Isolation and properties of the complex between the enhancer binding protein NIFA and the sensor NIFL.
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In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation. (+info)
Inhibition of iron-molybdenum cofactor biosynthesis by L127Delta NifH and evidence for a complex formation between L127Delta NifH and NifNE.
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Besides serving as the obligate electron donor to dinitrogenase during nitrogenase turnover, dinitrogenase reductase (NifH) is required for the biosynthesis of the iron-molybdenum cofactor (FeMo-co) and for the maturation of alpha(2)beta(2) apo-dinitrogenase (apo-dinitrogenase maturation). In an attempt to understand the role of NifH in FeMo-co biosynthesis, a site-specific altered form of NifH in which leucine at position 127 has been deleted, L127Delta, was employed in in vitro FeMo-co synthesis assays. This altered form of NifH has been shown to inhibit substrate reduction by the wild-type nitrogenase complex, forming a tight protein complex with dinitrogenase. The L127Delta NifH was found to inhibit in vitro FeMo-co synthesis by wild-type NifH as detected by the gamma gel shift assay. Increasing the concentration of NifNE and NifB-cofactor (NifB-co) relieved the inhibition of FeMo-co synthesis by L127Delta NifH. The formation of a complex of L127Delta NifH with NifNE was investigated by gel filtration chromatography. We herein report the formation of a complex between L127Delta NifH and NifNE in the presence of NifB-co. This work presents evidence for one of the possible roles for NifH in FeMo-co biosynthesis, i.e. the interaction of NifH with a NifNE.NifB-co complex. (+info)
Genetic analysis of nif regulatory genes by utilizing the yeast two-hybrid system detected formation of a NifL-NifA complex that is implicated in regulated expression of nif genes.
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In diazotrophic organisms, nitrogenase synthesis and activity are tightly regulated. Two genes, nifL and nifA, are implicated as playing a major role in this regulation. NifA is a transcriptional activator, and its activity is inhibited by NifL in response to availability of excess fixed nitrogen and high O(2) tension. It was postulated that NifL binds to NifA to inhibit NifA-mediated transcriptional activation of nif genes. Mutational analysis combined with transcriptional activation studies clearly is in agreement with the proposal that NifL interacts with NifA. However, several attempts to identify NifA-NifL interactions by using methods such as coimmunoprecipitations and chemical cross-linking experiments failed to detect direct interactions between these proteins. Here we have taken a genetic approach, the use of a yeast two-hybrid protein-protein interaction assay system, to investigate NifL interaction with NifA. A DNA fragment corresponding to the kinase-like domain of nifL was PCR amplified and was used to generate translation fusions with the DNA binding domain and the DNA activation domain of the yeast transcriptional activator GAL4 in yeast two-hybrid vectors. Similarly, a DNA fragment corresponding to the catalytic domain of nifA was PCR amplified and used to generate translation fusions with the DNA-binding domain and the DNA-activation domain of GAL4 in yeast two-hybrid vectors. After introducing appropriate plasmid combinations in yeast cells, the existance of direct interaction between NifA and NifL was analyzed with the MATCHMAKER yeast two-hybrid system by testing for the expression of lacZ and his3 genes. These analyses showed that the kinase-like domain of NifL directly interacts with the catalytic domain of NifA. (+info)
Pyruvate dehydrogenase from Azotobacter vinelandii. Properties of the N-terminally truncated enzyme.
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The pyruvate dehydrogenase multienzyme complex (PDHC) catalyses the oxidative decarboxylation of pyruvate and the subsequent acetylation of coenzyme A to acetyl-CoA. Previously, limited proteolysis experiments indicated that the N-terminal region of the homodimeric pyruvate dehydrogenase (E1p) from Azotobacter vinelandii could be involved in the binding of E1p to the core protein (E2p) [Hengeveld, A. F., Westphal, A. H. & de Kok, A. (1997) Eur J. Biochem. 250, 260-268]. To further investigate this hypothesis N-terminal deletion mutants of the E1p component of Azotobacter vinelandii pyruvate dehydrogenase complex were constructed and characterized. Up to nine N-terminal amino acids could be removed from E1p without effecting the properties of the enzyme. Truncation of up to 48 amino acids did not effect the expression or folding abilities of the enzyme, but the truncated enzymes could no longer interact with E2p. The 48 amino acid deletion mutant (E1pdelta48) is catalytically fully functional: it has a Vmax value identical to that of wild-type E1p, it can reductively acetylate the lipoamide group attached to the lipoyl domain of the core enzyme (E2p) and it forms a dimeric molecule. In contrast, the S0.5 for pyruvate is decreased. A heterodimer was constructed containing one subunit of wild-type E1p and one subunit of E1pdelta48. From the observation that the heterodimer was not able to bind to E2p, it is concluded that both N-terminal domains are needed for the binding of E1p to E2p. The interactions are thought to be mainly of an electrostatic nature involving negatively charged residues on the N-terminal domains of E1p and previously identified positively charged residues on the binding and catalytic domain of E2p. (+info)
In Azotobacter vinelandii, the E1 subunit of the pyruvate dehydrogenase complex binds fpr promoter region DNA and ferredoxin I.
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In Azotobacter vinelandii, deletion of the fdxA gene that encodes a well characterized seven-iron ferredoxin (FdI) is known to lead to overexpression of the FdI redox partner, NADPH:ferredoxin reductase (FPR). Previous studies have established that this is an oxidative stress response in which the fpr gene is transcriptionally activated to the same extent in response to either addition of the superoxide propagator paraquat to the cells or to fdxA deletion. In both cases, the activation occurs through a specific DNA sequence located upstream of the fpr gene. Here, we report the identification of the A. vinelandii protein that binds specifically to the paraquat activatable fpr promoter region as the E1 subunit of the pyruvate dehydrogenase complex (PDHE1), a central enzyme in aerobic respiration. Sequence analysis shows that PDHE1, which was not previously suspected to be a DNA-binding protein, has a helix-turn-helix motif. The data presented here further show that FdI binds specifically to the DNA-bound PDHE1. (+info)
Purification and biophysical characterization of a new [2Fe-2S] ferredoxin from Azotobacter vinelandii, a putative [Fe-S] cluster assembly/repair protein.
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During the purification of site-directed mutant variants of Azotobacter vinelandii ferredoxin I (FdI), a pink protein, which was not observed in native FdI preparations, appeared to associate specifically with variants that had mutations in ligands to FdI [Fe-S] clusters. That protein, which we designate FdIV, has now been purified. NH(2)-terminal sequence analysis revealed that the protein is the product of a previously described gene, herein designated fdxD, that is in the A. vinelandii iscSUA operon that encodes proteins involved in iron-sulfur cluster assembly or repair. An apoprotein molecular mass of 12,434.03 +/- 0.21 Da was determined by mass spectrometry consistent with the known gene sequence. The monomeric protein was shown to contain a single [2Fe-2S](2+/+) cluster by UV/visible, CD, and EPR spectroscopies with a reduction potential of -344 mV versus the standard hydrogen electrode. When overexpressed in Escherichia coli, recombinant FdIV holoprotein was successfully assembled. However, the polypeptide of the recombinant protein was modified in some way such that the apoprotein molecular mass increased by 52 Da. Antibodies raised against FdIV and EPR spectroscopy were used to examine the relative levels of FdIV and FdI in various A. vinelandii strains leading to the conclusion that FdIV levels appear to be specifically increased under conditions where another protein, NADPH:ferredoxin reductase is also up-regulated. In that case, the fpr gene is known to be activated in response to oxidative stress. This suggests that the fdxD gene and other genes in the iron-sulfur cluster assembly or repair operon might be similarly up-regulated in response to oxidative stress. (+info)
The basis of ammonium release in nifL mutants of Azotobacter vinelandii.
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In Azotobacter vinelandii, nitrogen fixation is regulated at the transcriptional level by an unusual two-component system encoded by nifLA. Certain mutations in nifL result in the bacterium releasing large quantities of ammonium into the medium, and earlier work suggested that this occurs by a mechanism that does not involve NifA, the activator of nif gene transcription. We have investigated a number of possible alternative mechanisms and find no evidence for their involvement in ammonium release. Enhancement of NifA-mediated transcription, on the other hand, by either elimination of nifL or overexpression of nifA, resulted in ammonium release, correlating with enhanced levels of nifH mRNA, raised levels of nitrogenase and acetylene-reducing activity, and increased concentrations of intracellular ammonium. Up to 35 mM ammonium can accumulate in the medium. Where measured, intracellular levels exceeded extracellular levels, indicating that rather than being actively transported, ammonium is lost from the cell passively, possibly by reversal of an NH(4)(+) uptake system. The data also indicate that in the wild type the bulk of NifA is inactivated by NifL during steady-state growth on dinitrogen. (+info)
Alteration of the reduction potential of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I.
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The [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E(0')) relative to other structurally similar ferredoxins. Previous attempts to raise that E(0') by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter the E(0') by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E(0') measurements show that all had altered E(0') relative to native FdI. For P21G FdI and I40Q FdI, the E(0') increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control of E(0'). Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E(0') while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a +168 mV change in E(0') while a -33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 A, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E(0'). Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change in E(0'). (+info)