Inhibition of iron-molybdenum cofactor biosynthesis by L127Delta NifH and evidence for a complex formation between L127Delta NifH and NifNE.
Besides serving as the obligate electron donor to dinitrogenase during nitrogenase turnover, dinitrogenase reductase (NifH) is required for the biosynthesis of the iron-molybdenum cofactor (FeMo-co) and for the maturation of alpha(2)beta(2) apo-dinitrogenase (apo-dinitrogenase maturation). In an attempt to understand the role of NifH in FeMo-co biosynthesis, a site-specific altered form of NifH in which leucine at position 127 has been deleted, L127Delta, was employed in in vitro FeMo-co synthesis assays. This altered form of NifH has been shown to inhibit substrate reduction by the wild-type nitrogenase complex, forming a tight protein complex with dinitrogenase. The L127Delta NifH was found to inhibit in vitro FeMo-co synthesis by wild-type NifH as detected by the gamma gel shift assay. Increasing the concentration of NifNE and NifB-cofactor (NifB-co) relieved the inhibition of FeMo-co synthesis by L127Delta NifH. The formation of a complex of L127Delta NifH with NifNE was investigated by gel filtration chromatography. We herein report the formation of a complex between L127Delta NifH and NifNE in the presence of NifB-co. This work presents evidence for one of the possible roles for NifH in FeMo-co biosynthesis, i.e. the interaction of NifH with a NifNE.NifB-co complex.
Rangaraj
P
P
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
Ryle
M J
MJ
Lanzilotta
W N
WN
Goodwin
P J
PJ
Dean
D R
DR
Shah
V K
VK
Ludden
P W
PW
D016948:Azotobacter vinelandii; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D017037:Dinitrogenase Reductase / Q000378:metabolism; D058085:Iron Compounds / Q000378:metabolism; D008983:Molybdoferredoxin / Q000096:biosynthesis* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases; D011485:Protein Binding
D001426:Bacterial Proteins; D017037:Dinitrogenase Reductase; D058085:Iron Compounds; D008983:Molybdoferredoxin; C085381:NifB cofactor; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase