title: Localization of a substrate binding site on the FeMo-cofactor in nitrogenase: trapping propargyl alcohol with an alpha-70-substituted MoFe protein.
abstract: Substitution of the MoFe protein alpha-70(Val) residue with Ala or Gly expands the substrate range of nitrogenase, allowing the reduction of larger alkynes, including propargyl alcohol (HC[triple bond]CCH(2)OH). Herein, we report characterization of the alpha-70(Val)(-->)(Ala) MoFe protein with propargyl alcohol trapped at the active site. The alpha-70(Ala) variant MoFe protein was rapidly frozen during reduction of propargyl alcohol, resulting in the conversion of the resting-state FeMo-cofactor EPR signal (S = 3/2 and g = [4.41, 3.60, 2.00]) to a new state (S = 1/2 and g = [2.123, 1.998, 1.986]). This EPR signal of the new state increased in intensity with increasing propargyl alcohol concentration, consistent with the binding of a single substrate. The EPR signal of the propargyl alcohol state showed temperature and microwave power dependencies markedly different from those of the classic FeMo-cofactor EPR signal, consistent with the difference in spin. The new state is analogous to that induced by the binding of the inhibitor CO ("lo CO" state) to FeMo-cofactor in the wild-type MoFe protein. The (13)C ENDOR spectrum of the alpha-70(Ala) MoFe protein with trapped (13)C-labeled propargyl alcohol exhibited three well-resolved (13)C doublets centered at the (13)C Larmor frequency with isotropic hyperfine couplings of approximately 3.2, approximately 1.4, and approximately 0.7 MHz, indicating that the alcohol (or a fragment) is coordinated to the cofactor. The results presented here localize the binding site of propargyl alcohol to one [4Fe-4S] face of FeMo-cofactor and indicate roles for the alpha-70(Val) residue in controlling FeMo-cofactor reactivity.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
authors.forename: Paul M C
authors.initials: PM
authors.lastname: Benton
authors.forename: Mikhail
authors.initials: M
authors.lastname: Laryukhin
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Mayer
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Dean
authors.forename: Lance C
authors.initials: LC
authors.lastname: Seefeldt
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000480:Alkynes / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D016948:Azotobacter vinelandii / Q000201:enzymology; D001665:Binding Sites; D002247:Carbon Isotopes; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008956:Models, Chemical; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000737:chemistry / Q000378:metabolism*; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D020005:Propanols / Q000737:chemistry / Q000378:metabolism*; D019097:Spin Trapping / Q000379:methods; D013379:Substrate Specificity; D014633:Valine / Q000737:chemistry / Q000378:metabolism
chemical_list: D000480:Alkynes; D002247:Carbon Isotopes; D008983:Molybdoferredoxin; D020005:Propanols; C028255:propargyl alcohol; D009591:Nitrogenase; D014633:Valine; D000409:Alanine