The Nitrogenase FeMo‐Cofactor Precursor Formed by NifB Protein: A Diamagnetic Cluster Containing Eight Iron Atoms

Abstract

The biological activation of N2 occurs at the FeMo-cofactor, a 7Fe-9S-Mo-C-homocitrate cluster. FeMo-cofactor formation involves assembly of a Fe6-8 -SX -C core precursor, NifB-co, which occurs on the NifB protein. Characterization of NifB-co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent [4Fe-4S] cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB-co and deliver it to the NifEN protein, upon which FeMo-cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB-co-loaded form provided an opportunity for Mössbauer analysis of NifB-co. The results indicate that NifB-co is a diamagnetic (S=0) 8-Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the (57) Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB-co is either a 4Fe(2+) -4Fe(3+) or 6Fe(2+) -2Fe(3+) cluster having valence-delocalized states.

Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB

“…Expression of functional NifB is absolutely required to engineer nitrogenase in eukaryotic organisms (e.g., plants). NifB catalyzes the formation of NifB-co, a unique [Fe–S] cluster intermediate in the biosynthesis of FeMo-co of nitrogenase ( Shah et al, 1994 ; Curatti et al, 2006 ; Wiig et al, 2012 ; Guo et al, 2016 ). All diazotrophs carry at least one nifB gene ( Dos Santos et al, 2012 ), and it is not likely that NifB-co can be produced by any other enzyme of plant origin ( Vicente and Dean, 2017 ).…”

“…A main hurdle to overcome in order to generate functional nitrogenase proteins is obtaining active NifB. NifB is an extremely O 2 -sensitive S -adenosyl methionine (SAM)–radical enzyme ( Curatti et al, 2006 ), that provides the key intermediate metal cluster (called NifB-co) in the biosynthesis of FeMo-co ( Shah et al, 1994 ; Guo et al, 2016 ). As NifB-co also serves as precursor for FeV-co in the vanadium nitrogenase and for FeFe-co in the iron-only nitrogenase, NifB is required for all biological nitrogen fixation activity in nature ( Bishop and Joerger, 1990 ; Curatti et al, 2007 ; Dos Santos et al, 2012 ).…”

Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB

“…NifB is an S -adenosyl methionine (SAM)-radical protein that converts [4Fe-4S] clusters into NifB-co, an [8Fe-9S-C] cluster that serves as precursor to FeMo-co, FeV-co and FeFe-co, thus catalyzing the first committed step in nitrogenase active-site cofactor biosynthesis ( Shah et al, 1994 ; Allen et al, 1995 ; Curatti et al, 2006 ; George et al, 2008 ; Wiig et al, 2012 ) (Supplementary Figure 1 ). In contrast to FeMo-co, NifB-co is a diamagnetic cluster containing two spectroscopically distinct Fe sites ( Guo et al, 2016 ).…”

Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae

“…The complete process of FeMo-co biosynthesis can be performed in vitro (5). A key enzyme for this process is NifB (6), which generates an [8Fe-9S-C] cluster called NifB-co (7, 8), which functions as obligate precursor to FeMo-co and also to the active-site cofactors of the other nitrogenase types (5) (Fig. 1 A ).…”

“…Current data indicate that NifB harbors a catalytic [4Fe-4S] S -adenosylmethionine (SAM)-coordinated cluster and 2 additional [4Fe-4S] accessory clusters. NifB uses radical SAM chemistry to initiate NifB-co formation, which is accomplished by fusing its accessory clusters and inserting one S and one C atom (6, 8, 10–12) (Fig. 1 B ).…”