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Vanadium nitrogenase of Azotobacter chroococcum. MgATP-dependent electron transfer within the protein complex.

ABSTRACT: The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2V) to the VFe protein (AclV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23 degrees C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145, 391-396]. Extrapolation of the dependence of kobs. on [MgATP] to infinite MgATP concentration gave k = 46 s-1 for the first-order electron-transfer reaction that occurs with the Ac2V MgATPAclV complex. MgATP binds with an apparent KD = 230 +/- 10 microM and MgADP acts as a competitive inhibitor with Ki = 30 +/- 5 microM. The Fe protein and VFe protein associate with k greater than or equal to 3 x 10(7) M-1.s-1. A comparison of the dependences of kobs. for electron transfer on protein concentrations for the vanadium nitrogenase from A. chroococcum with those for the molybdenum nitrogenase from K. pneumoniae [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex.

SUBMITTER: Thorneley RN

PROVIDER: S-EPMC1135657 | biostudies-other | 1989 Feb

REPOSITORIES: biostudies-other

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The vanadium-iron protein of vanadium nitrogenase from Azotobacter chroococcum contains an iron-vanadium cofactor.

Project description:N-Methylformamide extracts of acid-treated precipitated VFe protein of the V-nitrogenase of Azotobacter chroococcum are yellow-brown in colour and contain vanadium, iron and acid-labile sulphur in the approximate proportions 1:6:5. E.p.r. spectra of the extracts exhibit a weak signal with g values near 4.5, 3.6 and 2.0 characteristic of an S = 3/2 metal-containing centre. The N-methylformamide extracts activated the MoFe protein polypeptides from mutants of nitrogen-fixing bacteria unable to synthesize FeMoco, the active centre of Mo-nitrogenase. The active hybrid protein exhibited the characteristic substrate-reducing phenotype associated with the VFe protein except that it could not reduce N2 to NH3. The above data are interpreted as demonstrating the existence of an iron- and vanadium-containing cofactor, FeVaco, within the VFe protein. It is suggested that nitrogen fixation requires specific interactions between FeVaco or FeMoco and their respective polypeptides. The biosynthesis of these cofactors is discussed.

| S-EPMC1148848 | biostudies-other

Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase.

Project description:A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.

| S-EPMC1135427 | biostudies-other

The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein.

Project description:1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes for conventional nitrogenase (nifHDK) was separated into two components: an Fe-containing protein and a vanadoprotein. 2. The larger protein was purified to homogeneity by the criterion of electrophoresis of 10% (w/v) acrylamide gels in the presence of SDS. Two types of subunit, of Mr 50,000 and 55,000, were present in equal amounts. 3. The protein had an Mr of 210,000 and contained 2 V atoms, 23 Fe atoms and 20 acid-labile sulphide groups per molecule. The Mo content was less than 0.06 g-atom/mol. All the common amino acids were present, with a predominance of acidic residues. Ultracentrifugal analysis gave a maximum sedimentation coefficient of 9.7 S and a symmetrical boundary at 5 mg of protein X ml-1; dissociation occurred at lower concentrations. The specific activities (nmol of product/min per mg of protein), when assayed under optimum conditions with the complementary Fe protein from this strain, were 1348 for H2 evolution, 350 for NH3 formation and 608 for acetylene reduction. Activity was O2-labile, with a t1/2 of 40 s in air. At low temperatures the dithionite-reduced protein showed e.p.r. signals at g = 5.6, 4.35, 3.77 and 1.93, consistent with an S = 3/2 ground state with an additional S = 1/2 centre giving rise to the feature at g = 1.93. The u.v. spectra of dithionite-reduced and thionine-oxidized protein were very similar. Oxidation resulted in a general increase in absorbance in the visible region. The shoulder at 380 nm in the spectrum of reduced protein was replaced with shoulders near 330 nm and 420 nm on oxidation.

| S-EPMC1147972 | biostudies-other

The vanadium nitrogenase of Azotobacter chroococcum. Reduction of acetylene and ethylene to ethane.

Project description:1. The vanadium (V-) nitrogenase of Azobacter chroococcum transfers up to 7.4% of the electrons used in acetylene (C2H2) reduction for the formation of ethane (C2H6). The apparent Km for C2H2 (6 kPa) is the same for either ethylene (C2H4) or ethane (C2H6) formation and much higher than the reported Km values for C2H2 reduction to C2H4 by molybdenum (Mo-) nitrogenases. Reduction of C2H2 in 2H2O yields predominantly [cis-2H2]ethylene. 2. The ratio of electron flux yielding C2H6 to that yielding C2H4 (the C2H6/C2H4 ratio) is increased by raising the ratio of Fe protein to VFe protein and by increasing the assay temperature up to at least 40 degrees C. pH values above 7.5 decrease the C2H6/C2H4 ratio. 3. C2H4 and C2H6 formation from C2H2 by V-nitrogenase are not inhibited by H2. CO inhibits both processes much less strongly than it inhibits C2H4 formation from C2H2 with Mo-nitrogenase. 4. Although V-nitrogenase also catalyses the slow CO-sensitive reduction of C2H4 to C2H6, free C2H4 is not an intermediate in C2H6 formation from C2H2. 5. Propyne (CH3C identical to CH) is not reduced by the V-nitrogenase. 6. Some implications of these results for the mechanism of C2H6 formation by the V-nitrogenase are discussed.

| S-EPMC1148769 | biostudies-other

The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the Fe protein.

Project description:1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes of Monitrogenase (nifHDK) was associated with a V + Fe-containing protein and an Fe-containing protein [Robson, Eady, Richardson, Miller, Hawkins & Postgate (1986) Nature (London) 322, 388-390; Eady, Robson, Richardson, Miller & Hawkins (1987) Biochem. J. 244, 197-207]. 2. The Fe protein was purified to homogeneity by the criterion of Coomassie Blue staining after electrophoresis in 10% or 17% (w/v) polyacrylamide gels in the presence of SDS. One type of subunit, of Mr 32,000 +/- 2000, was found. 3. The native protein had an Mr of 62,500 +/- 2500 and contained approximately 4 Fe atoms and 4 acid-labile sulphide groups per molecule. The amino acid composition was similar to those of other purified Fe proteins, and, characteristically, tryptophan was absent. The specific activities (nmol of protein/min per mg of protein) when assayed under optimum conditions with the VFe protein from this strain were 1211 for H2 evolution under Ar, 337 for NH3 from N2 formation and 349 for C2H2 reduction. Activity of the Fe protein was O2-labile with a t1/2 of 36 s in air. At low temperatures the dithionite-reduced protein exhibited e.p.r. signals consistent with the presence of both S = 1/2 and S = 3/2 spin states. These signals were similar to those given by other nitrogenase Fe proteins, as were the changes in their line shape that occurred in the presence of MgATP or MgADP. The absorbance spectra showed that an increase in absorption occurred in the visible range on reversible oxidation of the dithionite-reduced protein. The oxidized-minus-reduced epsilon 420 was 6000 M-1.cm-1.

| S-EPMC1135386 | biostudies-other

Hydrazine is a product of dinitrogen reduction by the vanadium-nitrogenase from Azotobacter chroococcum.

Project description:During the enzymic reduction of N2 to NH3 by Mo-nitrogenase, free hydrazine (N2H4) is not detectable, but an enzyme-bound intermediate can be made to yield N2H4 by quenching the enzyme during turnover [Thorneley, Eady & Lowe (1978) Nature (London) 272, 557-558]. In contrast, we show here that the V-nitrogenase of Azotobacter chroococcum produces a small but significant amount of free N2H4 (up to 0.5% of the electron flux resulting in N2 reduction) as a product of the reduction of N2. The amount of N2H4 formed increased 15-fold on increasing the assay temperature from 20 degrees C to 40 degrees C. Activity cross-reactions between nitrogenase components of Mo- and V-nitrogenases showed that the formation of free N2H4 was associated with the VFe protein. These data provide the first direct evidence for an enzyme intermediate at the four-electron-reduced level during the reduction of N2 by V-nitrogenase.

| S-EPMC1151257 | biostudies-other

Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum.

Project description:In crude cell-free extracts of Azotobacter chroococcum, nitrogenase was much less sensitive to irreversible inactivation by O2 than was the purified enzyme. When nitrogenase was partially purified by anaerobic discontinuous sucrose-density-gradient centrifugation, O2-tolerance was retained. This preparation was considerably enriched in four polypeptides, three of which were derived from the Mo-Fe(molybdenum-iron) protein and Fe (iron) protein of nitrogenase. The fourth was purified to homogeneity and shown to be an iron-sulphur protein (mol.wt. 14000) probably containing a 2Fe--2S centre. When this protein was added to purified nitrogenase, the enzyme was rendered O2-tolerant, through stabilization was Mg2+-dependent. The isolated O2-tolerant nitrogenase was an equimolar stoicheiometric complex between the MO--Fe, Fe and protective proteins. It is likely that the formation of this complex in vivo is the mechanism of 'conformational protection' in this organism.

| S-EPMC1161196 | biostudies-other

Electron transfer within nitrogenase: evidence for a deficit-spending mechanism.

Project description:The reduction of substrates catalyzed by nitrogenase utilizes an electron transfer (ET) chain comprised of three metalloclusters distributed between the two component proteins, designated as the Fe protein and the MoFe protein. The flow of electrons through these three metalloclusters involves ET from the [4Fe-4S] cluster located within the Fe protein to an [8Fe-7S] cluster, called the P cluster, located within the MoFe protein and ET from the P cluster to the active site [7Fe-9S-X-Mo-homocitrate] cluster called FeMo-cofactor, also located within the MoFe protein. The order of these two electron transfer events, the relevant oxidation states of the P-cluster, and the role(s) of ATP, which is obligatory for ET, remain unknown. In the present work, the electron transfer process was examined by stopped-flow spectrophotometry using the wild-type MoFe protein and two variant MoFe proteins, one having the β-188(Ser) residue substituted by cysteine and the other having the β-153(Cys) residue deleted. The data support a "deficit-spending" model of electron transfer where the first event (rate constant 168 s(-1)) is ET from the P cluster to FeMo-cofactor and the second, "backfill", event is fast ET (rate constant >1700 s(-1)) from the Fe protein [4Fe-4S] cluster to the oxidized P cluster. Changes in osmotic pressure reveal that the first electron transfer is conformationally gated, whereas the second is not. The data for the β-153(Cys) deletion MoFe protein variant provide an argument against an alternative two-step "hopping" ET model that reverses the two ET steps, with the Fe protein first transferring an electron to the P cluster, which in turn transfers an electron to FeMo-cofactor. The roles for ATP binding and hydrolysis in controlling the ET reactions were examined using βγ-methylene-ATP as a prehydrolysis ATP analogue and ADP + AlF(4)(-) as a posthydrolysis analogue (a mimic of ADP + P(i)).

| S-EPMC3202676 | biostudies-literature

Electron Transfer in Nitrogenase.

Project description:Nitrogenase is the only enzyme capable of reducing N2 to NH3. This challenging reaction requires the coordinated transfer of multiple electrons from the reductase, Fe-protein, to the catalytic component, MoFe-protein, in an ATP-dependent fashion. In the last two decades, there have been significant advances in our understanding of how nitrogenase orchestrates electron transfer (ET) from the Fe-protein to the catalytic site of MoFe-protein and how energy from ATP hydrolysis transduces the ET processes. In this review, we summarize these advances, with focus on the structural and thermodynamic redox properties of nitrogenase component proteins and their complexes, as well as on new insights regarding the mechanism of ET reactions during catalysis and how they are coupled to ATP hydrolysis. We also discuss recently developed chemical, photochemical, and electrochemical methods for uncoupling substrate reduction from ATP hydrolysis, which may provide new avenues for studying the catalytic mechanism of nitrogenase.

| S-EPMC7466952 | biostudies-literature

Nitrogen transfer and cross-feeding between Azotobacter chroococcum and Paracoccus aminovorans promotes pyrene degradation.

Project description:Nitrogen is a limiting nutrient for degraders function in hydrocarbon-contaminated environments. Biological nitrogen fixation by diazotrophs is a natural solution for supplying bioavailable nitrogen. Here, we determined whether the diazotroph Azotobacter chroococcum HN can provide nitrogen to the polycyclic aromatic hydrocarbon-degrading bacterium Paracoccus aminovorans HPD-2 and further explored the synergistic interactions that facilitate pyrene degradation in nitrogen-deprived environments. We found that A. chroococcum HN and P. aminovorans HPD-2 grew and degraded pyrene more quickly in co-culture than in monoculture. Surface-enhanced Raman spectroscopy combined with 15N stable isotope probing (SERS - 15N SIP) demonstrated that A. chroococcum HN provided nitrogen to P. aminovorans HPD-2. Metabolite analysis and feeding experiments confirmed that cross-feeding occurred between A. chroococcum HN and P. aminovorans HPD-2 during pyrene degradation. Transcriptomic and metabolomic analyses further revealed that co-culture significantly upregulated key pathways such as nitrogen fixation, aromatic compound degradation, protein export, and the TCA cycle in A. chroococcum HN and quorum sensing, aromatic compound degradation and ABC transporters in P. aminovorans HPD-2. Phenotypic and fluorescence in situ hybridization (FISH) assays demonstrated that A. chroococcum HN produced large amounts of biofilm and was located at the bottom of the biofilm in co-culture, whereas P. aminovorans HPD-2 attached to the surface layer and formed a bridge-like structure with A. chroococcum HN. This study demonstrates that distinct syntrophic interactions occur between A. chroococcum HN and P. aminovorans HPD-2 and provides support for their combined use in organic pollutant degradation in nitrogen-deprived environments.

| S-EPMC10689768 | biostudies-literature

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