Posttranslational regulation of nitrogenase in Rhodobacter capsulatus: existence of two independent regulatory effects of ammonium.

In the photosynthetic bacterium Rhodobacter capsulatus, nitrogenase activity is regulated by ADP-ribosylation of component II in response to the addition of ammonium to cultures or to the removal of light. The ammonium stimulus results in a fast and almost complete inhibition of the in vivo acetylene reduction activity, termed switch-off, which is reversed after the ammonium is exhausted. In the present study of the response of cells to ammonium, ADP-ribosylation of component II occurred but could not account for the extent and timing of the inhibition of activity. The presence of an additional response was confirmed with strains expressing mutant component II proteins; although these proteins are not a substrate for ADP-ribosylation, the strains continued to exhibit a switch-off response to ammonium. This second regulatory response of nitrogenase to ammonium was found to be synchronous with ADP-ribosylation and was responsible for the bulk of the observed effects on nitrogenase activity. In comparison, ADP-ribosylation in R. capsulatus was found to be relatively slow and incomplete but responded independently to both known stimuli, darkness and ammonium. Based on the in vitro nitrogenase activity of both the wild type and strains whose component II proteins cannot be ADP-ribosylated, it seems likely that the second response blocks either the ATP or the electron supply to nitrogenase.     

Mutagenesis studies of the FeSII protein of Azotobacter vinelandii: roles of histidine and lysine residues in the protection of nitrogenase from oxygen damage

The Azotobacter FeSII protein, also known as the Shethna protein, forms a protective complex with nitrogenase during periods when nitrogenase is exposed to oxygen. One possible mechanism for its action is an oxidation state-dependent conformational interaction with nitrogenase whereby the FeSII protein dissociates from the MoFe and Fe proteins of nitrogenase under reducing conditions. Herein we report the construction and characterization of five site-directed mutants of the FeSII protein (H12Q, H55Q, K14A, K15A, and the double mutant K14A/K15A) which were individually purified after being individually overexpressed in Escherichia coli. These mutant FeSII proteins maintain native-like assembly and orientation of the 2Fe-2S center on the basis of EPR and NMR spectroscopic characterization and their redox midpoint potentials, which are within 25 mV of that of the wild type protein. The abilities of the individual mutant proteins to protect nitrogenase were assessed by determining the remaining nitrogenase activities after adding each pure version back to extracts from an FeSII deletion strain, and then exposing the mixture to oxygen. In these assays, the H12Q mutant functioned as well as the wild type protein. However, mutation of His55, a few residues away from a cluster-liganding cysteine, results in much less efficient protection of nitrogenase. These results are consistent with pH titrations in both oxidation states, which show that His12 is insensitive to 2Fe-2S cluster oxidation state. His55's pK is weakly responsive to oxidation state, and the pK increase of 0. 16 pH unit upon 2Fe-2S cluster oxidation is indicative of ionization of another group between His55 and the 2Fe-2S cluster, which could modulate the FeSII protein's affinity for nitrogenase in a redox state-dependent manner. Both K14A and K15A mutant FeSII proteins partially lost their ability to protect nitrogenase, but the lysine double mutant lost almost all its protective ability. The nitrogenase component proteins in an Azotobacter strain bearing the double lysine mutation (in the chromosome) were degraded much more rapidly in vivo than those in the wild type strain under carbon substrate-limited conditions. These results indicate that the two lysines may have an important role in FeSII function, perhaps in the initial steps of recognizing the nitrogenase component proteins.     

Effect of nitrogenase compounds from mutant and wild-type strains of Azotobacter on the dilution effect of nitrogenase

Nitrogenase activity exhibits a dilution effect. Evidence is presented that the reason for the dilution effect is that one of the component proteins of nitrogenase is limiting in preparations of this enzyme. The limiting component appears to be the non-haem-iron-containing protein (also called fraction II, iron protein, azoferredoxin), which is equivalent to the enhancement factor for nitrogenase activity present in extracts of nitrogenaseless mutant 22R1. A mathematical function of specific activity is described that is useful in describing nitrogenase. It takes into account the dilution effect and the exponential nature of the relationship between nitrogenase activity and enzyme protein concentration.     

Nitrogenase in the archaebacterium Methanosarcina barkeri 227.

The discovery of nitrogen fixation in the archaebacterium Methanosarcina barkeri 227 raises questions concerning the similarity of archaebacterial nitrogenases to Mo and alternative nitrogenases in eubacteria. A scheme for achieving a 20- to 40-fold partial purification of nitrogenase components from strain 227 was developed by using protamine sulfate precipitation, followed by using a fast protein liquid chromatography apparatus operated inside an anaerobic glove box. As in eubacteria, the nitrogenase activity was resolved into two components. The component 1 analog had a molecular size of approximately 250 kDa, as estimated by gel filtration, and sodium dodecyl sulfate-polyacrylamide gels revealed two predominant bands with molecular sizes near 57 and 62 kDa, consistent with an alpha 2 beta 2 tetramer as in eubacterial component 1 proteins. For the component 2 analog, a molecular size of approximately 120 kDa was estimated by gel filtration, with a subunit molecular size near 31 kDa, indicating that the component 2 protein is a tetramer, in contrast to eubacterial component 2 proteins, which are dimers. Rates of C2H2 reduction by the nearly pure subunits were 1,000 nmol h-1 mg of protein-1, considerably lower than those for conventional Mo nitrogenases but similar to that of the non-Mo non-V nitrogenase from Azotobacter vinelandii. Strain 227 nitrogenase reduced N2 at a higher rate per electron than it reduced C2H2, also resembling the non-Mo non-V nitrogenase of A. vinelandii. Ethane was not produced from C2H2. NH4+ concentrations as low as 10 microM caused a transient inhibition of C2H2 reduction by strain 227 cells. Antiserum against component 2 Rhodospirillum rubrum nitrogenase was found to cross-react with component 2 from strain 227, and Western immunoblots using this antiserum showed no evidence for covalent modification of component 2. Also, extracts of strain 227 cells prepared before and after switch-off had virtually the same level of nitrogenase activity. In conclusion, the nitrogenase from strain 227 is similar in overall structure to the eubacterial nitrogenases and shows greatest similarity to alternative nitrogenases.     

Complementary functioning of the component proteins of nitrogenase from several bacteria

The nitrogenase proteins from eight organisms have been highly purified, and a survey of their cross-reactions shows that the nitrogenase proteins from a wide variety of organisms can interact with one another. An active cross-reaction is the complementary functioning of the MoFe protein and the Fe protein from different organisms. Of 64 possible combinations of component proteins, 8 yielded homologous nitrogenases (components from the same organism); 45 of the 56 possible heterologous crosses generated active hybrid nitrogenases; 4 heterologous crosses yielded no measurable nitrogenase activity but did form inactive tight-binding complexes; 6 crosses did not give measurable activity; and 1 cross was not made. All these crosses were assayed for acetylene reduction, and some also were assayed for ammonia formation, hydrogen evolution, and ATP hydrolysis activity. The activity generated by combining two complementary heterologous nitrogenase components depended on pH, component ratio, and protein concentration, the same factors that determine the activity of homologous nitrogenases. However, several crosses showed an unusual dependency on component ratio and protein concentration, and some cross-reactions showed interesting ATP hydrolysis activity.     

Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity.

Adenine nucleotide pools were measured in Rhodospirillum rubrum cultures that contained nitrogenase. The average energy charge [([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP])] was found to be 0.66 and 0.62 in glutamate-grown and N-limited cultures respectively. Treatment of glutamate-grown cells with darkness, ammonia, glutamine, carbonyl cyanide m-chlorophenylhydrazone, or phenazine methosulphate resulted in perturbations in the adenine nucleotide pools, and led to loss of whole-cell nitrogenase activity and modification in vivo of the Fe protein. Treatment of N-limited cells resulted in similar changes in adenine nucleotide pools but not enzyme modification. No correlations were found between changes in adenine nucleotide pools or ratios of these pools and switch-off of nitrogenase activity by Fe protein modification in vivo. Phenazine methosulphate inhibited whole-cell activity at low concentrations. The effect on nitrogenase activity was apparently independent of Fe protein modification.     

Nif- Hup- mutants of Rhizobium japonicum.

Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity.     

Regulation of nitrogen fixation (nif) genes of Azospirillum brasilense by nifA and ntr (gln) type gene products

Abstract Eight Nif⁻ mutants of Azospirillum brasilense were obtained by N -nitrosoguanidine mutagenesis and isolated by growth on glutamate medium. Three of these mutants had no nitrogenase activity, possessed no nitrogenase structural proteins and were complemented by Klebsiella pneumoniae nifA . Evidence will be presented that one of these mutants is defective in a nifA type regulatory gene but the other two were also complemented by K. pneumoniae ntrC and may be ntrC⁻ -type mutants. A fourth mutant was defective in the MoFe component protein of nitrogenase.     

Docking of nitrogenase iron- and molybdenum-iron proteins for electron transfer and MgATP hydrolysis: the role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein.

Docking of the nitrogenase component proteins, the iron protein (FeP) and the molybdenum-iron protein (MoFeP), is required for MgATP hydrolysis, electron transfer between the component proteins, and substrate reductions catalyzed by nitrogenase. The present work examines the function of 3 charged amino acids, Arg 140, Glu 141, and Lys 143, of the Azotobacter vinelandii FeP in nitrogenase component protein docking. The function of these amino acids was probed by changing each to the neutral amino acid glutamine using site-directed mutagenesis. The altered FePs were expressed in A. vinelandii in place of the wild-type FeP. Changing Glu 141 to Gln (E141Q) had no adverse effects on the function of nitrogenase in whole cells, indicating that this charged residue is not essential to nitrogenase function. In contrast, changing Arg 140 or Lys 143 to Gln (R140Q and K143Q) resulted in a significant decrease in nitrogenase activity, suggesting that these charged amino acid residues play an important role in some function of the FeP. The function of each amino acid was deduced by analysis of the properties of the purified R140Q and K143Q FePs. Both altered proteins were found to support reduced substrate reduction rates when coupled to wild-type MoFeP. Detailed analysis revealed that changing these residues to Gln resulted in a dramatic reduction in the affinity of the altered FeP for binding to the MoFeP. This was deduced in FeP titration, NaCl inhibition, and MoFeP protection from Fe2+ chelation experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the nifQ gene product in the incorporation of molybdenum into nitrogenase in Klebsiella pneumoniae

NifQ- mutants of Klebsiella pneumoniae are defective in nitrogen fixation due to an elevated requirement for molybdenum. When millimolar concentrations of molybdate were added to the medium, the effects of the nifQ mutations were suppressed. NifQ- mutants were not impaired in the uptake of molybdate, but molybdate accumulation was defective in these mutants. All of the nif-coded proteins were present in NifQ- cells derepressed in the absence of molybdenum. Molybdenum-activatable nitrogenase component I was found at the same level observed in the wild type. Molybdenum, thus, does not play a role in nif expression or in the short-term stability of nif-coded proteins. The defect in NifQ- mutants was in the incorporation of molybdenum into nitrogenase component I. The nifQ gene product acts together with the products of nifB, nifN, and nifE in the biosynthesis of the iron-molybdenum cofactor of nitrogenase.     

Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs.

A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.     

Stimulation by light of nitrogenase synthesis in cells of Rhodopseudomonas capsulata growing in N-limited continuous cultures

In cells of Rhodopseudomonas capsulata growing in nitrogen-limited continuous culture the nitrogenase-specific activity was found to be closely dependent on the light intensity. As light intensity, measured with a photodiode immersed in the culture, was varied stepwise from 1000 to 7000 lux, the nitrogenase activity, measured at steady state, increased gradually up to 5-fold. Shifting light intensity from 1200 to 7000 lux resulted in a sharp rise in nitrogenase activity which doubled within the first two hours. The determination by immunoassays of the intracellular levels of each nitrogenase component revealed that the light-dependent stimulation of nitrogenase activity was correlated with the accumulation of the nitrogenase enzyme inside the cells. Under high illumination, nitrogenase represented up to 40% of the cytoplasmic proteins. The specific activities of each component in intact cells, calculated on the basis of their relative concentration in the cells and on in vivo nitrogenase assays, appeared roughly constant and hardly affected by changes of light intensity. The specific activity of the Fe protein was about 7-fold higher in intact cells than in the purified state. The ratio of the two nitrogenase components remained fairly constant and close to one, irrespective of the light intensity to which cells were exposed. These results demonstrate that in nitrogen-limited grown cells of Rps. capsulata light brings about an induction or a derepression of nitrogenase synthesis the extent of which is dependent on light intensity.     

Two Forms of Nitrogenase from the Photosynthetic Bacterium Rhodospirillum rubrum

Acetylene reduction by nitrogenase from Rhodospirillum rubrum, unlike that by other nitrogenases, was recently found by other investigators to require an activation of the iron protein of nitrogenase by an activating system comprising a chromatophore membrane component, adenosine 5'-triphosphate (ATP), and divalent metal ions. In an extension of this work, we observed that the same activating system was also required for nitrogenase-linked H(2) evolution. However, we found that, depending on their nitrogen nutrition regime, R. rubrum cells produced two forms of nitrogenase that differed in their Fe protein components. Cells whose nitrogen supply was totally exhausted before harvest yielded predominantly a form of nitrogenase (A) whose enzymatic activity was not governed by the activating system, whereas cells supplied up to harvest time with N(2) or glutamate yielded predominantly a form of nitrogenase (R) whose enzymatic activity was regulated by the activating system. An unexpected finding was the rapid (less than 10 min in some cases) intracellular conversion of nitrogenase A to nitrogenase R brought about by the addition to nitrogen-starved cells of glutamine, asparagine, or, particularly, ammonia. This finding suggests that mechanisms other than de novo protein synthesis were involved in the conversion of nitrogenase A to the R form. The molecular weights of the Fe protein and Mo-Fe protein components from nitrogenases A and R were the same. However, nitrogenase A appeared to be larger in size, because it had more Fe protein units per Mo-Fe protein than did nitrogenase R. A distinguishing property of the Fe protein from nitrogenase R was its ATP requirement. When combined with the Mo-Fe protein (from either nitrogenase A or nitrogenase R), the R form of Fe protein required a lower ATP concentration but bound or utilized more ATP molecules during acetylene reduction than did the A form of Fe protein. No differences between the Fe proteins from the two forms of nitrogenase were found in the electron paramagnetic resonance spectrum, midpoint oxidation-reduction potential, or sensitivity to iron chelators.     

Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases.

The nucleotide sequences of the Acinetobacter calcoaceticus benABC genes encoding a multicomponent oxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate 1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfer component, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with related sequences, including those for the multicomponent toluate, toluene, benzene, and naphthalene 1,2-dioxygenases, indicated that the similarly sized subunits of the hydroxylase components were derived from a common ancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the alpha-subunits of all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(II) ion. The less conserved beta-subunits of the hydroxylases may be responsible for determining substrate specificity. Each dioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoate dioxygenase, encoded by benC, and the corresponding protein of the toluate 1,2-dioxygenase, encoded by xylZ, were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminal region which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certain monooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfer systems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adenine dinucleotide binding were identified.     

Nitrogenase of Klebsiella pneumoniae. Interaction of the component proteins studied by ultracentrifugation

Sedimentation-velocity analyses of mixtures of the component proteins of nitrogenase of Klebsiella pneumoniae at a 1:1 molar ratio, showed a single peak of sedimentation coefficient (12.4S) considerably greater than that obtained for the larger (Fe+Mo-containing) protein centrifuged alone (10.4S). When the ratio exceeded 1:1 (the smaller Fe-containing protein in excess) an additional peak corresponding in sedimentation coefficient (about 4.5S) to free Fe-containing protein appeared. When proteins, which had been inactivated by exposure to air were used, no interaction occurred. Na(2)S(2)O(4) at 2mm both reversed and prevented interaction between the two proteins; sedimentation coefficients corresponded to those of the proteins when centrifuged alone. These results demonstrate the formation of a complex between the nitrogenase proteins, and, together with data of activity titration curves, are consistent with the formulation of the nitrogenase complex of K. pneumoniae as (Fe-containing protein)-(Fe+Mo-containing protein).     

Quinoprotein d-glucose dehydrogenase in Acinetobacter calcoaceticus LMD 79.41: the membrane-bound enzyme is distinct from the soluble enzyme

Abstract The hifH gene from Klebsiella pneumoniae , which codes for the Fe protein component of nitrogenase, was used as a probe to detect nifH homologues in total cellular DNA from 13 obligate methane oxidizing bacteria. All but one of those strains that had previously been shown capable of fixing dinitrogen contained sequences homologous to nifH . DNAs from three of the six non-diazotrophic strains were also found to possess such homology.     

Carotenoids provide the major antioxidant defence in the globally significant N2-fixing marine cyanobacterium Trichodesmium

Photosynthetic oxygen-evolving microorganisms contend with continuous self-production of molecular oxygen and reactive oxygen species. The deleterious effects of reactive oxygen species are exacerbated for cyanobacterial nitrogen-fixers (diazotrophs) due to the innate sensitivity of nitrogenase to oxygen. This renders incompatible the processes of oxygen-evolving photosynthesis and N-fixation. We examined total antioxidative potential of various diazotrophic and non-diazotrophic cyanobacteria. We focused on Trichodesmium spp., a bloom-forming marine diazotroph that contributes significantly to global nitrogen fixation. Among the species tested, Trichodesmium possessed the highest antioxidant activity. Moreover, while proteins constituted the dominant antioxidative component of all other cyanobacteria tested, Trichodesmium was unique in that small-molecule natural products provided the majority of antioxidant activity, while proteins constituted only 13% of total antioxidant activity. Bioassay-guided fractionation followed by high-performance liquid chromatography profiling of antioxidant purified fractions identified the highly potent antioxidant all- trans -β-carotene, and small amounts of 9- cis -β-carotene and retinyl palmitate. Search of the Trichodesmium genome identified protein sequences homologous to key enzymes in the β-carotene to retinyl palmitate biosynthetic pathway, including 33–37% identity to lecithin retinol acyltransferase. The present study demonstrates the importance of carotenoids in Trichodesmium 's arsenal of defensive compounds against oxidative damage and protection of nitrogenase from oxygen and its radicals.     

Nucleotide sequence of the gene coding for the nitrogenase iron protein from Klebsiella pneumoniae

We report the complete DNA sequence of the Klebsiella pneumoniae nifH gene, the gene which codes for component 2 (Fe protein or nitrogenase reductase) of the nitrogenase enzyme complex. The amino acid sequence of the K. pneumoniae nitrogenase Fe protein is deduced from the DNA sequence. The K. pneumoniae Fe protein contains 292 amino acids, has a Mr = 31,753, and contains 9 cysteine residues. We compare the amino acid sequence of the K. pneumoniae protein with available amino acid sequence data on nitrogenase Fe proteins from two other species, Clostridium pasteurianum and Azotobacter vinelandii. The C. pasteurianum Fe protein, for which the complete sequence is known, shows 67% homology with the K. pneumoniae Fe protein. Extensive regions of strong conservation (90-95%) are found, while other regions show relatively poor conservation (30-35%). It is suggested that these strongly conserved regions are of special importance to the function of this enzyme, and the findings are discussed in the light of evolutionary theories on the origin of nif genes.     

The nucleotide sequence of the uvrD gene of E. coli.

The nucleotide sequence of a cloned section of the E. coli chromosome containing the uvrD gene has been determined. The coding region for the UvrD protein consists of 2,160 nucleotides which would direct the synthesis of a polypeptide 720 amino acids long with a calculated molecular weight of 82 kd. The predicted amino acid sequence of the UvrD protein has been compared with the amino acid sequences of other known adenine nucleotide binding proteins and a common sequence has been identified, thought to contribute towards adenine nucleotide binding.     

Nitrogenase Inhibition in Nodules from Pea Plants Grown Under Salt Stress Occurs at the Physiological Level and can be Alleviated by B and Ca

In order to shed new light on the mechanisms of salt-mediated symbiotic N₂-fixation inhibition, the effect of salt stress (75 mM) on N₂-fixation in pea root nodules induced by R. leguminosarum was studied at the gene expression, protein production and enzymatic activity levels. Acetylene reduction assays for nitrogenase activity showed no activity in salt-stressed plants. To know whether salt inhibits N₂-fixing activity at a molecular or at a physiological level, expression of the nifH gene, encoding the nitrogenase reductase component of the nitrogenase enzyme was analyzed by RT-PCR analysis of total RNA extracted from nodulated roots. The nifH messenger RNA was present both in plants grown in the presence and absence of salt, although a reduction was observed in salt-stressed plants. Similar results were obtained for the immunodetection of the nitrogenase reductase protein in Western-blot assays, indicating that nitrogen fixation failed mainly at physiological level. Given that nutrient imbalance is a typical effect of salt stress in plants and that Fe is a prosthetic component of nitrogenase reductase and other proteins required by symbiotic N₂-fixation, as leghemoglobin, plants were analyzed for Fe contents by atomic absorption and the results confirmed that Fe levels were severely reduced in nodules developed in salt-stressed plants. In a previous papers (El-Hamdaoui et al., 2003b), we have shown that supplementing inoculated legumes with boron (B) and calcium (Ca) prevents nitrogen fixation decline under saline conditions stress. Analysis of salt-stressed nodules fed with extra B and Ca indicated that Fe content and nitrogenase activity was similar to that of non-stressed plants. These results indicate a linkage between Fe deprivation and salt-mediated failure of nitrogen fixation, which is prevented by B and Ca leading to increase of salt tolerance.