ATP synthetase associated with the nitrogenase of Azotobacter vinelandii.

Preparations of nitrogenase from $\text{Azotobacter vinelandii}$ show an ATP synthetase activity when incubated in the presence of ADP, phosphate, ammonium chloride and an oxidizing agent. The synthesis is linked to an oxidation-reduction and the activity parallels nitrogenase activity through purification and in a step gradient sedimentation. The reductive dephosphorylation of nitrogen fixation may possibly be reversed to yield an oxidative phosphorylation.     

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.     

Relationships between nitrogenase,glutamine synthetase,glutamine, and energy charge in Azotobacter vinelandii

When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.Abbreviations GS glutamine synthetase - GS-S(Mg) Mg²⁺ dependent synthetic activity of GS - GS-T(Mn) Mn²⁺ dependent transferase activity of GS     

Inhibition of nitrogenase activity by ammonium chloride in Azotobacter vinelandii.

In Azotobacter vinelandii cells, the short-term inhibition of nitrogenase activity by NH4Cl was found to depend on several factors. The first factor is the dissolved oxygen concentration during the assay of nitrogenase. When cells are incubated with low concentrations of oxygen, nitrogenase activity is low and ammonia inhibits strongly. With more oxygen, nitrogenase activity increases. Cells incubated with an optimum amount of oxygen have maximum nitrogenase activity, and the extent of inhibition by ammonia is small. With higher amounts of oxygen, the nitrogenase activity of the cells is decreased and strongly inhibited by ammonia. The second factor found to be important for the inhibition of nitrogenase activity by NH4Cl was the pH of the medium. At a low pH, NH4+ inhibits more strongly than at a higher pH. The third factor that influenced the extent of ammonia inhibition was the respiration rate of the cells. When cells are grown with excess oxygen, the respiration rate of the cells is high and inhibition of nitrogenase activity by ammonia is small. Cells grown under oxygen-limited conditions have a low respiration rate and NH4Cl inhibition of nitrogenase activity is strong. Our results explain the contradictory reports described in the literature for the NH4Cl inhibition of nitrogenase in A. vinelandii.     

Cellular ATP levels and nitrogenase switchoff upon oxygen stress in chemostat cultures of Azotobacter vinelandii.

When Azotobacter vinelandii, growing diazotrophically in chemostat culture, was subjected to sudden increases in the ambient oxygen concentration (oxygen stress), nitrogenase activity was switched off and cellular ATP pools decreased at rates depending on the stress level. Following a fast decrease, the ATP pool approached a lower level. When the stress was released, these effects were reversed. The reversible decrease of the ATP pool upon oxygen stress could also be observed with cultures assimilating ammonium and, at the same time, fixing dinitrogen because of growth at a high C/N ratio but not with cultures growing only at the expense of ammonium. When strains OP and UW136 of A. vinelandii were subjected to long-term increases in ambient oxygen, the sizes of cellular ATP pools eventually started to increase to the level before stress and diazotrophic growth resumed. The cytochrome d-deficient mutant MK5 of A. vinelandii, however, impaired in aerotolerant diazotrophic growth, was unable to recover from stress on the basis of its ATP pool. The results suggest that adaptation to higher ambient oxygen depends on increased ATP synthesis requiring increased electron flow through the entire respiratory chain, which is possible only in combination with the more active, yet possibly uncoupled, branch terminated by cytochrome d. It is proposed that the decrease of the cellular ATP level under oxygen stress resulted from the increased energy and electron donor requirement of nitrogenase in reacting with oxygen.     

Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium.

We have constructed a strain of Azotobacter vinelandii which has deletions in the genes for both the molybdenum (Mo) and vanadium (V) nitrogenases. This strain fixed nitrogen in medium that did not contain Mo or V. Growth and nitrogenase activity were inhibited by Mo and V. In highly purified medium, growth was limited by iron. Addition of other metals (Co, Cr, Cu, Mn, Ni, Re, Ti, W, and Zn) did not stimulate growth. Like the V-nitrogenase, the nitrogenase synthesized by the double deletion strain reduced acetylene to both ethylene and ethane (C2H6/C2H4 ratio, 0.046). There was an approximately 10-fold increase in ethane production when Mo was added to the deletion strain grown in medium lacking Mo and V. This change in reactivity may be due to the incorporation of an Mo-containing cofactor into the nitrogenase synthesized by the double-deletion strain. A strain synthesizing the V-nitrogenase did not show a similar increase in ethane production. The growth characteristics of the double-deletion strain, together with the metal composition reported for a nitrogenase isolated from a tungstate-tolerant strain lacking genes for the molydenum enzyme grown in the absence of Mo and V (J. R. Chisnell, R. Premakumar, and P. E. Bishop, J. Bacteriol. 170:27-33, 1988) show that A. vinelandii can synthesize a nitrogenase which lacks both Mo and V. Reduction of dinitrogen by nitrogenase can therefore occur at a center lacking both these metals.     

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.     

Azotobacter vinelandii vanadium nitrogenase: formaldehyde is a product of catalyzed HCN reduction, and excess ammonia arises directly from catalyzed azide reduction

The Mo-nitrogenase-catalyzed reduction of both cyanide and azide results in the production of excess NH3, which is an amount of NH3 over and above that expected to be formed from the well-recognized reactions. Several suggestions about the possible sources of excess NH3 have been made, but previous attempts to characterize these reactions have met with either limited (or no) success or controversy. Because V-nitrogenase has a propensity to release partially reduced intermediates, e.g., N2H4 during N2 reduction, it was selected to probe the reduction of cyanide and azide. Sensitive assay procedures were developed and employed to monitor the production of either HCHO or CH3OH (its further two-electron-reduced product) from HCN. Like Mo-nitrogenase, V-nitrogenase suffered electron-flux inhibition by CN- (but was much less sensitive than Mo-nitrogenase), but unlike the case for Mo-nitrogenase, MgATP hydrolysis was also inhibited by CN-. V-Nitrogenase also released more of the four-electron-reduced intermediate, CH3NH2, than did Mo-nitrogenase. At high NaCN concentrations, V-nitrogenase directed a significant percentage of electron flux into excess NH3, and under these conditions, substantial amounts of HCHO, but no CH3OH, were detected for the first time. With azide, in contrast to the case for Mo-nitrogenase, both total electron flux and MgATP hydrolysis with V-nitrogenase were inhibited. V-Nitrogenase, unlike Mo-nitrogenase, showed no preference between the two-electron reduction to N2-plus-NH3 and the six-electron reduction to N2H4-plus-NH3. V-Nitrogenase formed more excess NH3, but reduction of the N2 produced by the two-electron reduction of N3(-) was not its source. Rather, it was formed directly by the eight-electron reduction of N3(-). Unlike Mo-nitrogenase, CO could not completely eliminate either cyanide or azide reduction by V-nitrogenase. CO did, however, eliminate the inhibition of both electron flux and MgATP hydrolysis by CN-, but not that caused by azide. These different responses to CO suggest different sites or modes of interaction for these two substrates with V-nitrogenase.     

Stoichiometry and spectral properties of the MoFe cofactor and noncofactor redox centers in the MoFe protein of nitrogenase from Azotobacter vinelandii

Reductive EPR and optical titrations of oxidized MoFe protein using reduced methyl viologen as reductant were used to quantitate the stoichiometry of the various spectroscopically and electrochemically distinct redox centers in the oxidized MoFe protein. Three centers were found to correlate with the EPR signal development (MoFe cofactor centers), and three centers were found to be independent of the EPR signal (P clusters) but to demonstrate distinct optical and kinetic properties. Oxidative EPR and optical titrations of reduced MoFe protein are reported which support the presence of three P-cluster centers. The optical titrations show a distinct change in kinetic behavior between the MoFe cofactor and P-cluster centers. Controlled potential coulometry demonstrates that incremental oxidation of reduced protein by methylene blue, thionine, or indigodisulfonate occurs specifically at three P-cluster sites. Subsequent oxidation by methylene blue and thionine (but not indigodisulfonate) causes the EPR signal to disappear. Three P-cluster sites, two EPR sites, and one presently uncharacterized site are suggested by the results of this study.     

Quantitative relations for the repression of nitrogenase synthesis in Azotobacter vinelandii by ammonia

A method is described which allows the quantitative determination of small ammonia concentrations in the culture of nitrogen-fixing microorganisms. With this method the ammonia concentration range was estimated in which repression of nitrogenase synthesis in Azotobacter vinelandii occurs. Both in batch and continuous cultures there was no repression below 10 μM, whereas nitrogenase synthesis stopped completely if the ammonia concentration in the medium exceeded 25 μM.     

A reinvestigation of the pre-steady-state ATPase activity of the nitrogenase from Azotobacter vinelandii.

The pre-steady-state ATPase activity of nitrogenase has been reinvestigated. The exceptionally high burst in the hydrolysis of MgATP by the nitrogenase from Azotobacter vinelandii communicated by Cordewener et al. (1987) [Cordewener J., ten Asbroek A., Wassink H., Eady R. R., Haaker H. & Veeger C. (1987) Eur. J. Biochem. 162, 265-270] was found to be caused by an apparatus artefact. A second possible artefact in the determination of the stoichiometry of the pre-steady-state ATPase activity of nitrogenase was observed. Acid-quenched mixtures of dithionite-reduced MoFe or Fe protein of Azotobacter vinelandii nitrogenase and MgATP contained phosphate above the background level. It is proposed that due to this reaction, quenched reaction mixtures of nitrogenase and MgATP may contain phosphate in addition to the phosphate released by the ATPase activity of the nitrogenase complex. It was feasible to monitor MgATP-dependent pre-steady-state proton production by the absorbance change at 572 nm of the pH indicator o-cresolsulfonaphthalein in a weakly buffered solution. At 5.6 degrees C, a pre-steady-state phase of H+ production was observed, with a first-order rate constant of 2.2 s-1, whereas electron transfer occurred with a first-order rate constant of 4.9 s-1. At 20.0 degrees C, MgATP-dependent H+ production and electron transfer in the pre-steady-state phase were characterized by observed rate constants of 9.4 s-1 and 104 s-1, respectively. The stopped-flow technique failed to detect a burst in the release of protons by the dye-oxidized nitrogenase complex. It is concluded that the hydrolysis rate of MgATP, as judged by proton release, is lower than the rate of electron transfer from the Fe protein to the MoFe protein.     

The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 3. Stoichiometry and function of the individual components.

Labelling studies with N-ETHYLMALEIMIDE SHOW THAT EITHER IN THE PRESENCE OF Mg2+, thiamine pyrophosphate (TPP) and pyruvate or in the presence of NADH the overall activity of the pyruvate dehydrogenase complex from Azotobacter vinelandii is inhibited without much inhibition of the partial reactions. The complex undergoes a conformational change upon incubation with NADH. The inhibition by bromopyruvate is less specific. Specific incorporation of a fluorescent maleimide derivative was observed on the two transacetylase isoenzymes. Binding studies with a similar spin label analogue show that 3 molecules/FAD are incorporated by incubation of pyruvate, Mg2+ and TPP, whereas 2 molecules/FAD are incorporated via incubation with NADH. The spin label spectra support the idea that in the complex the active centres of the component enzymes are connected by rapid rotation of the lipoyl moiety. Three acetyl groups are incorporated in the complex by incubation with [2-14C]pyruvate. Time-dependent incorporation supports the view that the two transacetylase isoenzymes react in non-identical ways with the pyruvate dehydrogenase components of the complex. The results show that the complex contains 2 low-molecular-weight transacetylase molecules and 4 molecules of the high-molecular-weight isoenzyme. Mn2+-binding studies show that the complex binds 10 ions, with different affinities. 2 Mn2+ ions are bound with a 20-fold higher affinity than the remaining 8 Mn2+ ions. The latter 8 ions bind with equal affinities and are thought to reflect binding to the pyruvate dehydrogenase components of the complex. It is concluded that the complex contains 8 pyruvate dehydrogenase molecules, 4 high-molecular-weight transacetylase molecules, 2 low-molecular-weight transacetylase molecules and 1 dimeric (2-FAD-containing) symmetric molecule of lipoamide dehydrogenase. Evidence comes from pyruvate-dependent inactivation and labelling studies that the pyruvate dehydrogenase components contain either an - SH group or an S-S bridge which participates in the hydroxyethyl transfer to the transacetylase components.     

ADP-ribosylation of dinitrogenase reductase from Clostridium pasteurianum prevents its inhibition of nitrogenase from Azotobacter vinelandii.

Rates of protein synthesis were measured in vivo [corrected] in the lung and heart from fed rats exposed to hyperoxia (less than or equal to 95% O2) for either 6 or 24 h. Protein synthesis rates were depressed by 16-32% compared with normoxic controls in these tissues. The inhibition in both tissues was greatest after 24 h hyperoxic exposure. The decreased fractional rates of synthesis in both tissues were related to changes in ribosomal activity rather than capacity. The fall in synthesis rate per ribosome was greatest in both tissues when the exposure period was increased to 24 h. The possible mechanism(s) involved in hyperoxia-induced depression of protein synthesis are discussed.     

Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii.

A second alternative nitrogenase complex (nitrogenase 3) was purified from a nifHDK deletion strain of Azotobacter vinelandii. The active complex is made up of two components, dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase 3 contains two protein subunits (alpha, Mr 58,000, and beta, Mr 50,000) which assemble into at least two active configurations: alpha 2 beta 2 (dinitrogenase 3s) and alpha 1 beta 2 (dinitrogenase 3F). Dinitrogenase 3s contains 24 Fe and 18 acid-labile S2-ions per Mr 216,000, and dinitrogenase 3F contains 11 Fe and 9 acid-labile S2-ions per Mr 158,000. Dinitrogenase reductase 3 is composed of two protein subunits of identical Mr (32,500) and contains four Fe and four acid-labile S2- ions per Mr 65,000. On two-dimensional gels, the protein subunits of the nitrogenase 3 complex comigrated with the four Mo-, V-, and NH4+-repressible proteins originally designated as N2ase B: the nitrogenase hypothesized to exist in the alternative N2 fixation system first described in 1980 (P.E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980). Neutron activation analysis indicated that the nitrogenase 3 complex lacked significant amounts of Mo, V, Cr, Re, and W. Some Zn, however, was found in the dinitrogenase 3S and dinitrogenase 3F preparations. The pattern of substrate reduction efficiency was H+ greater than N2 greater than C2H2. The maximum specific activity found for N2 reduction was 38 nmol of NH3 per min per mg of protein (dinitrogenase 3S). Nitrogenase 3 was found to be extremely sensitive to O2, and activities could not be reproducibly maintained during freezing and thawing.