NUCLEOTIDE-BINDING BY THE NITROGENASE FE PROTEIN - A P-31 NMR-STUDY OF ADP AND ATP INTERACTIONS WITH THE FE PROTEIN OF KLEBSIELLA-PNEUMONIAE

Investigation of the interaction of MgADP(-) and MgATP(2-) with the Fe protein of Klebsiella pneumoniae nitrogenase by P-31 NMR showed that the adenine nucleotides are reversibly bound in slow exchange with fre e nucleotides. Dissociation of the MgADP(-)-Fe protein complex was slo w enough to enable its isolation by gel filtration, thus permitting th e 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 ferricy anide ion eliminated exchange between bound and free ADP, indicating t hat the intact iron sulphur cluster, located 20 Angstrom from the bind ing sites, is required for the reversible binding of MgADP(-). A chang e in conformation on controlled oxidation of Kp2 with indigocarmine in creased the chemical shift of the beta phosphate resonance of bound Mg ADP(-). Both oxidized and reduced conformers were observed transiently in the absence of dithionite. The P-31 resonances of both the beta an d gamma phosphates of bound MgATP(2-) indicated major changes in envir onment and labilization of both groups on binding to the Fe protein. H ighly purified Kp2 slowly hydrolysed ATP, resulting in mixtures of bou nd nucleotides. Partial occupation of Kp2 MgATP(2-)-binding sites (N=1 .9+/-0.2, K-d=145 mu M) in concentrated protein solutions was demonstr ated by flow dialysis. Scatchard plots of data for bound and free liga nd obtained after equilibration with Kp2 were linear and no co-operati ve interactions were detected. We conclude that MgADP(-) stabilizes th e 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.