title: Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.
abstract: Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.
authors.affiliation: Howard Hughes Medical Institute, California Institute of Technology, Pasadena, United States.|Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Spatzal
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Perez
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States.|Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, United States.
authors.forename: James B
authors.initials: JB
authors.lastname: Howard
authors.affiliation: Howard Hughes Medical Institute, California Institute of Technology, Pasadena, United States.|Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States.
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Rees
mesh_terms: D016948:Azotobacter vinelandii / Q000737:chemistry / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003485:Cyanates / Q000378:metabolism; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000737:chemistry / Q000378:metabolism*; D009591:Nitrogenase / Q000378:metabolism*; D011487:Protein Conformation; D012643:Selenium / Q000378:metabolism*; D018036:Selenium Compounds / Q000378:metabolism
chemical_list: D003485:Cyanates; D008983:Molybdoferredoxin; D018036:Selenium Compounds; C053721:selenocyanic acid; D009591:Nitrogenase; D012643:Selenium
keywords: Azotobacter vinelandii; biochemistry; biophysics; iron sulfur proteins; metalloproteins; nitrogen fixation; structural biology