nitrogenase and tetrafluoroaluminate

The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP-AlF(4)(-) structure.

Topics: Adenosine Diphosphate; Aluminum Compounds; Azotobacter vinelandii; Cross-Linking Reagents; Crystallography, X-Ray; Enzyme Stability; Ethyldimethylaminopropyl Carbodiimide; Fluorides; Glycine; Molybdoferredoxin; Multienzyme Complexes; Nitrogenase; Nonheme Iron Proteins; Protein Binding; Static Electricity

The hydrolysis of ATP to ADP and P(i) is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron (Fe) protein and the molybdenum-iron (MoFe) protein. The oxidation state of nitrogenase was found to greatly influence the nucleotide hydrolysis rates. MgATP hydrolysis rates were 20 times higher under dithionite reducing conditions (approximately 4,000 nmol of MgADP formed per min/mg of Fe protein) as compared with indigo disulfonate oxidizing conditions (200 nmol of MgADP formed per min/mg of Fe protein). In contrast, MgGTP, MgITP, and MgUTP hydrolysis rates were significantly higher under oxidizing conditions (1,400-2,000 nmol of MgNDP formed per min/mg of Fe protein) as compared with reducing conditions (80-230 nmol of MgNDP formed per min/mg of Fe protein). The K(m) values for MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330-540 microM) for both the reduced and oxidized states of nitrogenase. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF(4)(-) resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF(4)(-) x MgNDP. The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed.

Topics: Adenosine Triphosphate; Aluminum Compounds; Azotobacter vinelandii; Bacterial Proteins; Clostridium; Dithionite; Fluorides; Guanosine Triphosphate; Inosine Triphosphate; Kinetics; Molybdoferredoxin; Nitrogenase; Nucleotides; Oxidation-Reduction; Oxidoreductases; Uridine Triphosphate

When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumoniae nitrogenase are incubated with MgADP and AlF4(-) in the presence of dithionite as a reducing agent, a stable putative transition-state complex is produced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, the EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S=3/2 EPR signal arising from the dithionite reduced state of the MoFe cofactor centre of the protein. This is consistent with the [Fe4S4] centre of the Fe protein in the complex being oxidized, and similar observations have been made with the complex of Azotobacter vinelandii [Spee, Arendsen, Wassink, Marritt, Hagen and Haaker (1998) FEBS Lett. 432, 55-58]. No satisfactory explanation for the fate of the electrons lost by Kp2 has been forthcoming. However, we report here that during the preparation of the MgADP-AlF4 K. pneumoniae complex under argon, H2 was evolved in amounts corresponding to one half of the FeMoco content of the Kp1 (FeMoco is the likely catalytic site of nitrogenase with a composition Mo:Fe7:S9:homocitrate). This is surprising, since activity is observed during incubation in the absence of MgATP, normally regarded as being essential for nitrogenase function, and in the presence of MgADP, a strong competitive inhibitor of nitrogenase. The formation of H2 by nitrogenase in the absence of AlF4(-) was also observed in reaction mixtures containing MgADP but not MgATP. The reaction showed saturation kinetics when Kp1 was titrated with increasing amounts of Kp2 and, at saturation, the amount of H2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H2 on [MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase since MgADP supports a very low rate of hydrogen evolution.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aluminum Compounds; Argon; Catalytic Domain; Dithionite; Electron Spin Resonance Spectroscopy; Electrons; Fluorides; Hydrogen; Iron; Iron-Sulfur Proteins; Kinetics; Klebsiella pneumoniae; Molybdoferredoxin; Nitrogenase; Oxidation-Reduction; Protons; Reducing Agents; Tricarboxylic Acids