB acquires N₂-reducing capability only after conversion of the precursor K-cluster (2x[Fe₄S₄]) to an L-cluster. Both L-cluster-bound NifB (NifB^L) and NifEN (NifEN^L) catalyze ATP-independent N₂ reduction in vitro when supplied with a chemical reductant or photoexcited quantum dots. Moreover, these L-cluster-containing proteins support in vivo N₂-fixation in NifH-deficient E. coli strains, where the low-potential ferredoxin YfhL serves as an essential physiological electron donor. The intrinsic reactivity of the L-cluster toward N₂ supports an evolutionary model in which primordial nitrogenase was a simpler, one-component, NifEN^L-like enzyme that preceded the modern, high-efficiency two-component system; whereas the shared L-cluster topology found in ancient nondiazotrophic enzymes like methyl-CoM reductase and methylthio-alkane reductase further implies that the L-cluster may represent an evolutionary link among nitrogen, carbon, and sulfur biogeochemical cycles.