title: Assembly scaffold NifEN: A structural and functional homolog of the nitrogenase catalytic component.
abstract: NifEN is a biosynthetic scaffold for the cofactor of Mo-nitrogenase (designated the M-cluster). Previous studies have revealed the sequence and structural homology between NifEN and NifDK, the catalytic component of nitrogenase. However, direct proof for the functional homology between the two proteins has remained elusive. Here we show that, upon maturation of a cofactor precursor (designated the L-cluster) on NifEN, the cluster species extracted from NifEN is spectroscopically equivalent and functionally interchangeable with the native M-cluster extracted from NifDK. Both extracted clusters display nearly indistinguishable EPR features, X-ray absorption spectroscopy/extended X-ray absorption fine structure (XAS/EXAFS) spectra and reconstitution activities, firmly establishing the M-cluster-bound NifEN (designated NifEN(M)) as the only protein other than NifDK to house the unique nitrogenase cofactor. Iron chelation experiments demonstrate a relocation of the cluster from the surface to its binding site within NifEN(M) upon maturation, which parallels the insertion of M-cluster into an analogous binding site in NifDK, whereas metal analyses suggest an asymmetric conformation of NifEN(M) with an M-cluster in one αβ-half and an empty cluster-binding site in the other αβ-half, which led to the proposal of a stepwise assembly mechanism of the M-cluster in the two αβ-dimers of NifEN. Perhaps most importantly, NifEN(M) displays comparable ATP-independent substrate-reducing profiles to those of NifDK, which establishes the M-cluster-bound αβ-dimer of NifEN(M) as a structural and functional mimic of one catalytic αβ-half of NifDK while suggesting the potential of this protein as a useful tool for further investigations of the mechanistic details of nitrogenase.
authors: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900;
authors: Aaron W
authors: AW
authors: Fay
authors: Department of Chemistry, Stanford University, Stanford, CA 94305;
authors: Michael A
authors: MA
authors: Blank
authors: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900;
authors: Johannes G
authors: JG
authors: Rebelein
authors: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900;
authors: Chi Chung
authors: CC
authors: Lee
authors: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900; Department of Chemistry, University of California, Irvine, CA 92697-2025; hodgson@slac.stanford.edu mribbe@uci.edu hedman@slac.stanford.edu yilinh@uci.edu.
authors: Markus W
authors: MW
authors: Ribbe
authors: Stanford Synchrotron Radiation Lightsource, Stanford Linear Accelerator Center, Stanford University, Menlo Park, CA 94025 hodgson@slac.stanford.edu mribbe@uci.edu hedman@slac.stanford.edu yilinh@uci.edu.
authors: Britt
authors: B
authors: Hedman
authors: Department of Chemistry, Stanford University, Stanford, CA 94305; hodgson@slac.stanford.edu mribbe@uci.edu hedman@slac.stanford.edu yilinh@uci.edu.
authors: Keith O
authors: KO
authors: Hodgson
authors: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900; hodgson@slac.stanford.edu mribbe@uci.edu hedman@slac.stanford.edu yilinh@uci.edu.
authors: Yilin
authors: Y
authors: Hu
mesh_terms: D016948:Azotobacter vinelandii / Q000737:chemistry* / Q000201:enzymology; D020134:Catalytic Domain; D003067:Coenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007502:Iron Chelating Agents / Q000737:chemistry; D008982:Molybdenum / Q000737:chemistry* / Q000378:metabolism; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009591:Nitrogenase / Q000737:chemistry* / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003067:Coenzymes; D007502:Iron Chelating Agents; D008983:Molybdoferredoxin; D021122:Protein Subunits; D008982:Molybdenum; D007501:Iron; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase
keywords: NifEN; assembly; catalysis; functional homolog; nitrogenase