In vitro biosynthesis of iron-molybdenum cofactor and maturation of the nif-encoded apodinitrogenase. Effect of substitution for NifH with site-specifically altered forms of NifH.
NifH has three different roles in the nitrogenase enzyme system. Apart from serving as the physiological electron donor to dinitrogenase, NifH is involved in iron-molybdenum cofactor (FeMo-co) biosynthesis and in maturation of the FeMo-co-deficient form of apodinitrogenase to a FeMo-co-activable form (apodinitrogenase maturation). The exact roles of NifH in these processes are not well understood. In the present study, the features of NifH required for the aforementioned processes have been investigated by the use of site-specifically altered forms of the enzyme. The ability of six altered forms of NifH inactive in substrate reduction (K15R, D39N, D43N, L127Delta, D129E, and F135Y) to function in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions was investigated. We report that the ability of NifH to bind and not hydrolyze MgATP is required for it to function in these processes. We also present evidence that the ability of NifH to function in these processes is not dictated by the properties known to be required for its function in electron transfer to dinitrogenase. Evidence toward the existence of separate, overlapping sites on NifH for each of its functions (substrate reduction, FeMo-co biosynthesis, and apodinitrogenase maturation) is presented.
Rangaraj
P
P
Department of Biochemistry and Center for the Study of Nitrogen Fixation, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
Ryle
M J
MJ
Lanzilotta
W N
WN
Ludden
P W
PW
Shah
V K
VK
D000255:Adenosine Triphosphate / Q000378:metabolism; D016948:Azotobacter vinelandii; D004579:Electron Transport; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000096:biosynthesis* / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000235:genetics* / Q000378:metabolism*; D010088:Oxidoreductases; D011487:Protein Conformation
D008983:Molybdoferredoxin; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase