nitrogenase and aluminum-fluoride

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-methylanthraniloy

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aluminum Compounds; Beryllium; Binding Sites; Catalysis; Electron Transport; Fluorides; Iron; Klebsiella pneumoniae; Magnetic Resonance Spectroscopy; Models, Chemical; Nitrogenase; Protein Binding; Protein Conformation; Spectrophotometry; Time Factors; Ultraviolet Rays

Nitrogenase is a two-component metalloenzyme that catalyzes a MgATP hydrolysis driven reduction of substrates. Aluminum fluoride plus MgADP inhibits nitrogenase by stabilizing an intermediate of the on-enzyme MgATP hydrolysis reaction. We report here the redox properties and electron paramagnetic resonance (EPR) signals of the aluminum fluoride-MgADP stabilized nitrogenase complex of Azotobacter vinelandii. Complex formation lowers the midpoint potential of the [4Fe-4S] cluster in the Fe protein. Also, the two-electron reaction of the unique [8Fe-7S] cluster in the MoFe protein is split in two one-electron reactions both with lower midpoint potentials. Furthermore, a change in spin-state of the two-electron oxidized [8Fe-7S] cluster is observed. The implications of these findings for the mechanism of MgATP hydrolysis driven electron transport within the nitrogenase protein complex are discussed.

Topics: Adenosine Diphosphate; Adenosine Triphosphatases; Aluminum Compounds; Azotobacter vinelandii; Electron Spin Resonance Spectroscopy; Fluorides; Hydrolysis; Iron-Sulfur Proteins; Metalloproteins; Molybdenum; Nitrogenase; Oxidation-Reduction; Potentiometry; Protein Conformation

The coupling of ATP hydrolysis to electron transfer by the enzyme nitrogenase during biological nitrogen fixation is an important example of a nucleotide-dependent transduction mechanism. The crystal structure has been determined for the complex between the Fe-protein and MoFe-protein components of nitrogenase stabilized by ADP x AIF4-, previously used as a nucleoside triphosphate analogue in nucleotide-switch proteins. The structure reveals that the dimeric Fe-protein has undergone substantial conformational changes. The beta-phosphate and AIF4- groups are stabilized through intersubunit contacts that are critical for catalysis and the redox centre is repositioned to facilitate electron transfer. Interactions in the nitrogenase complex have broad implications for signal and energy transduction mechanisms in multiprotein complexes.

Topics: Adenosine Diphosphate; Aluminum Compounds; Azotobacter vinelandii; Crystallography, X-Ray; Enzyme Stability; Fluorides; Hydrolysis; Models, Molecular; Molybdoferredoxin; Nitrogenase; Oxidoreductases; Protein Binding; Protein Conformation; Signal Transduction

A stable complex is formed between the nitrogenase proteins of Azotobacter vinelandii, aluminium fluoride and MgADP. All nitrogenase activities are inhibited. The complex formation was found to be reversible. An incubation at 50 degrees C recovers nitrogenase activity. The complex has been characterized with respect to protein and nucleotide composition and redox state of the metal-sulfur clusters. Based on the inhibition by aluminium fluoride together with MgADP, it is proposed that a stable transition state complex with nitrogenase is isolated.

Topics: Adenosine Diphosphate; Aluminum Compounds; Azotobacter vinelandii; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Fluorides; Magnesium Chloride; Nitrogenase; Phosphates; Sodium Chloride

Coupling of ATP hydrolysis to electron transfer in nitrogenase has properties similar to nucleotide-dependent switch proteins. Aluminum fluoride, a powerful inhibitor of some switch proteins, is a progressive, slowly reversible (t1/2 for reversal > 21 h) inhibitor of nitrogenase that requires both component proteins (Fe-protein and MoFe-protein) and nucleotide (either ATP or ADP). The pseudo first-order inhibition is dependent on the aluminum fluoride species, AlF4, and is linear with [Al] concentration (nonsaturating) at a pH optimum near 7.1-7.3. The inhibitor appears to react with the transient complex of the two component proteins and nucleotide. Although ADP can support the AlF inhibition, the rate of inhibition is more than 30-fold greater with ATP, which suggests the reactive conformation more closely resembles ATP hydrolysis. Conditions that increase enzymic turnover (protein concentration and component ratio) also increase the rate of inhibition, while ionic strength which slows enzymic activity spares the inhibition. The inhibited protein was isolated by gel filtration chromatography and found to be an AlF4-ADP-Fe-protein.MoFe-protein complex with the ratio of 2:1 that is consistent with two active sites per MoFe-protein alpha 2 beta 2 tetramer. Hence, inhibition by AlF4 is the stabilization of a complex that no longer hydrolyzes ATP or reduces substrates. We propose that AlF-ADP is tightly bound only in Fe-protein conformations obtained in the complex with MoFe-protein. Ligands (including Arg-46) at the base of a flexible flap on the Fe-protein could immobilize MoFe-protein--Fe-protein interface, thereby preventing dissociation of the complex.

Topics: Adenine Nucleotides; Aluminum Compounds; Enzyme Inhibitors; Fluorides; Metalloproteins; Models, Molecular; Nitrogenase; Protein Conformation