Open AccessComparative In‐Vivo and In‐Vitro 99 Mo‐Time‐Differential‐Perturbed‐Angular‐Correlation Studies on the Nitrogenase Mofe Protein and on Other Mo Species of Different N 2 ‐Fixing Bacteria
Author(s) -
Müller Achim,
Suer Waltraud,
Pohlmann Carsten,
Schneider Klaus,
Thies WolfGerolf,
Appel Helmut
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00311.x
Subject(s) - nitrogenase , in vivo , in vitro , chemistry , physics , differential (mechanical device) , biology , biochemistry , nitrogen , nitrogen fixation , quantum mechanics , thermodynamics , microbiology and biotechnology
Klebsiella pneumoniae, Azotobacter vinelandii and Rhodobacter capsulatus were cultivated in media containing 99 MoO 2‐ 4 . The distribution of 99 Mo in cells grown under conditions of repression and derepression of nitrogenase synthesis, was investigated by anion‐exchange (DEAE‐Sephacel) chromatography. Cells of K. pneumoniae took up MoO 2‐ 4 only under conditions of derepression of nitrogenase thus serving the formation of the FeMo cofactor of the MoFe protein (Kp1) as the predominant Mo‐containing species. In the case of A. vinelandii , under diazotrophic growth conditions, molybdenum was preferably incorporated into the nitrogenase MoFe protein (Av1). However, if excess amounts of molybdate were present in the medium, molybdenum was also bound to the Mo‐storage protein. In the presence of 20 mM NH 4 + , conditions which completely repress nitrogenase formation, molybdenum accumulated in the Mo‐storage protein exclusively. This protein proved to be unstable towards DEAE‐Sephacel, apparently releasing all the molybdenum in form of MoO 2‐ 4 during the fractionation procedure. R. capsulatus contained, in addition to the MoFe protein (Rc1), significant amounts of other not‐yet‐identified Mo species, which partially are formed under conditions of both, repression and derepression of nitrogenase. The Mo centers of all these compounds were characterized by measuring the nuclear quadrupole interaction of the process 99 Mo(β − ) 99 Tc using time differential perturbed angular correlation spectroscopy. The quadrupole coupling constant ( V Q ) determined for the Mo center in MoFe proteins was consistently in the range 66–81 MHz. The values of the coupling constants determined with intact cells and with the isolated, partially purified, MoFe proteins were in very good agreement. For the Mo‐storage protein of A. vinelandii , a quadrupole coupling constant of approximately 180 MHz was determined by measurements performed with nitrogenase‐repressed cells as well as with gel‐filtered cell‐free extracts. Our work proves that the relevant study of hyperfine interactions allows the identification of the MoFe protein and also other Mo proteins in vivo as well as in vitro.