title: Optimization of FeMoco maturation on NifEN.
abstract: Mo-nitrogenase catalyzes the reduction of dinitrogen to ammonia at the cofactor (i.e., FeMoco) site of its MoFe protein component. Biosynthesis of FeMoco involves NifEN, a scaffold protein that hosts the maturation of a precursor to a mature FeMoco before it is delivered to the target location in the MoFe protein. Previously, we have shown that the NifEN-bound precursor could be converted in vitro to a fully complemented "FeMoco" in the presence of 2 mM dithionite. However, such a conversion was incomplete, and Mo was only loosely associated with the NifEN-bound "FeMoco". Here we report the optimized maturation of the NifEN-associated precursor in 20 mM dithionite. Activity analyses show that upon the optimal conversion of precursor to "FeMoco", NifEN is capable of activating a FeMoco-deficient form of MoFe protein to the same extent as the isolated FeMoco. Furthermore, EPR and XAS/EXAFS analyses reveal the presence of a tightly organized Mo site in NifEN-bound "FeMoco", which allows the observation of a FeMoco-like S = 3/2 EPR signal and the modeling of a NifEN-bound "FeMoco" that adopts a conformation very similar to that of the MoFe protein-associated FeMoco. The sensitivity of FeMoco maturation to dithionite concentration suggests an essential role of redox chemistry in this process, and the optimal potential of dithionite solution could serve as a guideline for future identification of in vivo electron donors for FeMoco maturation.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA.
authors.forename: Janice M
authors.initials: JM
authors.lastname: Yoshizawa
authors.forename: Michael A
authors.initials: MA
authors.lastname: Blank
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Fay
authors.forename: Chi Chung
authors.initials: CC
authors.lastname: Lee
authors.forename: Jared A
authors.initials: JA
authors.lastname: Wiig
authors.forename: Yilin
authors.initials: Y
authors.lastname: Hu
authors.forename: Keith O
authors.initials: KO
authors.lastname: Hodgson
authors.forename: Britt
authors.initials: B
authors.lastname: Hedman
authors.forename: Markus W
authors.initials: MW
authors.lastname: Ribbe
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004227:Dithionite / Q000737:chemistry; D005798:Genes, Bacterial; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D008983:Molybdoferredoxin; D046912:Multiprotein Complexes; D004227:Dithionite