title: Electron spin echo envelope modulation spectroscopic analysis of altered nitrogenase MoFe proteins from Azotobacter vinelandii.
abstract: Electron spin echo envelope modulation (ESEEM) spectroscopy was used to study changes in the polypeptide environment of the FeMo-cofactor that were elicited by amino-acid substitutions within the nitrogenase MoFe protein alpha-subunit. A previous ESEEM study [Thomann et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6620] detected modulation arising from nitrogen coupled to the S = 3/2 spin system of the FeMo-cofactor (Fe7S9Mo:homocitrate). Such modulation was found to be sensitive to the substitution of alpha-195His by alpha-195Asn as indicated by whole-cell ESEEM analysis of mutant strains from Azotobacter vinelandii. Subsequent structural studies revealed that the alpha-195His residue does not provide direct N-coordination to the cluster but is within hydrogen-bonding distance of one of a set of three sulfides that bridge the FeMo-cofactor subcluster fragments. In the present work, the ESEEM analysis is extended to both partially purified alpha-195Asn MoFe protein and purified MoFe protein from an additional mutant strain in which alpha-195His is replaced by alpha-195Gln. The dramatic decrease in the intensity of the ESEEM signal resulting from the alpha-195Asn substitution in whole cells was confirmed for the case of the isolated alpha-195Asn MoFe protein. In contrast, substitution of alpha-195His by alpha-195Gln caused no detectable change in the modulation. Simulations of the alpha-195His and alpha-195Gln ESEEM data give quadrupole parameters of e2qQ = 2.2 MHz and eta = 0.5.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208.
authors.forename: V J
authors.initials: VJ
authors.lastname: DeRose
authors.forename: C H
authors.initials: CH
authors.lastname: Kim
authors.forename: W E
authors.initials: WE
authors.lastname: Newton
authors.forename: D R
authors.initials: DR
authors.lastname: Dean
authors.forename: B M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005973:Glutamine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation
chemical_list: D008983:Molybdoferredoxin; D005973:Glutamine; D006639:Histidine; D009591:Nitrogenase