Van Stappen, CaseyJimenez-Vicente, EmilioPerez-Gonzalez, AnaYang, Zhi-YongSeefeldt, Lance C.DeBeer, SerenaDean, Dennis R.Decamps, Laure2022-07-212022-07-212022-03-242041-6520http://hdl.handle.net/10919/111311Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD(2)K(2) polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD(2)K(2). P-cluster maturation involves the iron protein NifH(2) as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD(2)K(2) species bearing immature P-clusters were isolated from an Azotobacter vinelandii strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD(2)K(2) complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S](2+/+) clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.application/pdfenCreative Commons Attribution-NonCommercial 4.0 Internationalx-ray-absorptionelectron-paramagnetic-resiron-sulfur proteinsapo-mofe proteinescherichia-colisynthetic analogsgenetic-analysisfe proteineprspectroscopyArticle - Refereedhttps://doi.org/10.1039/d1sc06418e1312354328782041-6539