Electron spin echo envelope modulation spectroscopic analysis of altered nitrogenase MoFe proteins from Azotobacter vinelandii. Academic Article uri icon

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)

published proceedings

  • Biochemistry

author list (cited authors)

  • DeRose, V. J., Kim, C. H., Newton, W. E., Dean, D. R., & Hoffman, B. M.

citation count

  • 31

complete list of authors

  • DeRose, VJ||Kim, CH||Newton, WE||Dean, DR||Hoffman, BM

publication date

  • March 1995