title: 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.
abstract: Stable inactive 2 : 1 complexes of the Klebsiella pneumoniae nitrogenase components (Kp2/Kp1) were prepared with ADP or the fluorescent ADP analogue, 2'(3')-O-[N-methylanthraniloyl] ADP and AlF(4)(-) or BeF(3)(-) ions. By analogy with published crystallographic data [Schindelin et al. (1997) Nature 387, 370-376)], we suggest that the metal fluoride ions replaced phosphate at the two ATP-binding sites of the iron protein, Kp2. The beryllium (BeF(x)) and aluminium (AlF(4)(-)) containing complexes are proposed to correspond to the ATP-bound state and the hydrolytic transition states, respectively, by analogy with the equivalent complexes of myosin [Fisher et al. (1995) Biochemistry 34, 8960-8972]. (31)P 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 step that caused broadening of the (31)P NMR signals and, in the case of the AlF4- complex, slow hydrolysis of some of the excess ADP to AMP and inorganic phosphate. The purified BeFx complex contained 3.8 +/- 0.1 MgADP per mol Kp1. With the AlF(4)(-) complex, MgAMP and adenosine (from MgAMP hydrolysis) replaced part of the bound MgADP although four AlF(4)(-) ions were retained, demonstrating that full occupancy by MgADP is not required for the stability of the complex. The fluorescence emission maximum of 2'(3')-O-[N-methylanthraniloyl] 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 yield of bound 2'(3')-O-[N-methylanthraniloyl] ADP was enhanced by 40% in the AlF(4)(-) complex relative to the solvent but no increase in fluorescence was observed in the BeFx complex. Resonance energy transfer from conserved tyrosine residues located in proximity to the Kp2 nucleotide-binding pocket was marked in the AlF(4)(-) complex but minimal in the BeFx fluoride complex, illustrating a clear conformational difference in the Fe protein of the two complexes. Our data indicate that complex formation during the nitrogenase catalytic cycle is a multistep process involving at least four conformational states of Kp2: similar to the free Fe protein; as initially complexed with detectable (31)P NMR; as detected in mature complexes with no detectable (31)P NMR; in the AlF(4)(-) complex in which an altered tyrosine interaction permits resonance energy transfer with 2'(3')-O-[N-methylanthraniloyl] ADP.
authors.affiliation: Ecovale Research, Harrisville, New Hampshire, USA.
authors.forename: R W
authors.initials: RW
authors.lastname: Miller
authors.forename: R R
authors.initials: RR
authors.lastname: Eady
authors.forename: S A
authors.initials: SA
authors.lastname: Fairhurst
authors.forename: C A
authors.initials: CA
authors.lastname: Gormal
authors.forename: B E
authors.initials: BE
authors.lastname: Smith
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D017607:Aluminum Compounds / Q000494:pharmacology*; D001608:Beryllium / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D004579:Electron Transport; D005459:Fluorides / Q000737:chemistry* / Q000494:pharmacology*; D007501:Iron / Q000737:chemistry*; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009591:Nitrogenase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D017607:Aluminum Compounds; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D007501:Iron; D009591:Nitrogenase; D001608:Beryllium; D005459:Fluorides; C032311:aluminum fluoride