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Femo Cofactor Maturation on Nifen SPECIAL FEATURE FeMo cofactor maturation on NifEN SPECIAL FEATURE Yilin Hu*, Mary C. Corbett†, Aaron W. Fay*, Jerome A. Webber*, Keith O. Hodgson†‡§, Britt Hedman‡§, and Markus W. Ribbe*§ *Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900; †Department of Chemistry, Stanford University, Stanford, CA 94305; and ‡Stanford Synchrotron Radiation Laboratory, Stanford Linear Accellerator Center, Stanford University, 2575 Sand Hill Road, MS 69, Menlo Park, CA 94025-7015 Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved May 10, 2006 (received for review March 31, 2006) FeMo cofactor (FeMoco) biosynthesis is one of the most compli- Unlike the P cluster, FeMoco, which contains additional hetero- cated processes in metalloprotein biochemistry. Here we show that metal (Mo) and organic moiety (homocitrate), is first assembled on Mo and homocitrate are incorporated into the Fe͞S core of the a scaffold protein (17, 18) and then inserted into its destined FeMoco precursor while it is bound to NifEN and that the resulting location in MoFe protein (‘‘ex situ’’ assembly). Biosynthesis of fully complemented, FeMoco-like cluster is transformed into a FeMoco presumably starts with the production of the Fe͞Scoreby mature FeMoco upon transfer from NifEN to MoFe protein through NifB (encoded by nifB) (19, 20), which is then transferred to, and direct protein–protein interaction. Our findings not only clarify the further processed on, the ␣2␤2 tetrameric NifEN protein (17, 21). process of FeMoco maturation, but also provide useful insights into Sequence similarity between the respective subunit-encoding genes the other facets of nitrogenase chemistry. led to the proposal that NifE and NifN form a structurally homol- ogous complex to the MoFe protein (22, 23) and that, by analogy, biosynthesis ͉ nitrogenase NifEN also contains two types of metal cluster sites, one corre- sponding to the P-cluster site and the other to the FeMoco site of itrogenase is the key player in nature’s ingenious scheme to the MoFe protein (17, 21). The P-cluster analog was identified as Nconvert the inert atmospheric dinitrogen to the bioavailable a [4Fe-4S] cluster likely coordinated by conserved Cys residues at form of ammonia (for recent reviews, see refs. 1–8). The the NifE–NifN interface (21). The FeMoco analog had not been Mo-nitrogenase of Azotobacter vinelandii is composed of the iron captured on NifEN until an efficient one-step purification proce- (Fe) protein and the molybdenum–iron (MoFe) protein. The dure, which minimized the degradation of proteins and the conse- homodimeric Fe protein has one nucleotide-binding site per quent loss of metal clusters, was applied to a His-tagged form of the subunit and a single [4Fe-4S] cluster bridged between the two NifEN protein (17). In a so-called FeMoco maturation assay ␣ ␤ subunits. The 2 2-tetrameric MoFe protein contains two comprising NifEN, molybdate, homocitrate, Fe protein, MgATP unique metal clusters per ␣␤-subunit: the [8Fe-7S] P-cluster (9), and ⌬nifB MoFe protein, the FeMoco analog on NifEN was proven ␣␤ which is located at the -interface and ligated to six protein to be a FeMoco precursor by its ability to activate FeMoco-deficient residues; and the [Mo-7Fe-9S-X-homocitrate] (the identity of X ⌬nifB MoFe protein. Extended x-ray absorption fine structure is unknown but is considered to be C, O, or N; ref. 10) FeMo (EXAFS) analysis of the NifEN-bound precursor showed that it was BIOCHEMISTRY ␣ cofactor (FeMoco), which is situated within the -subunit and structurally similar to FeMoco except for the notable absence of Mo bound to only two protein residues and an exogenous homoci- (18). Therefore, FeMoco cannot be formed through condensation trate ligand. Both P-cluster and FeMoco are composed of of [Mo-3Fe-3S] and [4Fe-3S] partial cubanes; rather, the Fe͞S smaller substructures: the P-cluster comprises two [4Fe-4S] ␮ structure of FeMoco is formed first, possibly through condensation subclusters that share a 6-sulfide (9) and FeMoco consists of of smaller Fe͞S subclusters in a fashion similar to that proposed for [Mo-3Fe-3S] and [4Fe-3S] subcubanes that are bridged by three ␮ ␮ P-cluster assembly and, then, Mo and homocitrate are added to 2-sulfides and share a central 6-light atom (10). These metal complete the synthesis. Although instrumental in clarifying the clusters are essential for nitrogenase reaction, a process that trajectory of FeMoco biosynthesis, our original study left such involves ATP-dependent electron transfer from the [4Fe-4S] unanswered questions as: when Mo and homocitrate are inserted cluster of the Fe protein to the P-cluster of the MoFe protein and into the cluster; how FeMoco is transferred from NifEN to the finally to FeMoco where substrate reduction takes place, and MoFe protein; and what role the Fe protein and MgATP play in consequently become the major subjects in the vigorous studies FeMoco maturation. of nitrogenase catalysis (1–8). Meanwhile, there is an emerging The current study addresses these remaining questions by understanding of nitrogenase biosynthesis, in particular, P- following the final steps of FeMoco assembly in A. vinelandii cluster and FeMoco assembly in A. vinelandii, that is poised to ͞ further clarify the structure and function of these important using a similar biochemical spectroscopic strategy to that used previously (17, 18). Through this approach, we show that Mo and clusters while also serving as a paradigm for the field of complex ͞ metalloprotein biosynthesis (11, 12). homocitrate are incorporated into the Fe S core of the FeMoco The P cluster is a classical example of high-nuclearity clusters precursor while it is bound to NifEN and that the fully comple- containing only Fe and S, and it is likely assembled at its targeted mented cluster is subsequently transferred from NifEN to MoFe location (‘‘in situ’’ assembly). Using a biochemical͞spectroscopic protein through direct protein–protein interaction. The function approach, we identified the presence of two pairs of [4Fe-4S]-like clusters that likely represent P-cluster precursors in a FeMoco- Author contributions: Y.H., K.O.H., B.H., and M.W.R. designed research; Y.H., M.C.C., deficient MoFe protein purified from a nifH-deletion strain (13, A.W.F., J.A.W., and M.W.R. performed research; Y.H., M.C.C., K.O.H., B.H., and M.W.R. 14). This protein becomes catalytically active upon incubation analyzed data; and Y.H., M.C.C., K.O.H., B.H., and M.W.R. wrote the paper. with the deleted gene product, indicating that it might represent The authors declare no conflict of interest. a physiologically relevant intermediate during P-cluster assembly This article is a PNAS direct submission. (15). These results suggest that the P-cluster is formed through Abbreviations: FeMoco, FeMo cofactor; EXAFS, extended x-ray absorption fine structure; the fusion of its substructural units, a reaction mechanism that IDS, indigo disulfonate. is well known in synthetic inorganic chemistry (16) and partic- §To whom correspondence may be addressed. E-mail: [email protected], hogdson@ssrl. ularly appropriate considering the ‘‘modular’’ composition of the slac.stanford.edu, or [email protected]. P cluster. © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602647103 PNAS ͉ November 14, 2006 ͉ vol. 103 ͉ no. 46 ͉ 17119–17124 Downloaded by guest on September 30, 2021 Table 1. Metal contents of NifEN؅ Metal Protein Mo Fe NifEN Ͻ0.01 16.1 Ϯ 1.1 NifENcomplete 1.2 Ϯ 0.1 15.8 Ϯ 0.6 ⌬nifB NifEN Ͻ0.01 8.2 Ϯ 1.0 ⌬nifB NifENcomplete Ͻ0.01 7.7 Ϯ 0.1 NifENminus Mo͞homocitrate Ͻ0.01 14.9 Ϯ 0.1 NifENminus homocitrate 0.3 Ϯ 0.1 15.2 Ϯ 0.1 Fig. 1. Protein purification and visible region absorption spectroscopy. (A) NifENminus Mo Ͻ0.01 15.7 Ϯ 0.2 Coomassie blue-stained 10–20% gradient SDS͞PAGE of NifEN, ⌬nifB NifEN, NifENminus MgATP Ͻ0.01 15.6 Ϯ 0.2 complete ⌬ complete ␮ ␮ NifEN and nifB NifEN . Lane 1, 10 g protein standard; lane 2, 15 g NifENminus Fe protein Ͻ0.01 15.3 Ϯ 0.1 ␮ ⌬ ␮ purified NifEN; lane 3, 15 g of purified nifB NifEN; lane 4, 15 g of purified NifENapo Fe protein Ͻ0.01 14.8 Ϯ 0.2 NifENcomplete; lane 5, 15 ␮g of purified ⌬nifB NifENcomplete.(B) Visible region NifENA157S Fe protein 0.2 Ϯ 0.1 15.4 Ϯ 0.1 spectra of the same protein samples in A. Spectra of dithionite-reduced NifEN (1), M156C Fe protein Ϯ Ϯ ⌬nifB NifEN (2), NifENcomplete (3), and ⌬nifB NifENcomplete (4) are shown between NifEN 0.2 0.1 14.6 0.4 A157G Fe protein Ͻ Ϯ 350 and 550 nm. The samples were prepared at a concentration of 5 mg͞ml, as NifEN 0.01 16.1 1.0 described in Supporting Text, which is published as supporting information on NifENMgADP Ͻ0.01 16.4 Ϯ 1.1 the PNAS web site. NifENATP␥S Ͻ0.01 15.3 Ϯ 0.1 NifENAMPPNP Ͻ0.01 16.5 Ϯ 1.0 of Fe protein and MgATP in FeMoco maturation is described in Data are expressed as moles of metal per mole of protein. a companion paper (24). Results the formation of a FeMoco precursor and, accordingly, both ⌬nifB NifEN and ⌬nifB NifENcomplete are shown to have suffi- The FeMoco maturation assay, which was designed to test the cient Fe to form the permanent clusters alone: Ϸ8 mol Fe and capability of NifEN in FeMoco biosynthesis, comprises (i) no Mo per mol of protein (Table 1). Consistent with the metal NifEN, the source of FeMoco precursor; (ii) molybdate and analysis results, the visible region absorption spectra of these homocitrate, the constituents of FeMoco absent from the proteins reveal that the NifENcomplete spectrum (Fig.
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