Identity and function of an essential nitrogen ligand of the nitrogenase cofactor biosynthesis protein NifB
Lee A. Rettberg, Jarett Wilcoxen, Andrew J. Jasniewski, Chi Chung Lee, Kazuki Tanifuji, Yilin Hu, R. David Britt, Markus W. Ribbe
Abstract
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 [Fe 4 S 4 ] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe 8 S 9 C] 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 ( Ma NifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of Ma NifB 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 Ma NifB 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.