Transition state complexes of the Klebsiella pneumoniae nitrogenase proteins - Spectroscopic properties of aluminium fluoride-stabilized and beryllium fluoride-stabilized MgADP complexes reveal conformational differences of the Fe protein

Stable inactive 2 : 1 complexes of the Klebsiella pneumoniae nitrogenase co mponents (Kp2/Kp1) were prepared with ADP or the fluorescent ADP analogue, 2'(3')-O-[N-methylanthraniloyl] ADP and AlF4- or BeF3- ions. By analogy wit h published crystallographic data [Schindelin et al. (1997) Nature 387, 370 -376)], we suggest that the metal fluoride ions replaced phosphate at the t wo ATP-binding sites of the iron protein, Kp2. The beryllium (BeFx) and alu minium (AlF4-) containing complexes are proposed to correspond to the ATP-b ound state and the hydrolytic transition stares, respectively, by analogy w ith the equivalent complexes of myosin [Fisher et al. (1995) Biochemistry 3 4, 8960-8972]. P-31 NMR spectroscopy showed that during the initial stages of complex formation, MgADP bound to the complexed Kp2 in a manner similar to that reported for isolated Kp2. This process was followed by a second st ep that caused broadening of the P-31 NMR signals and, in the case of the A lF4- complex, slow hydrolysis of some of the excess ADP to AMP and inorgani c phosphate. The purified BeFx complex contained 3.8 +/- 0.1 MgADP per mol Kp1. With the AlF4- complex, MgAMP and adenosine (from MgAMP hydrolysis) re placed part of the bound MgADP although four AlF4- ions were retained, demo nstrating that full occupancy by MgADP is not required for the stability of the complex. The fluorescence emission maximum of 2'(3')-O-[N-methylanthra niloyl] ADP was blue-shifted by 6-8 nm in both metal fluoride complexes and polarization was 6-9 times that of the free analogue. The fluorescence yie ld of bound 2'(3 ')-O-[N-methylanthraniloyl] ADP was enhanced by 40% in the AlF4- complex relative to the solvent but no increase in fluorescence was observed in the BeFx complex. Resonance energy transfer from conserved tyro sine residues located in proximity to the Kp2 nucleotide-binding pocket was marked in the AlF4- complex but minimal in the BeFx fluoride complex, illu strating a clear conformational difference in the Fe protein of the two com plexes. Our data indicate that complex formation during the nitrogenase cat alytic cycle is a multistep process involving at least four conformational states of Kp2: similar to the free Fe protein; as initially complexed with detectable P-31 NMR; as detected in mature complexes with no detectable P-3 1 NMR; in the AlF4- complex in which an altered tyrosine interaction permit s resonance energy transfer with 2'(3')-O-[N-methyianthraniloyl] ADP.