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

ABSTRACT: 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.

SUBMITTER: Miller RW

PROVIDER: S-EPMC1135427 | biostudies-other | 1988 Dec

REPOSITORIES: biostudies-other

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The molybdenum and vanadium nitrogenases of Azotobacter chroococcum: effect of elevated temperature on N2 reduction.

Project description:During the reduction of N2 by V-nitrogenase at 30 degrees C, some hydrazine (N2H4) is formed as a product in addition to NH3 [Dilworth and Eady (1991) Biochem. J. 277, 465-468]. We show here the following. (1) That over the temperature range 30-45 degrees C the apparent Km for the reduction of N2 to yield these products is the same, but increases from 30 to 58 kPa of N2. On increasing the temperature from 45 degrees C to 50 degrees C, little change occurred in the rate of reduction of protons to H2; the rate of N2H4 production increased, but the rate of NH3 formation decreased 7-fold. (2) Temperature-shift experiments from 42 to 50 degrees C or from 50 to 42 degrees C showed that this selective loss of the ability to reduce N2 to NH3 was reversible. The effects we observe are consistent with the existence of different conformers of the VFe-protein at the two temperatures, that predominating at 50 degrees C being largely unable to reduce N2 to ammonia. (3) Measurement of the ratio between H2 evolution and N2 reduced to NH3 at N2 pressures up to 339 kPa for both Mo- and V-nitrogenases gave limiting H2/N2 values of 1.13 +/- 0.13 for Mo-nitrogenase and 3.50 +/- 0.03 for V-nitrogenase. Since for Mo-nitrogenase our measured value for the ratio at 339 kPa is the same as that derived by Simpson and Burris [(1984) Science 224, 1095-1097] at 5650 kPa, there appears to be little or no divergence from the predictions based on the apparent Km for N2. These data then suggest that there may be a fundamentally different mechanism for N2 binding to V-nitrogenase compared with Mo-nitrogenase. (4) We did not detect any N2H4 as a product of N2 reduction by Mo-nitrogenase over the temperature range investigated; however, at 50 degrees C this system reduced acetylene (C2H2) to yield some ethane (C2H6), in addition to ethylene (C2H4), a reaction normally associated with Mo-independent nitrogenases.

| S-EPMC1132180 | biostudies-other

Differential reduction of CO? by molybdenum and vanadium nitrogenases.

Project description:The molybdenum and vanadium nitrogenases are two homologous enzymes with distinct structural and catalytic features. Previously, it was demonstrated that the V?nitrogenase was nearly 700 times more active than its Mo counterpart in reducing CO to hydrocarbons. Herein, a similar discrepancy between the two nitrogenases in the reduction of CO2 is reported, with the V?nitrogenase being capable of reducing CO2 to CO, CD4, C2D4, and C2D6, and its Mo?counterpart only capable of reducing CO2 to CO. Furthermore, it is shown that the V?nitrogenase may direct the formation of CD4 in part via CO2-derived CO, but that it does not catalyze the formation of C2D4 and C2D6 along this route. The exciting observation of a V?nitrogenase-catalyzed C-C coupling with CO2 as the origin of the building blocks adds another interesting reaction to the catalytic repertoire of this unique enzyme system. The differential activities of the V and Mo?nitrogenases in CO2 reduction provide an important framework for systematic investigations of this reaction in the future.

| S-EPMC4240507 | biostudies-literature

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

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

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

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. 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

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

Project description: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.

| S-EPMC1135657 | biostudies-other

Molybdenum nitrogenase of Azotobacter chroococcum. Tight binding of MgADP to the MoFe protein.

Project description:The dye-oxidized or dithionite-reduced forms of the MoFe protein of molybdenum nitrogenase of Azotobacter chroococcum were shown to bind 2 mol of MgADP/mol of protein, as determined by column equilibrium techniques. The gel-filtration elution profile of unbound Mg[14C]ADP was not symmetrical, consistent with a low rate of dissociation from the protein. Symmetrical elution profiles were observed for the oxidized Fe protein of nitrogenase, which bound 2 mol of MgADP/mol of protein. The low rate of dissociation of MgADP from MoFe protein was shown by non-equilibrium column techniques, where 1 mol of MgADP/mol of MoFe protein remained tightly bound during chromatography. Very weak binding of MgATP (less than 0.01 mol of MgATP/mol of MoFe protein) to dye-oxidized but not to dithionite-reduced MoFe protein was observed. These results are discussed in terms of their relevance to the catalytic cycle of nitrogenase catalysis.

| S-EPMC1133492 | biostudies-other

The vanadium- and molybdenum-containing nitrogenases of Azotobacter chroococcum. Comparison of mid-point potentials and kinetics of reduction by sodium dithionite of the iron proteins with bound magnesium adenosine 5'-diphosphate.

Project description:The mid-point potentials of the Fe protein components (Ac2 and Ac2* respectively) of the Mo nitrogenase and V nitrogenase from Azotobacter chroococcum were determined in the presence of MgADP to be -450 mV (NHE) [Ac2(MgADP)2-Ac2*ox.(MgADP)2 couple] and -463 mV (NHE) [Ac2* (MgADP)2-Ac2*ox.(ADP)2 couple] at 23 degrees C at pH 7.2. These values are consistent with a flavodoxin characterized by Deistung & Thorneley [(1986) Biochem. J. 239, 69-75] with Em = -522 mV (NHE) being an effective electron donor to both the Mo nitrogenase and the V nitrogenase in vivo. Ac2*ox.(MgADP)2 and Ac2*ox.(MgADP)2 were reduced by SO2.- (formed by the predissociation of dithionite ion, S2O4(2-)) at similar rates, k = 4.7 X 10(6) +/- 0.5 X 10(6) M-1.s-1 and 3.2 X 10(6) +/- 0.2 X 10(6) M-1.s-1 respectively, indicating structural homology at the electron-transfer site associated with the [4Fe-4S] centre in these proteins.

| S-EPMC1148978 | biostudies-other

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