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

Lee A Rettberg,Chi Chung Lee,Jarett Wilcoxen,Yilin Hu,Markus W Ribbe,R David Britt,Andrew J Jasniewski,Kazuki Tanifuji

doi:10.1038/s41467-020-15627-9

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.

Highlights

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly
Previous studies of the NifB proteins from Azotobacter vinelandii and Methanosarcina acetivorans have led to the proposal that NifB utilizes a unique, radical SAM-dependent mechanism for carbide insertion concomitant with the transformation of the K-cluster to an [Fe8S9C] cluster, which represents both an 8Fe precursor and an all-full complement of iron (Fe) core of the M-cluster[13,14,15,16]
This process begins with an SN2-type transfer of a methyl group from a SAM molecule to the K-cluster (Supplementary Fig. 1a, ➀), and it is followed by hydrogen abstraction of the methyl group by a 5′-deoxyadenosyl (5′-dA) radical that is derived from the homolytic cleavage of a second SAM molecule (Supplementary Fig. 1a, ➁)

Summary

Introduction

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. 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. Previous studies of the NifB proteins from Azotobacter vinelandii and Methanosarcina acetivorans have led to the proposal that NifB utilizes a unique, radical SAM-dependent mechanism for carbide insertion concomitant with the transformation of the K-cluster to an [Fe8S9C] cluster (designated the Lcluster), which represents both an 8Fe precursor and an all-Fe core of the M-cluster[13,14,15,16]. These observations point to a dual function of His[43] in positioning/orienting the K1-cluster relative to the K2-cluster for proper coupling and deprotonating the initial carbon radical for carbide formation

Methods

Results

Conclusion