Regulation of nitrogen-fixation by different nitrogen sources in the marine non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067

The effect of various nitrogen sources on the synthesis and activity of nitrogenase was studied in the marine, non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067 grown under defined culture conditions. Cells grown with N₂ as the sole inorganic nitrogen source showed light-dependent nitrogenase activity (acetylene reduction). Nitrogenase activity in cells grown on N₂ was not suppressed after 7 h incubation with 2 mM NaNO₃ or 0.02 mM NH₄Cl. However, after 3 h of exposure to 0.5 mM of urea, nitrogenase was inactivated. Cells grown in medium containing 2 mM NaNO₃, 0.5 mM urea or 0.02 mM NH₄Cl completely lacked the ability to reduce acetylene. Western immunoblots tested with polyclonal antisera against the Fe-protein and the Mo–Fe protein, revealed the following: (1) both the Fe-protein and the Mo–Fe protein were synthesized in cells grown with N₂ as well as in cells grown with NaNO₃ or low concentration of NH₄Cl; (2) two bands (apparent molecular mass of 38 000 and 40 000) which cross-reacted with the antiserum to the Fe-protein, were found in nitrogen-fixing cells; (3) only one protein band, corresponding to the high molecular mass form of the Fe-protein, was found in cells grown with NaNO₃ or low concentration of NH₄Cl; (4) neither the Fe-protein nor the Mo–Fe protein was found in cells grown with urea; (5) the apparent molecular mass of the Fe-protein of Trichodesmium sp. NIBB1067 was about 5000 dalton higher than that of the heterocystous cyanobacterium, Anabaena cylindrica IAM-M1.     

A novel covalent modification of nitrogenase in a cyanobacterium

In extracts of the unicellular cyanobacterium Gloeothece, the Fe-protein of nitrogenase can be separated by SDS-PAGE into two antigenically identifiable components. Unlike the situation in photosynthetic bacteria such as Rhodospirillum rubrum, these two forms do not arise from covalent modification of the protein by ADP-ribosylation. Rather, the Fe-protein of Gloeothece nitrogenase is subjected to modification by palmitoylation.     

Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.     

Growth of Spirillum lipoferum at constant partial pressures of oxygen, and the properties of its nitrogenase in cell-free extracts.

Spirillum lipoferum, an N2-fixing organism, was grown at constant concentrations of dissolved O2. When supplied with NH4+ aerobically, its doubling time was 1 h; when it fixed N2 microaerophilically, its doubling time was 5-5 to 7 h and the optimal PO2 for growth was 0-005 to 0-007 atm. At its optimal PO2 for growth on N2, S. lipoferum assimilated 8 to 10 mg nitrogen/g carbon substrate used; its efficiency was less at higher PO2 levels. Nitrogenase in cell-free extracts required Mg2+ and Mn2+, and the Fe-protein was activated by Rhodospirillum rubrum activating factor. The nitrogenase had an optimal pH of 7-1 to 7-4 and an apparent Km for acetylene of 0-0036 atm. Extracts of S. lipoferum lost their nitrogenase activity on storage at -18 degrees C, and activity was restored by adding purified Fe-protein from other N2-fixing bacteria.     

柱孢鱼腥藻的铁蛋白与棕色固氮菌的钼铁蛋白的交叉互补作用

纯化的柱孢鱼腥藻铁蛋白能够与棕色固氮菌的钼铁蛋白有效地交叉反应,展现较高的活性。此异源交叉反应的乙炔还原比活及放氢比活,分别是蓝藻同源互补比活的83.8及66.7％。比较藻铁蛋白与菌钼铁蛋白异源交叉反应及藻固氮酶组分之间的同源反应的动力学特点时发现,铁蛋白对钼铁蛋白的最佳克分子比数前者(异源交叉反应)较后者(藻同源反应)为高,前者为5,后者为1;但反应的时间进程两者差别不大。     

Immunolocalization and Western Blot Analysis of Nitrogenase in Oscillatoria limosa During a Light-dark Cycle

Nitrogenase of the non-heterocystous nitrogen-fixing cyanobacterium Oscillatoria limosa was subjected to western blot analysis and immunogold electron microscopy using antisera raised against dinitrogenase (MoFe-protein, Component I) and dinitrogenase reductase (Fe-protein, Component II). O. limosa was grown diazotrophically under an alternating light-dark cycle (16–8h light-dark). Although nitrogenase activity (acetylene reduction) was found predominantly during the dark phase, being absent during most of the light period, immunogold electron microscopy revealed label of both subunits of nitrogenase in samples taken throughout the light-dark cycle. It was also shown that the nitrogenase label was distributed homogeneously in the cell and that it was present in every cell of every trichome whether fixing nitrogen or not. On average, 34 (± 6) gold particles μm^?2 thin section were detected. Nitrate-grown cells did not contain nitrogenase label. Western blot analysis of the Fe-protein in samples taken during the light phase, revealed a single band with an apparent molecular weight of 37 kDa. At the end of the light period, and during the dark phase when high nitrogenase activities were observed, an additional band of 36 kDa was found. The anti-MoFe-protein antiserum revealed a single band of 56 kDa which was present throughout the light-dark cycle. Nitrate-grown cells were not recognized by either antiserum. It is concluded that nitrogenase enzyme is present in O. limosa throughout the light-dark cycle but that the Fe-protein is modified (inactive form) during the light period when nitrogenase activity is absent.     

Analysis of the nifHDK operon and structure of the NifH protein from the unicellular, diazotrophic cyanobacterium, Cyanothece strain sp. ATCC 51142(1)

Cyanothece sp. ATCC 51142 is a unicellular, diazotrophic cyanobacterium that demonstrates diurnal rhythms for photosynthesis and N(2) fixation, with peaks of O(2) evolution and nitrogenase activity approximately 12 h out of phase. We cloned and sequenced the nifHDK operon, and determined that the amino acid sequences of all three proteins were highly conserved relative to those of other cyanobacteria and bacteria. However, the Fe-protein, encoded by the nifH gene, demonstrated two differences from the related protein in Azotobacter vinelandii, for which a 3-D structure has been determined. First, the Cyanothece Fe-protein contained a 37 amino acid extension at the N-terminus. This approximately 4 kDa addition to the protein appeared to fold as a separate domain, but remained a part of the active protein, as was verified by migration on acrylamide gels. In addition, the Cyanothece Fe-protein had amino acid differences at positions involved in formation of the Fe-protein dimer-dimer contacts in A. vinelandii nitrogenase. There were also changes in residues involved with interaction between the Fe-protein and the MoFe-protein when compared with A. vinelandii. Since the Cyanothece Fe-protein is quickly degraded after activity, it is suggested that the extension and the amino acid alterations were somehow involved in this degradative process.     

Regulation of carbon monoxide dehydrogenase and hydrogenase in Rhodospirillum rubrum: effects of CO and oxygen on synthesis and activity.

Exposure of the photosynthetic bacterium Rhodospirillum rubrum to carbon monoxide led to increased carbon monoxide dehydrogenase and hydrogenase activities due to de novo protein synthesis of both enzymes. Two-dimensional gels of [35S]methionine-pulse-labeled cells showed that induction of CO dehydrogenase synthesis was rapidly initiated (less than 5 min upon exposure to CO) and was inhibited by oxygen. Both CO dehydrogenase and the CO-induced hydrogenase were inactivated by oxygen in vivo and in vitro. In contrast to CO dehydrogenase, the CO-induced hydrogenase was 95% inactivated by heating at 70 degrees C for 5 min. Unlike other hydrogenases, this CO-induced hydrogenase was inhibited only 60% by a 100% CO gas phase.     

Nitrogenase of Azotobacter chroococcum. Kinetics of the reduction of oxidized iron-protein by sodium dithionite.

The kinetics of the reduction of oxidized Fe-protein of nitrogenase from Azotobacter chroococcum by sodium dithionite were studied by stopped-flow and rapid-freezing e.p.r. (electron-paramagnetic-resonance) spectroscopy. The appearance of the gav. = 1.94 e.p.r. signal (0.24 electron integrated intensity/mol) was associated with a one-electron reduction by SO2--with k greater than 10(8)M-1-S-1 at 23 degrees C. A value of k = 1.75s-1 was obtained for the rate of dissociation of S2O42- into 2SO2-- at 23 degrees C. Further reductions by SO2-- occurred in three slower phases with rate constants in the range 10(4) -10(6)M-1-S-1. These latter phases have no corresponding e.p.r. signal changes and are probably associated with enzymically inactive protein. The high rate of reduction by SO2-- of the Fe-protein alone (k greater than 10(8)M-1-S-1) relative to the rate of oxidation of the Fe-protein in the catalytically active Fe:Mo-Fe protein complex (k = 2.2 X 1O(2)s-1) and the observation that in the steady state the Fe-protein is substantially oxidized means that at normal assay concentrations another reaction must limit the rate of reduction of Fe-protein during turnover.     

Nitrogenase in free-living and symbiotic cyanobacteria: Immunoelectron microscopic localization

Abstract Nitrogenase (Fe-protein) was localized in the free-living cyanobacterium Anabaena cylindrica and in the cyanobionts of Cycas revoluta and Peltigera aphthosa , using colloidal gold as an immunocytochemical marker. The Fe-protein was found to be evenly distributed throughout the heterocyst cytoplasma in A. cylindrica and in both the cyanobionts, including multiple heterocysts of the C. revoluta cyanobiont. No label was observed in the vegetative cells of free-living A. cylindrica or of the cyanobionts, although the cyanobionts apparently live under microaerobic conditions.     

Identification of Propionate as an Endogenous CO2 Acceptor in Rhodospirillum rubrum and Properties of Purified Propionyl-Coenzyme A Carboxylase

A heat-stable endogenous CO(2) acceptor has been found in extracts of Rhodospirillum rubrum grown photoheterotrophically on acetate. Evidence is presented which suggests that this factor is propionic acid. Thus, paper and gas chromatographic analyses have indicated that propionic acid is present in boiled extracts prepared from R. rubrum cells. The products of (14)CO(2) fixation obtained with either the boiled extract or propionic acid as the CO(2) acceptor were identical and were identified as methylmalonic acid and succinic acid by paper chromatography. The enzyme which catalyzes the carboxylation of propionyl-coenzyme A (propionyl-CoA carboxylase) was purified from R. rubrum cells grown on acetate and its properties were studied. The enzyme is similar to propionyl-CoA carboxylases isolated from mammalian sources.     

Change in subunit composition of the iron protein of nitrogenase from Rhodospirillum rubrum during activation and inactivation of iron protein.

The subunit composition of the Fe protein of nitrogenase from Rhodospirillum rubrum during activation and inactivation was investigated. It was found that the upper subunit (on gel electrophoresis) of the two-subunit Fe protein was converted into the lower subunit during activation in vitro. When the Fe protein was inactivated in vivo by the addition of NH4Cl and alpha-oxoglutarate to the cells, a phosphate-labelled upper band appeared. During activation in vitro by the activating enzyme, some of the phosphate of the upper band remained with the protein and appeared in the lower band. Activations in vitro were performed on inactive Fe protein obtained from cells grown with glutamate as the nitrogen source. Both native and oxygen-denatured Fe protein exhibited the loss of upper band during treatment with activating enzyme.     

Identification of a second site compensatory mutation in the Fe-protein that allows diazotrophic growth of Azotobacter vinelandii UW97

Azotobacter vinelandii UW97 is defective in nitrogen fixation due to a replacement of serine at position 44 by phenylalanine in the Fe-protein [Pulakat, L., Hausman, B.S., Lei, S. and Gavini, N. (1996) J. Biol. Chem. 271, 1884-1889]. Serine residue 44 is located in a conserved domain that links the nucleotide binding site and the MoFe-protein docking surface of the Fe-protein. Therefore, it is possible that the loss of function by A. vinelandii UW97-Fe-protein may be caused by global conformational disruption or disruption of the conformational change upon MgATP binding. To determine whether it is possible to generate a functional nitrogenase complex via a compensating second site mutation(s) in the Fe-protein, we have attempted to isolate genetic revertants of A. vinelandii UW97 that can grow on nitrogen-free medium. One such revertant, designated A vinelandii BG9, encoded a Fe-protein that retained the Ser44Phe mutation and also had a second mutation that caused the replacement of a lysine at position 170 by glutamic acid. Lysine 170 is highly conserved and is located in a conserved region of the Fe-protein. This region is implicated in stabilizing the MgATP-induced conformation of the Fe-protein and in docking to the MoFe-protein. Further complementation analysis showed that the Fe-protein mutant that retained serine 44 but contained the substitution of lysine at position 170 by glutamic acid was also non-functional. Thus, neither Ser44Phe nor Lys170Glu mutants of Fe-protein were functional; however, the Fe-protein in A. vinelandii BG9 that contained both substitutions could support diazotrophic growth on the strain.     

MgATP-Bound and nucleotide-free structures of a nitrogenase protein complex between the Leu 127 Delta-Fe-protein and the MoFe-protein

A mutant form of the nitrogenase iron protein with a deletion of residue Leu 127, located in the switch II region of the nucleotide binding site, forms a tight, inactive complex with the nitrogenase molybdenum iron (MoFe) protein in the absence of nucleotide. The structure of this complex generated with proteins from Azotobacter vinelandii (designated the L127Delta-Av2-Av1 complex) has been crystallographically determined in the absence of nucleotide at 2.2 A resolution and with bound MgATP (introduced by soaking) at 3.0 A resolution. As observed in the structure of the complex between the wild-type A. vinelandii nitrogenase proteins stabilized with ADP.AlF(4-), the most significant conformational changes in the L127Delta complex occur in the Fe-protein component. While the interactions at the interface between the MoFe-protein and Fe-proteins are conserved in the two complexes, significant differences are evident at the subunit-subunit interface of the dimeric Fe-proteins, with the L127Delta-Av2 structure having a more open conformation than the wild-type Av2 in the complex stabilized by ADP.AlF(4-). Addition of MgATP to the L127Delta-Av2-Av1 complex results in a further increase in the separation between Fe-protein subunits so that the structure more closely resembles that of the wild-type, nucleotide-free, uncomplexed Fe-protein, rather than the Fe-protein conformation in the ADP.AlF(4-) complex. The L127Delta mutation precludes key interactions between the Fe-protein and nucleotide, especially, but not exclusively, in the region corresponding to the switch II region of G-proteins, where the deletion constrains Gly 128 and Asp 129 from forming hydrogen bonds to the gamma-phosphate and activating water for attack on this group, respectively. These alterations account for the inability of this mutant to support mechanistically productive ATP hydrolysis. The ability of the L127Delta-Av2-Av1 complex to bind MgATP demonstrates that dissociation of the nitrogenase complex is not required for nucleotide binding.     

Structural basis for VO<Superscript>2+</Superscript>-inhibition of nitrogenase activity: (B) pH-sensitive inner-sphere rearrangements in the <Superscript>1</Superscript>H-environment of the metal coordination site of the nitrogenase Fe–protein identified by ENDOR spectroscopy

The nitrogenase Fe-protein is the specific ATP-activated electron donor to the active site-containing nitrogenase MoFe-protein. It has been previously demonstrated that different VO(2+)-nucleotide coordination environments exist for the Fe-protein that depend on pH and are distinguishable by EPR spectroscopy. After having studied the nitrogenase 31P and 23Na superhyperfine structure for this system by electron nuclear double resonance (ENDOR) spectroscopy (Petersen et al. 2008 in J Biol Inorg Chem. doi:10.1007/s00775-008-0360-0), we here report on the 1H-interactions with the nucleotide-bound metal center after substitution of the natural diamagnetic metal Mg2+ with paramagnetic oxo-vanadium(IV). ENDOR spectra show a number of resonances arising from interactions of the VO2+ ion with protons. In the presence of reduced Fe-protein and VO2+ ADP, at least three sets of nonexchangeable protons are detected. At low pH the superhyperfine couplings of most of these are consistent with proton interactions originating from the nucleotide. There is no indication of 1H-resonances that exchange in D2O at neutral pH and could be assigned to inner-sphere hydroxyl coordination. Exchangeable hydroxyl protons in the inner coordination sphere with reduced Fe-protein are only found in the low pH form; based on their hyperfine tensor components these have been assigned to an axially coordinated hydroxyl water molecule. The pH-dependent alterations of the proton couplings that exchange in D2O suggest that they are partially caused by a rearrangement in the local hydroxyl coordination environment of the metal center. These rearrangements especially affect the apical metal position, where an axially coordinated water present at low pH is absent at neutral pH. Oxidation of the Fe-protein induced substantial changes in the electron-nucleus interactions. This indicates that the oxidation state of the iron-sulfur cluster has an important effect on the metal coordination environment at the nucleotide binding site of the Fe-protein. The distinct VO(2+)-nucleotide coordination structures with ADP and ATP and the redox state of the [4Fe-4S] cluster imply that VO2+ has a critical influence on the switch regions of the regulatory protein, and, taken together, this provides a plausible explanation for the inhibitory action of VO2+.     

The surface membrane charge of bacteria and its role in serine proteinase secretion by Bacillus subtilis cells

Univalent, bivalent and trivalent metal cations increase the fluorescence yield of 9-aminoacridine in the suspensions of chromatophores of the purple nonsulfur bacterium Rhodospirillum rubrum isolated thylakoid membranes and cells of cyanobacterium Anabaena variabilis, cells Bacillus subtilis. The active cation concentrations increase about in 10 times with the decrease of their valency by one. It points to the fact that the changes in 9-aminoacridine fluorescence serve for the monitoring of the surface charge of bacterial membranes. The negative surface charge of B. subtilis cells increases before the onset of the serine protease secretion. The metal cations stimulate the serine protease secretion by B. subtilis cells, the stimulating effect correlates with the action of cations on the 9-aminoacridine fluorescence yield. It is suggested that the surface charge of cytoplasmic membrane regulates the formation and release of serine protease by the cells of B. subtilis.     

Activation of Mg-ATP hydrolysis in isolated Rhodospirillum rubrum H+-ATPase

The effects of lauryl dimethylamine oxide on the Rhodospirillum rubrum H+-ATPase have been studied. This detergent activates Mg2+-dependent ATP hydrolysis in the isolated R. rubrum F0-F1 34-fold, whereas the Ca2+-ATPase activity is only slightly modified. ATPase activation by lauryl dimethylamine oxide enhances the effect on ATP hydrolysis exerted by free Mg2+ ions. Concentrations of free Mg2+ in the range of 0.025 mM favor activation while higher concentrations inhibit ATPase activity by approximately 70%. Steady-state kinetic analysis shows that lauryl dimethylamine oxide induces a complex kinetic behavior for Mg-ATP in the chromatophores, similar to the untreated F0-F1 complex. The initial rate value for Mg-ATP unisite catalysis was found to be 6.3 times higher (3.5 X 10(-3) mol Pi per mol R. rubrum F0-F1 per second) in the presence than in the absence of detergent, where the initial rate was 5.5 X 10(-4) mol Pi per mol R. rubrum F0-F1 per second. These experiments show that lauryl dimethylamine oxide shifts the cation requirement for ATP-hydrolysis of the isolated R. rubrum H+-ATPase from Ca2+ to Mg2+ and that it activates both multisite and unisite catalysis. Results are discussed in relation to the possibility of a regulatory role by Mg2+ ions on ATP hydrolysis expressed through subunit interactions.     

Nitrogen Fixation by Rhodospirillum rubrum Grown in Nitrogen-limited Continuous Culture

Cell-free extracts of the photosynthetic bacterium Rhodospirillum rubrum were inconsistent in reducing N(2). An internally illuminated fermentor, designed for the continuous culture of this organism on N(2) under nitrogen-limited conditions, produced cells which yielded cell extracts with consistent activity for cell-free N(2) fixation. A nitrogen-limited continuous culture, supplied ammonia rather than N(2), gave cell-free extracts with even more active N(2) fixation. Extracts of cells grown in the fermentor with glutamate nitrogen as the limiting nutrient in continuous culture did not reduce N(2), but whole cells fixed (15)N-enriched N(2). The discovery that cells from ammonia and glutamate nitrogen-limited continuous cultures are capable of N(2) reduction suggests that R. rubrum cells produce the N(2)-reducing enzymes in response to conditions of nitrogen deficiency rather than in response to the presence of N(2). Examination of the effect of the pN(2) on N(2) reduction by cell-free preparations of R. rubrum indicated that the K(N(2)) is approximately 0.071 atm. Cell-free extracts from R. rubrum were tested for their ability to reduce substrates other than N(2).     

Fe:S cluster ligands are the only cysteines required for nitrogenase Fe-protein activities

Serine substitutions for the five conserved cysteins (residues 38, 85, 97, 132, and 184) have been made in the Azotobacter vinelandii nitrogenase Fe-protein by site-specific mutagenesis. At least moderate levels of enzyme activity (greater than 10% of wild type enzyme) were found for enzymes with serine substitutions at residues 38, 85, and 184; whereas, no activity was detected for enzymes with serines at residues 97 and 132. This is consistent with cysteines 97 and 132 being the four ligands to the Fe:S cluster (two ligands from each of the two identical subunits). Although previous chemical modification studies had implicated these residues as ligands, the earlier results did not portend the new finding that of all the conserved cysteines only these 2 residues are required for a second function of the Fe-protein. Namely, if either cysteine 97 or 132 is replaced, it appears that a functional Fe:S cluster cannot be incorporated into the apo-Fe-protein. The consequence is that these altered Fe-proteins cannot participate either in substrate reduction or in the biosynthesis of FeMo-cofactor, a metallocofactor of the MoFe-protein. These results implicate the Fe:S center of Fe-protein in the biosynthesis mechanism as either a redox partner or Fe:S donor. Additional results suggest that the posttranslational modification of Fe-protein by nifM product is not the insertion of the Fe:S center.     

Expression of the activating enzyme and Fe protein of nitrogenase from Rhodospirillum rubrum.

Activating enzyme (AE) is responsible for the in vitro activation of inactive Fe protein of nitrogenase from Rhodospirillum rubrum cells cultured anaerobically with glutamate as the N source. The expression of Fe protein and AE was examined in R. rubrum cultured photosynthetically or aerobically on media containing malate as the carbon source. One of the following N sources was used in each culture: glutamate, glutamine, limiting ammonia, high ammonia, glutamate plus histidine, and high ammonia plus histidine. Chromatophores from every culture exhibited AE activity; activity was highest in glutamate-grown cells. Fe protein was observed by rocket immunoelectrophoresis in cultures with nitrogenase activity. Several Nif-, Gln-, and His- mutants of R. rubrum were assayed for AE activity, nitrogenase activity, and Fe protein. Every mutant expressed AE activity, and Fe protein was observed in those cultures with nitrogenase activity. AE from every preparation was O2 labile, and each O2-denatured AE preparation inhibited activation by active AE.