Thiol reactivity of the nitrogenase Fe-protein from Azotobacter vinelandii

A procedure has been developed to examine some of the functional roles of the 14 cysteinyl residues in the nitrogenase Fe-protein (Av2) from Azotobacter vinelandii. The reduced form of Av2 was alkylated with iodo[2-14C]acetic acid under a variety of experimental conditions, e.g. reaction in the presence of nucleotides, alpha,alpha'-dipyridyl and nucleotides, or denaturants. The labeled cysteinyl residues were identified and quantified using an analytical DEAE-Sepharose ion exchange chromatography peptide mapping technique based upon the known amino acid sequence (Hausinger, R. P., and Howard, J. B. (1982) J. Biol. Chem. 257, 2483-2490). From the results of the labeling experiments, the following features of the Av2 structure have been proposed. 1) Av2 contains no disulfides, hyperreactive thiols, or surface thiols as defined by reaction with iodoacetic acid. 2) Cysteines 97 and 132 are the probable ligands for the Av2 Fe:S center which is bound symmetrically between subunits. 3) MgATP partially protects cysteine 85 from carboxymethylation by iodoacetic acid and may be part of the nucleotide-binding site. 4) Of the five nonligand thiols only cysteines 5 and 184 are completely alkylated when Av2 is denatured in hexamethylphosphoramide, whereas all five nonligand thiols appear to rapidly exchange at the Fe:S center if the protein is denatured in the absence of alkylating reagents. 5) Both Av2 and apo-Av2 appear to undergo a reversible conformational change upon binding MgATP.     

Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii

Wild-type Azotobacter vinelandii strain UW was transformed with plasmid pDB12 to produce a species (LS10) unable to synthesize the structural proteins of component 1 and component 2 of native nitrogenase. A spontaneous mutant of this strain was isolated (LS15) which can grow by nitrogen fixation in the presence or absence of either Mo or W. It is proposed that LS15 fixes nitrogen solely by an alternative nitrogen-fixing system which previously has been hypothesized to exist in A. vinelandii. Under nitrogen-fixing conditions, LS15 synthesizes a protein similar to component 2 (Av2) of native nitrogenase in that it can complement native component 1 (Av1) for enzymatic activity. Isolation and characterization of this second component 2 shows it to be a 4Fe-4S protein of molecular mass about 62 kDa and is antigenically similar to Av2. This protein is also similar to Av2 in that in the reduced state it possesses a rhombic ESR spectrum in the g = 2 region, which changes to an axial spectrum upon addition of MgATP. It is suggested that this second Fe-protein is associated with the alternative nitrogen-fixing system in A. vinelandii.     

Kinetics of MgATP-dependent iron chelation from the Fe-protein of the Azotobacter vinelandii nitrogenase complex. Evidence for two states

Chelation of Fe from the Fe-protein component (Av2) of Azotobacter vinelandii nitrogenase has been investigated. The chelation, which requires MgATP binding by Av2, is best described as a two-exponential process. The rates for the two phases differed by approximately 10-fold and increased as the concentration of MgATP was increased. The rates for both phases were 50% of maximum at approximately 1.5 mM MgATP. At MgATP concentrations greater than 100 microM, the more rapid phase represented approximately 25% of the total Fe chelated from Av2. However, below 100 microM MgATP, the proportion of the faster phase decreased until at 20 microM MgATP, only a single phase could be detected. The properties of Av2 were studied at various stages of Fe chelation. The partially chelated protein was isolated from the reaction by gel filtration and was subjected to a second MgATP-dependent Fe chelation. Material isolated after the completion of the first phase regained biphasic kinetics in subsequent chelation reactions. However, if MgATP was present during the isolation of Av2, then only a single phase was observed in the subsequent chelation studies. In addition, the enzymatic activity of Av2 decreased concomitantly with total Fe chelation. To account for these observations, a model is presented in which Av2 exists in two conformers. Fe chelation is proposed to occur from either conformer but only when two MgATP are bound. Both conformers bind MgATP with the same affinity but are distinguished by a 10-fold difference in chelation rate. The two conformers are in equilibrium and can interconvert only in the absence of MgATP. That is, MgATP binding prevents the conversion of the two conformational states.     

On the prosthetic group(s) of component II from nitrogenase. EPR of the Fe-protein from Azotobacter vinelandii

The EPR spectrum of the reduced Fe-protein from nitrogenase has been reinvestigated. The dependences on temperature, microwave power, and microwave frequency all suggest that the observed signal represents a magnetically isolated [4Fe-4S]1+(2+;1+) cluster. Also, the signal can be simulated assuming a simple, g-strained S = 1/2 system. However, the integrated intensity amounts to no more than 0.2 spins per protein molecule. It is, therefore, impossible that Fe-protein preparations contain a single type of [4Fe-4S] cluster.     

Stability and ferrous iron content of the nitrogenase and its components from Azotobacter vinelandii

Summary Nitrogenase of Azotobacter vinelandii is sensitive to oxygen, and sensitivity develops during purification. Such inactivation is well prevented by 0.1% hydroquinone plus 0.01% ascorbate, which are also effective in preventing inactivation of enzyme on storage under H₂. Activity is proportional to ferrous iron content of crude sample of enzyme. On storage at 0°C, 0.3 M KCl inactivates the enzyme, while KCl stabilizes its components. Nitrogenase is not cold labile, while the components are cold labile; Fe, Mo-component is most stable at 22°C and Fe-component at 13.5°C. Nitrogenase substrates, except N₂, stabilize nitrogenase, but not the components.     

Electron microscopy of the Mo-Fe-protein from Azotobacter vinelandii nitrogenase

The quaternary structure of the Mo-Fe-protein from Azotobacter vinelandii has been studied by electron microscopy. A model of the molecule of the Mo-Fe-protein has been proposed: two alpha subunits are displaced relative to two beta subunits along a twofold axis, so the molecule can be characterized by the point-group pseudosymmetry 222. Computer averaging of the images showed that one of the projections of the molecule could be characterized by twofold rotational symmetry. Micrographs of nitrogenase recombined complex (Mo-Fe-protein + Fe-protein) have been obtained. They showed particles close in size and form to the Mo-Fe-protein molecule. Therefore, it has been proposed that the Fe-protein could be situated in the central cavity of Mo-Fe-protein.     

Iron-molybdenum cofactor biosynthesis in Azotobacter vinelandii requires the iron protein of nitrogenase

Nitrogenase is composed of two separately purified proteins called the Fe protein and the MoFe protein. In Azotobacter vinelandii the genes encoding these structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). The MoFe protein contains an ironmolybdenum cofactor (FeMo cofactor) whose biosynthesis involves the participation of at least five gene products, nifQ, nifB, nifN, nifE, and nifV. In this study an A. vinelandii mutant strain, which contains a defined deletion within the nifH (Fe protein) gene, was isolated and studied. This mutant is still able to accumulate significant amounts of MoFe protein subunits. However, extracts of this nifH deletion strain have only very low levels of MoFe protein acetylene reduction activity. Fully active MoFe protein can be reconstituted by simply adding isolated FeMo cofactor to the extracts. Fe protein is not necessary to stabilize or insert this preformed FeMo cofactor into the FeMo cofactor-deficient MoFe protein synthesized by the nifH deletion strain. Extracts of the nifH deletion strain can carry out molybdate and ATP-dependent in vitro FeMo cofactor biosynthesis provided Fe protein is added, demonstrating that they contain the products encoded by the FeMo cofactor biosynthetic genes. These data demonstrate that the Fe protein is physically required for the biosynthesis of FeMo cofactor in A. vinelandii.     

Purification of azotophore membranes containing the nitrogenase from Azotobacter vinelandii

Azotophore membranes containing nitrogenase have been purified in high yield from $\text{A. vinelandii}$ by differential and sucrose density gradient sedimentation. The purified preparations appeared as uniform vesicular membranes of 40–75 nm diameter containing the majority of the nitrogenase activity from these cells and were readily separated from the intracytoplasmic membranes containing the cytochromes of the respiratory electron transfer system. The yield and specific activity of azotophores from cells broken by mechanical or osmotic treatment were similar.     

Molecular weights of nitrogenase components from Azotobacter vinelandii.

Meniscus depletion sedimentation equilibrium ultracentrifuge experiments were performed on purified MoFe and Fe proteins of $\text{Azotobacter vinelandii}$. The MoFe protein was found to have a molecular weight of 245,000, using an experimentally confirmed partial specific volume of 0.73. The MoFe protein formed one band on sodium dodecyl sulfate gel electrophoresis and had a subunit molecular weight of 56,000. The subunit molecular weight from ultracentrifuge experiments in 8 M urea was 61,000. The molecular weight of the Fe protein was calculated to be 60,500 in meniscus depletion experiments. Similar experiments in 8 M urea solvent indicated a subunit molecular weight of 30,000. A subunit molecular weight of 33,000 was obtained from sodium dodecyl sulfate gel electrophoresis experiments.     

Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii

A new nitrogenase from Azotobacter vinelandii has been isolated and characterized. It consists of two proteins, one of which is almost identical with the Fe protein (component 2) of the conventional enzyme. The second protein (Av1'), however, has now been isolated and shown to differ completely from conventional component 1, i.e., the MoFe protein. This new protein consists of two polypeptides with a total molecular weight of around 200,000. In place of Mo and Fe it contains V and Fe with a V:Fe ratio of 1:13 +/- 3. The ESR spectrum of Av1' also differs from conventional component 1 in that lacks the g = 3.6 resonance that arises from the FeMo cofactor but contains an axial signal with gav less than 2 as well as inflections in the g = 4-6 region possibly arising from an S = 3/2 state. This new enzyme can reduce dinitrogen, protons, and acetylene but is only able to utilize 10-15% of its electrons for the reduction of acetylene.     

Solubilization of the iron molybdenum cofactor of Azotobacter vinelandii nitrogenase in dimethylformamide and acetonitrile

The iron molybdenum cofactor of Azotobacter vinelandii nitrogenase has been solubilized for the first time in dimethylformamide and acetonitrile. These solutions have the ability to reconstitute the inactive nitrogenase of the UW 45 mutant of A. vinelandii and exhibit an S = 3/2 EPR signal similar to that for the cofactor in N-methylformamide. Our ability to obtain solutions of FeMoco in these solvents seemingly refutes a previous hypothesis concerning the necessity of solvents with a dissociable proton for iron molybdenum cofactor solubility and should facilitate the spectroscopic characterization of this important species.     

Characterization of the gene for the Fe-protein of the vanadium dependent alternative nitrogenase of Azotobacter vinelandii and construction of a Tn5 mutant

Summary A sequence homologous to the conventional nifH gene has been cloned from a different region of the Azotobacter vinelandii genome. Tn5 insertions were obtained in this clone and the mutagenized plasmid was used for marker exchange with A. vinelandii strain CA12 (nifHDK) to obtain Tn5 mutants. These mutants exhibited a Nif^- phenotype in the presence of vanadium, unlike CA12 which was Nif⁺ on vanadium-containing medium. The gene in the cloned nifH-like region is therefore apparently involved in the vanadium dependent alternative pathway of nitrogen fixation. This gene, nifH2, has been sequenced and encodes a protein of 289 amino acids that is similar to nifH in nucleotide sequence, deduced amino acid sequence, predicted secondary structure and hydrophobicity profile. A second open reading frame downstream of nifH2 codes for a protein of 64 amino acids, similar to the ferredoxin (Fd)-like protein encoded downstream of nifH ^* in A. chroococcum. Sequence analysis suggests that the nifH2 and Fd-like genes are in a single operon.     

Resolution of two subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase

The Mo-Fe protein of Azotobacter vinelandii nitrogenase was fractionated on 9.5 M urea isoelectric focusing gels and gave three distinct bands (alpha', alpha", beta'). Protein focused on nondenaturing gels gave a single brown band, which when excised and refocused on a denaturing gel gave the three-band pattern. Partial trypsin digestion of the subunits and fractionation of the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the alpha' and alpha" polypeptide moieties were the same. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha' and beta' proteins with appropriate molecular weight standards indicated Mr = 61,000 and 57,000, respectively. This is consistent with an overall alpha 2 beta 2 mass of 236,000 daltons.     

On the formation of an oxygen-tolerant three-component nitrogenase complex from Azotobacter vinelandii

Conditions are defined in which the oxygen-labile nitrogenase components from Azotobacter vinelandii can be protected against oxygen inactivation by the so-called Fe/S protein II. It is demonstrated that oxygen protection can be achieved by complex formation of the three proteins. Complex formation was studied by gel chromatography. Only when the three proteins are in the oxidized state and MgCl2 is present, can an oxygen-tolerant complex be isolated. Quantitative SDS/polyacrylamide gel electrophoresis of such complexes, yielded an average ratio of nitrogenase component 2/nitrogenase component 1 (Av2/Av1) of 2.4 +/- 0.5. Protection by Fe/S protein II was correlated with the amount of [2 Fe-2S] clusters present in the protein and not by the amount of protein. Measurements of the amount of iron and sulfide of Fe/S protein II showed that the iron and sulfide content of the protein was variable. The maximum values found indicate that Fe/S protein II contains two [2Fe-2S] clusters per dimer of 26 kDa. Full protection by Fe/S protein II was obtained with a ratio of Fe/S protein II/Av1 of 1.1 +/- 0.2; the Fe/S protein II containing two [2Fe-2S] clusters per dimer of 26 kDa. When Fe/S protein II contains less [2Fe-2S] clusters, more protein is necessary to obtain full protection. The three-component nitrogenase complex is also oxygen stable in the presence of MgATP or MgADP. Analysis in the ultracentrifuge showed that the major fraction of the reconstituted complex has a sedimentation coefficient centered around 34S. A small fraction (less than 30%) sediments with values centered around 111 S. This suggests an average mass for the oxygen-stable nitrogenase complex of 1.5 MDa. Taking into account the determined stoichiometry of the individual proteins, the molecular composition of the oxygen-stable nitrogenase complex is presumably 4 molecules of AV1,8--12 molecules of aAV2 and 4--6 molecules of Fe/S protein II containing two [2Fe-2S] clusters per dimer of 26 kDa.