Dinitrogenase reductase- and MgATP-dependent maturation of apodinitrogenase from Azotobacter vinelandii.
(33/39)
The requirements for iron-molybdenum cofactor (FeMo-co) activation of apodinitrogenase from Azotobacter vinelandii strain UW97, which lacks dinitrogenase reductase activity as assayed by substrate reduction, have been examined. Activation of apodinitrogenase from strain UW97 by FeMo-co requires the addition of both dinitrogenase reductase and MgATP. When the same apodinitrogenase is pretreated with dinitrogenase reductase and MgATP and then partially purified, however, it does not require these components for activation by FeMo-co. This suggests that dinitrogenase reductase and MgATP are involved in processing apodinitrogenase to a FeMo-co activatable form. This processing step coincides with a change in the subunit composition of apodinitrogenase from alpha 2 beta 2 to a form with an additional subunit (gamma) attached. The apodinitrogenase with the associated gamma subunit is apparently the form of the protein that is competent for activation by FeMo-co. (+info)
Proteolytic degradation of dinitrogenase reductase from Anabaena variabilis (ATCC 29413) as a consequence of ATP depletion and impact of oxygen.
(34/39)
Both components of nitrogenase, dinitrogenase and dinitrogenase reductase, are rapidly inactivated by oxygen. To investigate the proteolytic degradation of dinitrogenase reductase irreversibly destroyed by high oxygen concentrations, we carried out in vitro experiments with heterocyst extracts from Anabaena variabilis ATCC 29413. The results indicate a direct dependence of degradation on the applied oxygen concentration. Although the degrees of degradation were similar for both the modified and unmodified subunits of dinitrogenase reductase, there was a significant difference with respect to the cleavage products observed. The pattern of effective protease inhibitors suggests the involvement of serine proteases with chymotrypsin- and trypsin-like specificity. A protective effect was obtained by saturation of the nucleotide binding sites of dinitrogenase reductase with either ATP or ADP. As shown by gel filtration experiments, the adenylates prevented the nitrogenase subunits from extensive noncovalent aggregation, which is usually considered evidence for a denaturing process. The in vitro degradation of dinitrogenase reductase is discussed in connection with previous reports on degradation of nitrogenase in cyanobacteria under oxygen stress and/or starvation. (+info)
Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium.
(35/39)
Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions. (+info)
Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.
(36/39)
The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent alpha2beta2gamma2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum- or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an alpha2 dimer containing an Fe4S4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H2 very well but reduces N2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane. (+info)
The role of NAD+ as a signal during nitrogenase switch-off in Rhodospirillum rubrum.
(37/39)
The role of NAD+ in the metabolic regulation of nitrogenase, the 'switch-off' effect, in Rhodospirillum rubrum has been studied. We now show that the decrease in nitrogenase activity upon addition of NAD+ to R. rubrum is due to modification of dinitrogenase reductase. There was no effect when NAD+ was added to a mutant of R. rubrum devoid of dinitrogenase reductase ADP-ribosyltransferase, indicating that NAD+ 'switch-off' is an effect of the same regulatory system as ammonium 'switch-off'. We also show that oxaloacetate and alpha-ketoglutarate function as 'switch-off' effectors. On the other hand beta-hydroxybutyrate has the opposite effect by shortening the 'switch-off' period. Furthermore, by using an inhibitor of glutamate synthase the role of this enzyme in 'switch-off' was investigated. The results are discussed in relation to our proposal that changes in the concentration of NAD+ are involved in initiating 'switch-off'. (+info)
NAD-dependent cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum.
(38/39)
Chemical cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum has been investigated with a cross-linking system utilizing two reagents, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and sulfo-N-hydroxysuccinimide. Cross-linking between dinitrogenase reductase and DRAT requires the presence of NAD, the cellular ADP-ribose donor, or a NAD analog containing an unmodified nicotinamide group, such as nicotinamide hypoxanthine dinucleotide. NADP, which will not replace NAD in the modification reaction, does support cross-linking between dinitrogenase reductase and DRAT. The DRAT-catalyzed ADP-ribosylation of dinitrogenase reductase is inhibited by sodium chloride, as is the cross-linking between dinitrogenase reductase and DRAT, suggesting that ionic interactions are required for the association of these two proteins. Cross-linking is specific for native, unmodified dinitrogenase reductase, in that both oxygen-denatured and ADP-ribosylated dinitrogenase reductase fail to form a cross-linked complex with DRAT. The ADP-bound and adenine nucleotide-free states of dinitrogenase reductase form cross-linked complexes with DRAT; however, cross-linking is inhibited when dinitrogenase reductase is in its ATP-bound state. (+info)
Pleiotropic effects of potassium deficiency in a heterocystous, nitrogen-fixing cyanobacterium, Anabaena torulosa.
(39/39)
Omission of potassium from the growth medium caused multiple metabolic impairments and resulted in cessation of growth of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anabaena torulosa, during both diazotrophic and nitrogen-supplemented growth. Prominent defects observed during potassium deprivation were: (i) the loss of photosynthetic pigments, (ii) impairment of photosynthetic functions, (iii) reduced synthesis of dinitrogenase reductase (Fe-protein), (iv) inhibition of nitrogenase activity, and (v) specific qualitative modifications of protein synthesis leading to the repression of twelve polypeptides and synthesis and accumulation of nine novel polypeptides. The observed metabolic defects were reversible, and growth arrested under prolonged potassium deficiency was fully restored upon re-addition of potassium. Such pleiotropic effects of potassium deficiency demonstrate that apart from its well-known requirement for pH and turgor homeostasis, K+ plays other vital specific roles in cyanobacterial growth and metabolism. (+info)