The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein.
We have examined three strains of Azotobacter vinelandii, which contain defined deletions within the nifH, nifB, or nifE genes. All three strains accumulate inactive FeMo cofactor-deficient forms of the MoFe protein of nitrogenase. These forms can be activated in vitro by addition of isolated FeMo cofactor in N-methylformamide. Although the phenotypes of these strains are superficially the same, our characterizations demonstrate that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is quite different from that synthesized by either the delta nifB or delta nifE strains. These differences include the following: 1) the activation of the delta nifH protein requires MgATP, whereas the activation of the delta nifB and delta nifE proteins does not; 2) the delta nifH extracts can be activated with FeMo cofactor to wild-type levels of activity, whereas delta nifB and delta nifE extracts cannot; 3) the delta nifH protein is markedly less heat stable than the delta nifB and delta nifE proteins; and 4) the migration of the delta nifH protein on native gels is very different when compared with delta nifB and delta nifE, which look like each other. These data can be explained if the nifB and nifE gene products are only involved in FeMo cofactor biosynthesis, whereas the nifH gene product is involved in both the initial synthesis of FeMo cofactor and in the insertion of preformed FeMo cofactor into the MoFe protein. A model is presented that suggests that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is the one that normally participates in MoFe protein assembly in wild-type cells.
Tal
S
S
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Chun
T W
TW
Gavini
N
N
Burgess
B K
BK
D000255:Adenosine Triphosphate / Q000032:analysis; D001395:Azotobacter / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D008983:Molybdoferredoxin / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D008983:Molybdoferredoxin; D000255:Adenosine Triphosphate