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Abstract
NifB is an essential radical S-adenosylmethionine (SAM) enzyme for nitrogenase cofactor assembly. Previous studies show that NifB couples a putative pair of [Fe4S4] modules (designated K1 and K2) into an [Fe8S9C] cofactor precursor concomitant with radical SAM-dependent carbide insertion through the action of its SAM-binding [Fe4S4] module. However, the coordination and function of the NifB cluster modules remain unknown. Here, we use continuous wave and pulse electron paramagnetic resonance spectroscopy to show that K1- and K2-modules are 3-cysteine-coordinated [Fe4S4] clusters, with a histidine-derived nitrogen serving as the fourth ligand to K1 that is lost upon K1/K2-coupling. Further, we demonstrate that coexistence of SAM/K2-modules is a prerequisite for methyltransfer to K2 and hydrogen abstraction from the K2-associated methyl by a 5′-deoxyadenosyl radical. These results establish an important framework for mechanistic explorations of NifB while highlighting the utility of a synthetic-cluster-based reconstitution approach employed herein in functional analyses of iron–sulfur (FeS) enzymes.
Highlights
NifB is an essential radical S-adenosylmethionine (SAM) enzyme for nitrogenase cofactor assembly
Characterization of the NifB proteins from Azotobacter vinelandii[11,12,13] and Methanosarcina acetivorans[14] has unveiled a radical SAM-dependent mechanism employed by NifB for carbide insertion, which begins with methyltransfer in an SN2type mechanism from SAM to a putative [Fe4S4] cluster pair (Supplementary Fig. 1b, i)
Continuous wave (CW)- and pulse electron paramagnetic resonance (EPR) analyses reveal that both K1- and K2modules are coordinated by 3 Cys ligands, with a His residue providing an additional nitrogen ligand to the K1 module that is lost upon coupling of the K1- and K2-modules into an 8Fe core; whereas biochemical experiments further demonstrate that coexistence of the SAM- and K2-modules is a prerequisite for methyltransfer to the K2-derived sulfide atom and the subsequent hydrogen abstraction from the K2-associated methyl group by a 5′-dA radical
Summary
Establishing the 4Fe nature of the NifB-associated clusters. A series of variants—each carrying one of the three proposed 4Fe modules—was generated on the template of the NifB protein from M. acetivorans (designated MaNifB). Upon in vitro reconstitution with this synthetic [Fe4S4] compound, each of the three MaNifB variants has an S = 1/2 electron paramagnetic resonance (EPR) signal that is characteristic of a [Fe4S4]+ cluster when the protein is reduced by dithionite (Fig. 1c; see Supplementary Table 1 for Fe contents, spin concentrations and protein concentrations of EPR samples) Consistent with their different origins, the simulated spectra have distinct g values from one another (MaNifBSAM: g = [2.017 1.924 1.910]; MaNifBK1: g = [2.050 1.905 1.900]; MaNifBK2: g = [2.044 1.933 1.886]; see Supplementary Fig. 4 and Supplementary Table 2 for simulation parameters), and the contributions of most EPR features of the individual modules can be identified in the spectrum of the wild-type MaNifB (Fig. 1c). While there are numerous EPR measured hyperfine and quadrupole couplings from nitrogenous ligands to Fe sites, a majority of them are from histidine ligated sites, including those found in the various Rieske-type [Fe2S2]
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