Electron spin echo envelope modulation spectroscopic analysis of altered nitrogenase MoFe proteins from Azotobacter vinelandii.
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)
DeRose
V J
VJ
Department of Chemistry, Northwestern University, Evanston, Illinois 60208.
Kim
C H
CH
Newton
W E
WE
Dean
D R
DR
Hoffman
B M
BM
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
D008983:Molybdoferredoxin; D005973:Glutamine; D006639:Histidine; D009591:Nitrogenase