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PubMed1985

Complex formation and O2 sensitivity of Azotobacter vinelandii nitrogenase and its component proteins.

The O2 stability of the MoFe protein, the Fe protein, a 1:1 mixture of these proteins, and a 1:1 mixture in the presence of the Azotobacter vinelandii FeS-II protein has been studied as a function of time under controlled O2 partial pressures. The Fe protein is much more sensitive to O2 exposure than is the MoFe protein. The presence of the FeS-II protein at a 1:1 ratio with the component proteins measurably increases the O2 stability of the MoFe and Fe proteins. O2 inactivation of the MoFe protein was studied in some detail and found to be quite complex. At least three partially overlapping reactions are suggested. The first is the reversible oxidation of the metal clusters of the MoFe protein to the combined extent of 12 electrons with full retention of activity. The second phase consists primarily of activity loss with little increase in the extent of reversible oxidation. The third phase continues to decrease the protein activity but is also accompanied by formation of a g = 2.0 EPR signal and more extensive oxidation. Ultracentrifugation studies of the FeS-II protein at a 1:1:1 ratio with the Fe and MoFe proteins do not support the formation of the Bulen complex. The formation of other O2-stable complexes is discussed.

Azotobacter↗

PubMed1986

A comparative carbon-13, nitrogen-15, and phosphorus-31 nuclear magnetic resonance study on the flavodoxins from Clostridium MP, Megasphaera elsdenii, and Azotobacter vinelandii.

The flavodoxins from Megasphaera elsdenii, Clostridium MP, and Azotobacter vinelandii were studied by 13C, 15N, and 31P NMR techniques by using various selectivity enriched oxidized riboflavin 5'-phosphate (FMN) derivatives. It is shown that the pi electron distribution in protein-bound flavin differs from that of free flavin and depends also on the apoflavoprotein used. In the oxidized state Clostridium MP and M. elsdenii flavodoxins are very similar with respect to specific hydrogen bond interaction between FMN and the apoprotein and the electronic structure of flavin. A. vinelandii flavodoxin differs from these flavodoxins in both respects, but it also differs from Desulfovibrio vulgaris flavodoxin. The similarities between A. vinelandii and D. vulgaris flavodoxins are greater than the similarities with the other two flavodoxins. The differences in the pi electron distribution in the FMN of reduced flavodoxins from A. vinelandii and D. vulgaris are even greater, but the hydrogen bond patterns between the reduced flavins and the apoflavodoxins are very similar. In the reduced state all flavodoxins studied contain an ionized prosthetic group and the isoalloxazine ring is in a planar conformation. The results are compared with existing three-dimensional data and discussed with respect to the various possible mesomeric structures in protein-bound FMN. The results are also discussed in light of the proposed hypothesis that specific hydrogen bonding to the protein-bound flavin determines the specific biological activity of a particular flavoprotein.

Azotobacter↗

PubMed1986

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.

Azotobacter↗

PubMed1987

Characterization of the metal clusters in the nitrogenase molybdenum-iron and vanadium-iron proteins of Azotobacter vinelandii using magnetic circular dichroism spectroscopy.

Low-temperature magnetic circular dichroism (MCD) spectroscopy has been used to investigate the metal clusters in the conventional nitrogenase MoFe protein and alternative VFe protein from Azotobacter vinelandii. In the dithionite-reduced state, the MCD spectrum of the MoFe protein is extremely similar to that previously observed for the S = 3/2 spin state of the M clusters in the MoFe protein of Klebsiella pneumoniae. A paramagnetic cluster with an S = 3/2 ground state is also responsible for the temperature-dependent MCD transitions of dithionite-reduced VFe protein. However, the electronic and magnetic properties of this cluster are quite distinct from those of M centers in conventional nitrogenase. When these proteins are oxidized with thionine, the MoFe protein exhibits MCD spectra and magnetization characteristics identical with those observed for the P clusters in K. pneumoniae, while those of the VFe protein are only similar. However, the paramagnetism in the thionine-oxidized VFe protein, like the conventional enzyme, probably arises from an S = 5/2 spin system with near-axial symmetry and a negative zero-field splitting. Novel clusters with electronic, magnetic, and redox properties similar to those of conventional P clusters are, therefore, present in the VFe protein.

Azotobacter↗

PubMed1989

Kinetics and mechanism of the reaction of cyanide with molybdenum nitrogenase from Azotobacter vinelandii.

The steady-state kinetic behavior of the six-electron reduction of N2 by nitrogenase is known to differ markedly from the six-electron reduction of cyanide in two ways. First, on extrapolation to infinite concentration of cyanide, the H2 evolution reaction is almost completely suppressed whereas at extrapolated infinite concentration of N2, H2 evolution continues. Second, as the ratio of the Fe protein to the MoFe protein increases, the reduction of N2 is favored over H2 evolution, whereas the reduction of cyanide becomes less favored relative to H2 evolution. We have extended these steady-state experiments with Azotobacter vinelandii nitrogenase to include a third observation, that the six-electron reduction of N2 is favored over H2 evolution at high total protein concentrations whereas cyanide reduction is less favored over H2 evolution at high total protein concentrations. All three steady-state observations can be explained by a model whereby cyanide is proposed to bind to a redox state of the MoFe protein more oxidized than that reactive toward H2 evolution and N2 reduction. To test this model, we have examined the pre-steady-state kinetic behavior of both cyanide reduction by A. vinelandii nitrogenase and cyanide inhibition of total electron flow through nitrogenase. The data show that in the presence or absence of cyanide there is a short lag of 100 ms before H2 is detected, followed by a linear phase of H2 evolution lasting for about 3 s, during which time no effects of cyanide are observable. After 3 s electron flow is finally inhibited by cyanide, and the cyanide reduction product CH4 is finally formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Azotobacter↗

PubMed1990

Iron environment in ferritin with large amounts of phosphate, from Azotobacter vinelandii and horse spleen, analyzed using extended X-ray absorption fine structure (EXAFS).

The iron core of proteins in the ferritin family displays structural variations that include phosphate content as well as the number and the degree of ordering of the iron atoms. Earlier studies had shown that ferritin iron cores naturally high in phosphate, e.g., Azotobacter vinelandii (AV) ferritin (Fe:P ratio = 1:1.7), had decreased long-range order. Here, the influence of phosphate on the local structure around iron in ferritin cores is reported, comparing the EXAFS of AV ferritin, reconstituted ferritin [the protein coats of horse spleen ferritin mixed with Fe(II) with and without phosphate at pH 7] (Fe:P ratio = 1:0.25), and native horse spleen ferritin (Fe:P ratio = 1:0.125); reconstituted horse spleen ferritin without phosphate was indistinguishable from native horse spleen ferritin (HSF) in the analysis. In contrast, when the phosphate content was high in AV ferritin and horse spleen ferritin reconstituted with phosphate, the average iron atom had five to six phosphorus neighbors at 3.17 A. Moreover, the number of detectable iron neighbors was lower when phosphate was high or present during reconstitution (2-3 vs 5-6), and the interatomic distance was longer (3.50 vs 3.03 A), indicating that some phosphate bridges neighboring iron atoms. However, the decrease in the number of detectable iron-iron neighbors compared to HSF and the higher number of Fe-P interactions relative to Fe-Fe interactions suggest that some phosphate ligands were chain termini, or blocked crystal growth, and/or introduced defects which contributed both to the long-range disorder and to altered redox properties previously observed in AV ferritin.

Animals↗

PubMed1980

Role of magnesium adenosine 5'-triphosphate in the hydrogen evolution reaction catalyzed by nitrogenase from Azotobacter vinelandii.

We have investigated the role of MgATP in the reaction catalyzed by nitrogenase from Azotobacter vinelandii. There is a rapid burst of ATP hydrolysis in the pre-steady-state reaction that occurs on the same time scale as the electron transfer from dinitrogenase reductase to dinitrogenase. This burst corresponds to two ATP's hydrolyzed per electron transferred between the two proteins. Two MgATP molecules are bound to dinitrogenase reductase with dissociation constants of 430 microM and 220 microM. Investigation of the effect of MgATP concentration on the pre-steady-state kinetics of electron transfer from dinitrogenase reductase to dinitrogenase showed that there are two MgATP's required for this reaction, and the Km values are 220 microM and 970 microM. These values are similar to the dissociation constants for MgATP from dinitrogenase reductase and indicate that electron transfer between the two proteins is substantially slower than the binding and dissociation of MgATP from dinitrogenase reductase. The Km values for MgATP in steady-state H2 evolution were 390 microM and 30 microM. The decrease in the value of the second Km indicates that a slow, irreversible step occurs after the electron transfer from dinitrogenase reductase to dinitrogenase. It is possible to predict quantitatively the steady-state kinetics from the pre-steady-state kinetics, and this shows that the MgATP dependence of electron transfer is sufficient to account for effects of MgATP concentration on the steady-state H2 evolution catalyzed by nitrogenase. The hydrolysis of two ATP molecules when an electron is transferred between the two proteins of the nitrogenase system is sufficient to account for all of the ATP hydrolysis occurring in the steady-state reaction. The simplified scheme proposed to account for the MgATP dependency of the nitrogenase reaction indicates that the only role of MgATP is in support of the electron transfer from dinitrogenase reductase to dinitrogenase.

Adenosine Triphosphate↗

PubMed1980

Redox and spectroscopic properties of oxidized MoFe protein from Azotobacter vinelandii.

The MoFe protein from Azotobacter vinelandii undergoes a six-electron oxidation by various organic dye oxidants with full retention of initial activity. Reduction of the oxidized protein by S2O42- and by controlled potential electrolysis indicates the presence of two reduction regions at -290 and -480 mV, each requiring three electrons for complete reaction. Control of the oxidation conditions provides a means for preparing two distinct MoFe protein species selectively oxidized by three electrons. Selective reduction of the redox region at -290 mV causes development of the EPR signal associated with fully reduced MoFe protein while reduction at -480 mV produces a change in the visible spectrum but has no effect on the EPR signal intensity. Kinetic differences for reduction of the two redox regions indicate that the cofactor region undergoes a more rapid reaction with reductant than the other metal redox sites.

Azotobacter↗

PubMed2003

Surface changes and role of buried water molecules during the sulfane sulfur transfer in rhodanese from Azotobacter vinelandii: a fluorescence quenching and nuclear magnetic relaxation dispersion spectroscopic study.

The Azotobacter vinelandii rhodanese is a sulfurtransferase enzyme that catalyzes the transfer of the outer sulfur atom from thiosulfate to cyanide. Recently, investigations by NMR relaxation on the (15)N-enriched protein reported that interdomain contacts are rigidly maintained upon the sulfane sulfur transfer from the enzyme to the substrate. The modality of the enzymatic mechanism is then confined to a surface interaction, including dynamics of water molecules buried in the tertiary structure. Thus, investigations have been carried out by fluorescence, circular dichroism, and nuclear magnetic relaxation dispersion measurements. The comparison of circular dichroism spectra of the persulfurated enzyme and the sulfur-free form indicated that small changes occur. Fluorescence quenching studies have been performed to evaluate the conformational changes during catalysis using the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid, and acrylamide, iodide, and cesium ions as quenchers. Changes in exchange dynamics of water molecules buried in the structure with bulk water, observed by nuclear magnetic relaxation dispersion, are due to local conformational transitions, likely involving residues around the active site, and are consistent with the global correlation time found by (15)N relaxation. These results, taken together, provide important information for elucidating the conformational features of the mechanism of action of the enzyme either in the role of a selective donor of a sulfur atom to small-sized substrates (i.e., to cyanide, transforming it into thiocyanate) or in the role of sulfur insertase for the formation of the Fe(2)S(2) iron-sulfur cluster in sulfur-deprived ferredoxins.

Azotobacter vinelandii↗

PubMed2003

Mechanisms of redox-coupled proton transfer in proteins: role of the proximal proline in reactions of the [3Fe-4S] cluster in Azotobacter vinelandii ferredoxin I.

The 7Fe ferredoxin from Azotobacter vinelandii (AvFdI) contains a [3Fe-4S](+/0) cluster that binds a single proton in its reduced level. Although the cluster is buried, and therefore inaccessible to solvent, proton transfer from solvent to the cluster is fast. The kinetics and energetics of the coupled electron-proton transfer reaction at the cluster have been analyzed in detail by protein-film voltammetry, to reveal that proton transfer is mediated by the mobile carboxylate of an adjacent surface residue, aspartate-15, the pK of which is sensitive to the charge on the cluster. This paper examines the role of a nearby proline residue, proline-50, in proton transfer and its coupling to electron transfer. In the P50A and P50G mutants, a water molecule has entered the cluster binding region; it is hydrogen bonded to the backbone amide of residue-50 and to the Asp-15 carboxylate, and it is approximately 4 A from the closest sulfur atom of the cluster. Despite the water molecule linking the cluster more directly to the solvent, proton transfer is not accelerated. A detailed analysis reveals that Asp-15 remains a central part of the mechanism. However, the electrostatic coupling between cluster and carboxylate is almost completely quenched, so that cluster reduction no longer induces such a favorable shift in the carboxylate pK, and protonation of the base no longer induces a significant shift in the pK of the cluster. The electrostatic coupling is crucial for maintaining the efficiency of proton transfer both to and from the cluster, over a range of pH values.

Amino Acid Substitution↗

PubMed2004

Differentiation of acetylene-reduction sites by stereoselective proton addition during Azotobacter vinelandii nitrogenase-catalyzed C2D2 reduction.

The interactions of acetylene with its binding site(s) on the FeMo cofactor of the MoFe protein of Azotobacter vinelandii nitrogenase were probed using C(2)D(2). Specifically, the effects of changing the C(2)D(2) concentration, electron flux, pH, or the individual presence of N(2), ethylene, or CO on the formation of both cis- and trans-1,2-ethylene-d(2) from C(2)D(2) were measured. A hypothesis, involving two acetylene-reduction sites, was developed to explain the changes observed in the stereoselective protonation during both substrate-concentration-dependent and electron-flux-dependent C(2)D(2) reduction. One of these sites is a higher-affinity acetylene-binding site that produces only cis-1,2-ethylene-d(2) from C(2)D(2). The other is a lower-affinity acetylene-binding site, which produces both cis- and trans-1,2-ethylene-d(2). Added N(2) specifically inhibited the production of cis-1,2-ethylene-d(2) from C(2)D(2), which indicates that N(2) binds to (and is reduced at) the higher-affinity acetylene-binding site. High concentrations of added ethylene behaved like very high concentrations of acetylene and inhibited both the electron flux flowing through the enzyme and cis-isomer formation. Added CO, at very low concentrations, did not affect the relative distribution of cis- and trans-isomers, indicating a separate CO-binding site. The results of pH-dependence experiments showed that substrate inhibition at high C(2)D(2) concentrations is enhanced under acidic conditions but is absent under basic conditions and suggest that a low proton flux has a similar impact to that of a low electron flux; both inhibit cis-1,2-ethylene-d(2) formation selectively. Apparently, the factors affecting stereoselective protonation during C(2)D(2) reduction could be the same as those that perturb protonation of the FeMo cofactor when acetylene is reduced. The observed nitrogenase-catalyzed production of ethylene-d(1) from C(2)D(2) implicates a reversible protonation step in the mechanistic pathway.

Acetylene↗

PubMed2004

Formation of on- and off-pathway intermediates in the folding kinetics of Azotobacter vinelandii apoflavodoxin.

The folding kinetics of the 179-residue Azotobacter vinelandii apoflavodoxin, which has an alpha-beta parallel topology, have been followed by stopped-flow experiments monitored by fluorescence intensity and anisotropy. Single-jump and interrupted refolding experiments show that the refolding kinetics involve four processes yielding native molecules. Interrupted unfolding experiments show that the two slowest folding processes are due to Xaa-Pro peptide bond isomerization in unfolded apoflavodoxin. The denaturant dependence of the folding kinetics is complex. Under strongly unfolding conditions (>2.5 M GuHCl), single exponential kinetics are observed. The slope of the chevron plot changes between 3 and 5 M denaturant, and no additional unfolding process is observed. This reveals the presence of two consecutive transition states on a linear pathway that surround a high-energy on-pathway intermediate. Under refolding conditions, two processes are observed for the folding of apoflavodoxin molecules with native Xaa-Pro peptide bond conformations, which implies the population of an intermediate. The slowest of these two processes becomes faster with increasing denaturant concentration, meaning that an unfolding step is rate-limiting for folding of the majority of apoflavodoxin molecules. It is shown that the intermediate that populates during refolding is off-pathway. The experimental data obtained on apoflavodoxin folding are consistent with the linear folding mechanism I(off) <==> U <==> I(on) <== > N, the off-pathway intermediate being the molten globule one that also populates during equilibrium denaturation of apoflavodoxin. The presence of such on-pathway and off-pathway intermediates in the folding kinetics of alpha-beta parallel proteins is apparently governed by protein topology.

Apoproteins↗

PubMed2006

Redox-dependent structural changes in the Azotobacter vinelandii bacterioferritin: new insights into the ferroxidase and iron transport mechanism.

In this work, we report the X-ray crystal structure of the aerobically isolated (oxidized) and the anaerobic dithionite-reduced (at pH 8.0) forms of the native Azotobacter vinelandii bacterioferritin to 2.7 and 2.0 A resolution, respectively. Iron K-edge multiple anomalous dispersion (MAD) experiments unequivocally identified the presence of three independent iron-containing sites within the protein structure. Specifically, a dinuclear (ferroxidase) site, a b-type heme site, and the binding of a single iron atom at the four-fold molecular axis of the protein shell were observed. In addition to the novel observation of iron at the four-fold pore, these data also reveal that the oxidized form of the protein has a symmetrical ferroxidase site containing two five-coordinate iron atoms. Each iron atom is ligated by four carboxylate oxygen atoms and a single histidyl nitrogen atom. A single water molecule is found within hydrogen bonding distance of the ferroxidase site that bridges the two iron atoms on the side opposite the histidine ligands. Chemical reduction of the protein under anaerobic conditions results in an increase in the average Fe-Fe distance in the ferroxidase site from approximately 3.5 to approximately 4.0 A and the loss of one of the ligands, H130. In addition, there is significant movement of the bridging water molecule and several other amino acid side chains in the vicinity of the ferroxidase site and along the D helix to the three-fold symmetry axis. In contrast to previous work, the higher-resolution data for the dithionite-reduced structure suggest that the heme may be bound in multiple conformations. Taken together, these data allow a molecular movie of the ferroxidase gating mechanism to be developed and provide further insight into the iron uptake and/or release and mineralization mechanism of bacterioferritins in general.

Azotobacter vinelandii↗

PubMed2006

Electron exchange between Fe(II)-horse spleen ferritin and Co(III)/Mn(III) reconstituted horse spleen and Azotobacter vinelandii ferritins.

Azotobacter vinelandii bacterioferritin (AvBF) containing 800-1500 Co or Mn atoms as Co(III) and Mn(III) oxyhydroxide cores (Co-AvBF, Mn-AvBF) was synthesized by the same procedure used previously for horse spleen ferritin (HoSF). The kinetics of reduction of Co-AvBF and Mn-AvBF by ascorbic acid are first-order in each reactant. The rate constant for the reduction of Mn-AvBF (8.52 M(-1) min(-1)) is approximately 12 times larger than that for Co-AvBF (0.72 M(-1) min(-1)), which is consistent with a previous observation that Mn-HoSF is reduced approximately 10-fold faster than Co-HoSF [Zhang, B. et al. (2005) Inorg. Chem. 44, 3738-3745]. The rates of reduction of M-AvBF (M = Co and Mn) are more than twice that for the reduction of the corresponding M-HoSF. HoSF containing reduced Fe(II) cores (Fe(II)-HoSF), prepared by methyl viologen and CO, also reduces M-HoSF and M-AvBF species, with both cores remaining within ferritin, suggesting that electrons transfer through the ferritin shell. Electron transfer from Fe(II)-HoSF to Co-AvBF occurs at a rate approximately 3 times faster than that to Co-HoSF, indicating that the Co cores in AvBF are more accessible to reduction than the Co cores in HoSF. The presence of nonconductive (SiO2) or conductive (gold) surfaces known to bind ferritins enhances the rate of electron transfer. A more than approximately 4-fold increase in the apparent reaction rate is observed in the presence of gold. Although both surfaces (SiO2 and gold) enhance reaction by providing binding sites for molecular interaction, results show that ferritins with different mineral cores bound to a gold surface transfer electrons through the gold substrate so that direct contact of the reacting molecules is not required.

Animals↗

PubMed1996

Reduction of cyclic and acyclic diazene derivates by Azotobacter vinelandii nitrogenase: diazirine and trans-dimethyldiazene.

Nitrogenase reduces N2 to NH3, but the mechanistic details are unclear. Diazene (N2H2), a proposed 2e-/2H+ intermediate on the reduction pathway, is labile under typical enzyme assay conditions, and no firm evidence is available on whether or not it can be reduced by or inhibit nitrogenase. In this paper, we compare the interactions of Azotobacter vinelandii (Av) nitrogenase with two diazene analogues: diazirine, a photolabile diazene containing the azo (-N=N-) group in a strained, three-membered ring, and trans-dimethyldiazene, a diazene containing an unstrained trans-disubstituted N=N bond. Diazirine is reduced by nitrogenase under specific conditions to methane, methylamine, and ammonia in a ratio of ca. 1:2:4-5 with a Km value for all three products similar (0.05-0.09 mM) to that of dinitrogen (0.06-0.12 mM). The Km value of diazirine does not depend on the ratio of nitrogenase Fe protein (Av2) to nitrogenase MoFe protein (Av1) at Av2:Av1 ratios of 0.71 and 14.9. Diazirine potently and competitively inhibits acetylene reduction by Av nitrogenase with Ki = 0.03 mM and is predicted to inhibit H2 evolution completely at pressures >> Km. The experimental Henry's Law constant (1.50 M/atm) determined for trans-dimethyldiazene in H2O shows that it has about 20-fold higher solubility than diazirine in water at 30 degrees C. trans-Dimethyldiazene is reduced by nitrogenase under specific conditions to ammonia, methane and methylamine in a ratio of ca. 1:1:1 with Km values for the three products of 0.51-0.58 M. The product ratio does not change significantly when the component ratio (Av2:Av1 ) is varied over 2.06-13.62. trans-Dimethyldiazene reduction is inhibited noncompetitively by CO and C2H2 with Ki values of ca. 0.0008 and 0.006 atm, respectively. The results are discussed with respect to the stereoelectronic differences between the two azo substrates. A "random-edge" reduction is compared with alternative schemes for the diazirine reduction. For trans-dimethyldiazene, initial C-N cleavage is proposed to yield CH4 and a bound CH3N2H species, which is then reduced to CH3NH2 and NH3.

Acetylene↗

PubMed1996

Isolation of two forms of the nitrogenase VFe protein from Azotobacter vinelandii.

When Q-Sepharose was used in the purification of the V nitrogenase proteins from Azotobacter vinelandii, an increase in resolution was observed that resulted in a separation of the nitrogenase component 1 protein (Av1') into two forms, labeled Av1'A and Av1'B. Even though both forms possessed the same enzymatic behavior, Av1'A exhibited a lower specific activity and migrated during gel filtration with an apparent lower molecular weight than Av1'B. Furthermore, SDS-polyacrylamide gel electrophoresis showed different relative compositions of the two major subunits of both forms, with Av1'A possessing a trimer (alpha beta 2) pattern compared to the more typical tetramer (alpha 2 beta 2) pattern found for Av1'B. Metal analysis indicated a V-to-Fe ratio of 1:19 for Av1'A and 1:15 (or 2:30) for Av1'B, while acid-labile sulfide analysis showed that Av1'A possessed about half as much sulfide as Av1'B. EPR spectroscopy revealed that both proteins retained the S = 3/2 and S = 1/2 signals observed in earlier isolations, with an additional S = 1/2 signal present in the spectrum of protein A. These results suggest that Av1'A is an incomplete form of the VFe protein, containing only one cofactor and one P cluster with an additional [Fe4-S4]-like cluster. The presence of a V storage protein in A. vinelandii was also investigated. Although no V storage protein was found, two V-binding proteins were observed.

Azotobacter vinelandii↗

PubMed1996

Oxidative titration of the nitrogenase VFe protein from Azotobacter vinelandii: an example of redox-gated electron flow.

The nitrogenase VFe protein of Azotobacter vinelandii (Av1') has been shown to exist in two forms called Av1'A, which has a primary alpha beta 2 trimeric structure, and Av1'B, which has an alpha 2 beta 2 tetrameric structure [Blanchard, C. Z., & Hales, B. J. (1996) Biochemistry 35, 472-478]. Both forms exhibit S = 5/2 EPR signals in the as-isolated state that may be assigned to 1-equiv-oxidized P clusters (P+). These signals are abolished by enzymatic reduction with the component 2 protein (Av2'). Stepwise oxidative titrations of enzymatically reduced Av1'B result in the restoration of the S = 5/2 P+ signals and the concurrent decrease of the S = 3/2 vanadium cofactor signal. Further oxidation results in the appearance of an integer spin signal assigned to the 2-equiv-oxidized P cluster (P2+). Unlike the analogous signal previously observed in Mo nitrogenase component 1 (Av1), which arises from an excited state, the integer spin P2+ signal in Av1'B originates from a ground-state doublet. Similar oxidative titrations of enzymatically reduced Av1'A show redox behavior dramatically different from that of Av1'B, as monitored by EPR spectroscopy. We observed spectral evidence for a redox-induced intramolecular electron transfer between the reduced P cluster and the oxidized FeV cofactor cluster during the titrations.

Azotobacter vinelandii↗

PubMed1996

Carbohydrate fluxes into alginate biosynthesis in Azotobacter vinelandii NCIB 8789: NMR investigations of the triose pools.

The metabolite flux of carbohydrates through primary catabolism and into hexose resynthesis has been investigated for alginic acid biosynthesis in Azotobacter vinelandii. To do these studies, we fed the microorganism a variety of 13C-labeled glucose precursors, including [U-13C6]glucose. The incorporations of the precursors were determined by 1D 13C-NMR, 2D 13C-DQF-COSY, and inverse triple-quantum correlation experiments. The results clearly show that the entire catabolism of hexose is through the Entner-Doudoroff (E-D) pathway and that the triose pools are in equilibrium. Reentry into gluconeogenesis prior to alginate synthesis occurs totally from the glyceraldehyde 3-phosphate generated by the E-D pathway. The obligatory intermediacy of triose intermediates in alginate biosynthesis was proved. The experiments and results presented in this paper constitute a new method for distinguishing the E-D pathway from glycolysis in bacteria.

Alginates↗