Nucleotide binding by the nitrogenase Fe protein: a 31P NMR study of ADP and ATP interactions with the Fe protein of Klebsiella pneumoniae.
Investigation of the interaction of MgADP- and MgATP2- with the Fe protein of Klebsiella pneumoniae nitrogenase by 31P NMR showed that the adenine nucleotides are reversibly bound in slow exchange with free nucleotides. Dissociation of the MgADP--Fe protein complex was slow enough to enable its isolation by gel filtration, thus permitting the assignment of resonances to bound nucleotides. Spectra of ADP bound to Kp2 were similar to spectra of ADP bound to the myosin motor domain. Oxidative inactivation of a Kp2-MgADP- complex with excess ferricyanide ion eliminated exchange between bound and free ADP, indicating that the intact iron sulphur cluster, located 20 A from the binding sites, is required for the reversible binding of MgADP-. A change in conformation on controlled oxidation of Kp2 with indigocarmine increased the chemical shift of the beta phosphate resonance of bound MgADP-. Both oxidized and reduced conformers were observed transiently in the absence of dithionite. The 31P resonances of both the beta and gamma phosphates of bound MgATP2- indicated major changes in environment and labilization of both groups on binding to the Fe protein. Highly purified Kp2 slowly hydrolysed ATP, resulting in mixtures of bound nucleotides. Partial occupation of Kp2 MgATP2--binding sites (N=1.9+/-0.2, Kd=145 microM) in concentrated protein solutions was demonstrated by flow dialysis. Scatchard plots of data for bound and free ligand obtained after equilibration with Kp2 were linear and no co-operative interactions were detected. We conclude that MgADP- stabilizes the oxidized Fe protein conformer and this conformation in turn triggers the dissociation of the Fe protein from the MoFe protein in the rate-limiting step of the overall process of dinitrogen reduction.
Miller
R W
RW
The Nitrogen Fixation Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, U.K. rwmiller@top.monad.net
Eady
R R
RR
Gormal
C
C
Fairhurst
S A
SA
Smith
B E
BE
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D004795:Enzyme Stability; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D009591:Nitrogenase / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D010758:Phosphorus / Q000737:chemistry; D011487:Protein Conformation
D010758:Phosphorus; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase