title: Molecular insights into nitrogenase FeMoco insertion: TRP-444 of MoFe protein alpha-subunit locks FeMoco in its binding site.
abstract: Biosynthesis of the FeMo cofactor (FeMoco) of nitrogenase MoFe protein is arguably one of the most complex processes in metalloprotein biochemistry. Here we investigate the role of a MoFe protein residue (Trp-alpha444) in the final step of FeMoco assembly, which involves the insertion of FeMoco into its binding site. Mutations of this aromatic residue to small uncharged ones result in significantly decreased levels of FeMoco insertion/retention and drastically reduced activities of MoFe proteins, suggesting that Trp-alpha444 may lock the FeMoco tightly in its binding site through the sterically restricting effect of its bulky, aromatic side chain. Additionally, these mutations cause partial conversion of the P-cluster to a more open conformation, indicating a potential connection between FeMoco insertion and P-cluster assembly. Our results provide some of the initial molecular insights into the FeMoco insertion process and, moreover, have useful implications for the overall scheme of nitrogenase assembly.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, USA.
authors.forename: Yilin
authors.initials: Y
authors.lastname: Hu
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Fay
authors.forename: Benedikt
authors.initials: B
authors.lastname: Schmid
authors.forename: Beshoie
authors.initials: B
authors.lastname: Makar
authors.forename: Markus W
authors.initials: MW
authors.lastname: Ribbe
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000378:metabolism; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry*; D008958:Models, Molecular; D008982:Molybdenum / Q000737:chemistry*; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D009591:Nitrogenase / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D008983:Molybdoferredoxin; D008982:Molybdenum; D014364:Tryptophan; D007501:Iron; D009591:Nitrogenase