title: Trapping H- bound to the nitrogenase FeMo-cofactor active site during H2 evolution: characterization by ENDOR spectroscopy.
abstract: We here show that the iron-molybdenum (FeMo)-cofactor of the nitrogenase alpha-70(Ile) molybdenum-iron (MoFe) protein variant accumulates a novel S = (1)/(2) state that can be trapped during the reduction of protons to H(2). (1,2)H-ENDOR measurements disclose the presence of two protons/hydrides (H(+/)(-)) whose hyperfine tensors have been determined from two-dimensional field-frequency (1)H ENDOR plots. The two H(+/)(-) have large isotropic hyperfine couplings, A(iso)( )() approximately 23 MHz, which shows they are bound to the cofactor. The favored analysis for these plots indicates that the two H(+/)(-) have the same principal values, which indicates that they are chemically equivalent. The tensors are further related to each other by a permutation of the tensor components, which indicates an underlying symmetry of binding relative to the cofactor. At present, no model for the structure of the iron-molybdenum (FeMo)-cofactor in the S = (1)/(2) state trapped during the reduction of H(+) can be shown unequivocally to satisfy all of the constraints generated by the ENDOR analysis. The data disfavors any model that involves protonation of sulfides, and thus suggests that the intermediate instead contains two chemically equivalent bound hydrides; it appears unlikely that these are terminal monohydrides.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
authors.forename: Robert Y
authors.initials: RY
authors.lastname: Igarashi
authors.forename: Mikhail
authors.initials: M
authors.lastname: Laryukhin
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Dos Santos
authors.forename: Hong-In
authors.initials: HI
authors.lastname: Lee
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Dean
authors.forename: Lance C
authors.initials: LC
authors.lastname: Seefeldt
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D006859:Hydrogen / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000378:metabolism; D009591:Nitrogenase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011522:Protons; D013440:Sulfides / Q000737:chemistry
chemical_list: D008983:Molybdoferredoxin; D011522:Protons; D013440:Sulfides; D006859:Hydrogen; D009591:Nitrogenase