Minimal ATP‐Independent N ₂ ‐Reducing Systems Defined by L‐Cluster‐Bound Nitrogenase Assembly Platforms

Abstract

ABSTRACT The Mo‐nitrogenase, which consists of a reductase component (NifH) and a catalytic component (NifDK), catalyzes ATP‐dependent reduction of N 2 to NH 3 at its active‐site M‐cluster ([( R ‐homocitrate)MoFe 7 S 9 C]). A complex metallocofactor, the M‐cluster is assembled through NifB‐mediated formation of the intermediate L‐cluster ([Fe 8 S 9 C]), 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 N 2 ‐reducing activity. Such a function is strictly dependent on the L‐cluster, as NifB acquires N 2 ‐reducing capability only after conversion of the precursor K‐cluster (2x[Fe 4 S 4 ]) to an L‐cluster. Both L‐cluster‐bound NifB (NifB L ) and NifEN (NifEN L ) catalyze ATP‐independent N 2 reduction in vitro when supplied with a chemical reductant or photoexcited quantum dots. Moreover, these L‐cluster‐containing proteins support in vivo N 2 ‐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 N 2 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.