title: Electrophoretic studies on the assembly of the nitrogenase molybdenum-iron protein from the Klebsiella pneumoniae nifD and nifK gene products.
abstract: The electrophoretic properties of the molybdenum-iron (MoFe) protein component of nitrogenase and an iron-molybdenum cofactor (FeMoco)-reactivatable apoMoFe protein from Klebsiella pneumoniae were examined under anaerobic ([O2] < 5 ppm), nondenaturing conditions. In wild type K. pneumoniae extracts, two immunoreactive species migrating more slowly than purified MoFe protein were detected using anti-MoFe protein antibodies. The uppermost species comigrates with the apoMoFe protein produced by a K. pneumoniae mutant unable to synthesize FeMoco (UN106) and by Escherichia coli harboring the plasmids pVL222+pVL15 (nifHDKTYUSWZM+A). In vitro FeMoco titration of the UN106 and pVL222+pVL15 extracts increases the electrophoretic mobility of the apoMoFe protein to that of purified MoFe protein in a two-step process giving rise to a species of intermediate mobility between the apo- and holoMoFe proteins. Two-dimensional gel electrophoresis showed that a 20-kDa peptide is associated with the apoMoFe protein and with the intermediate species, but not with the holoMoFe protein. N-terminal sequencing identified this associated peptide as the nifY gene product, which we propose is acting as a temporary enforcer of the apoMoFe protein structure required for cofactor binding that is released upon FeMoco activation. This FeMoco-induced mobility shift was used to characterize the mutant apoMoFe proteins produced in E. coli as a result of deleting the various nitrogen fixation (nif) genes from the plasmid pVL222. E. coli extracts bearing plasmids deleted in nifH, nifS, nifTYUM, or nifWZM exhibit less than 10% of the apoMoFe protein activity of derepressed UN106 and contain an immunoreactive species whose electrophoretic mobility is increased upon addition of FeMoco from that of apoMoFe protein to that of holoMoFe protein in a single step. Anaerobic nondenaturing gel electrophoresis of 55Fe-labeled E. coli extracts followed by autoradiography showed that these inactive apoMoFe species do not contain iron, indicating that the P-clusters are absent. We therefore propose that NifH, S, U, W, Z, and M are all involved, to varying degrees, in P-cluster assembly. In addition, the presence of the P-clusters does appear to be necessary for the two-step FeMoco activation of the apoMoFe protein to occur.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: T C
authors.initials: TC
authors.lastname: White
authors.forename: G S
authors.initials: GS
authors.lastname: Harris
authors.forename: W H
authors.initials: WH
authors.lastname: Orme-Johnson
mesh_terms: D000595:Amino Acid Sequence; D000693:Anaerobiosis; D015153:Blotting, Western; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D009591:Nitrogenase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids
chemical_list: D008983:Molybdoferredoxin; D009591:Nitrogenase