Identity and function of an essential nitrogen ligand of the nitrogenase cofactor biosynthesis protein NifB

Abstract

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. Previously, a nitrogen ligand was shown to be involved in coupling a pair of [Fe₄S₄] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe₈S₉C] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive. Here, we use combined mutagenesis and pulse electron paramagnetic resonance analyses to establish histidine-43 of Methanosarcina acetivorans NifB (MaNifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of MaNifB to serve as a source for cofactor maturation upon substitution of histidine-43 with alanine; whereas x-ray absorption spectroscopy/extended x-ray fine structure experiments further suggest formation of an intermediate that lacks the cofactor core arrangement in this MaNifB variant. These results point to dual functions of histidine-43 in structurally assisting the proper coupling between K1 and K2 and concurrently facilitating carbide formation via deprotonation of the initial carbon radical.

NifB is a radical SAM enzyme involved in the biosynthesis of the Mo-nitrogenase cofactor, which is responsible for the ambient conversion of N₂ to NH₃. Here, the authors identify and uncover the function of a His43 residue as an essential nitrogen ligand of NifB in cofactor biosynthesis.