The nifY product of Klebsiella pneumoniae is associated with apodinitrogenase and dissociates upon activation with the iron-molybdenum cofactor

Abstract

Apodinitrogenase, which lacks the iron-molybdenum cofactor at its active site, is an oligomer that contains an additional protein not found in the active dinitrogenase tetramer. This associated protein in Kiebsiella pneumoniae is shown to be the product of the nifY gene. When apodinitrogenase is activated by the addition of the iron-molybdenum cofactor, NifY dissociates from the apodinitrogenase complex. The conditions for this dissociation are described. Finally, there are aspects of the dissociation and insertion process in K. pneumoniae that are different from that in Azotobacter vinelandii. (18).The active site of component I, the iron-molybdenum cofactor (FeMo-co), is synthesized by the nifgene products, including those of nifQ, -B, -V, -N, -E, and -H (18). Many mutations in nifB and nifNE result in strains that are unable to fix N2 (13) and accumulate a form of component I without the active site. Addition of purified FeMo-co to this apocomponent I (Apo I) in vitro yields an enzyme that is catalytically active (15).When Apo I was purified from a nifB Azotobacter vinelandii mutant, another protein of approximately 20 kDa copurified with it (11). A variety of efforts to dissociate this protein without destroying Apo I proved unsuccessful, demonstrating that the complex was very tight (11).Lacking either a good genetic or biochemical perspective on this associated protein from A. vinelandii, we examined the Apo I from Kiebsiella pneumoniae, reasoning that if it was important to the biochemistry of nitrogenase, a similar factor might be detected in that organism also. In fact, a previous publication describing the purification of Apo I from K pneumoniae had already reported the presence of a 20-kDa contaminant (5). Very recently, NifY has been detected in partially purified samples of Apo I from K pneumoniae, but the nature and role of this association were unclear (19). In this report, we show that the protein associated with pure Apo I from K pneumoniae is the product of nifY. We further characterize the nature and role of the proteins associated with Apo I from both K pneumoniae and A. vinelandii.The initial indication that the associated factor in K pneumoniae is NifY came from sequence analysis of the purified protein.To isolate the protein, we purified Apo I from K pneumoniae UN1217 (nifN4536) through the hy-* Corresponding author.droxylapatite column step as outlined previously (11), except that K pneumoniae Apo I was eluted from the DEAEcellulose column at 0.25 M NaCl. The reactivatible fractions were then analyzed on an alkyl superose fast protein liquid chromatography column and eluted at 0.63 M (NH4)2SO4 in 0.025 M MOPS (morpholinepropanesulfonic acid) (pH 7.4) containing 1.7 mM Na2S204. At this point, the Apo I protein is homogeneously pure. To isolate the associated protein from Apo I, the resulting protein preparation was separated on a sodium dodecyl sulfate-13% polyacrylamide gel electrophoresis (SDS-PAGE) gel prerun with 0.25% (wt/vol) thioglycolate in the buffer and transferred to an Immobil...

Cloning and Mutational Analysis of the γ Gene fromAzotobacter vinelandii Defines a New Family of Proteins Capable of Metallocluster Binding and Protein Stabilization

“…The actual role of NifY in the A. vinelandii nif system is not known. However, K. pneumoniae NifY is found instead of ␥ as the third subunit in the hexameric apodinitrogenase (10,15). NifX and VnfX are proteins involved in the biosyntheses of FeMo-co and FeV-co, respectively (8,32).…”

“…Azotobacter vinelandii or Klebsiella pneumoniae strains with mutations in nifB, nifN, or nifE produce a FeMo-co-deficient hexameric (␣ 2 ␤ 2 ␥ 2 ) apodinitrogenase that can be activated in vitro by the simple addition of purified FeMo-co (9,10). On the other hand, apodinitrogenase from ⌬nifH mutants has a tetrameric composition (␣ 2 ␤ 2 ) and requires some type of NifH-and MgATP-dependent maturation that, in turn, promotes the association of the ␥ subunit and leads to the form that is competent for FeMo-co activation (11,12).…”

“…In K. pneumoniae the third subunit in the hexameric apodinitrogenase is the product of the nifY gene (10,15). However, this is not the case in A. vinelandii, where the ␥-encoding gene is not in the nif gene clusters.…”

The FeMoco-deficient MoFe Protein Produced by a nifHDeletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

“…FeMoco-deficient, but P-cluster containing MoFe proteins have proved to be useful for the study of two major aspects of the nitrogenase research, the maturation of MoFe protein (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and the features of the P-cluster (10, 19 -21). Two types of 100% FeMoco-deficient MoFe proteins, presumably different catalytically and structurally, have been isolated and characterized.…”

The NifX Protein is Involved in the Final Stages of FeMo-cofactor Transport to the MoFe Protein

“…It may also be possible that the NifX stabilizes the apo-dinitrogenase in a conformation suitable for FeMoco insertion through its interaction with NifK. There have been earlier speculations concerning the overlapping or redundant roles of the proteins involved in the FeMoco biosynthetic pathway, for example, the role of NifY was found to be similar to that of NafY in studies involving the FeMoco biosynthetic pathway in K. pneumoniae and A. vinelandii [49] . However, this occurrence has not been yet explainable and future studies that investigate the FeMoco biosynthetic pathway in detail may lead to meaningful answers.…”