Analysis of steady state Fe and MoFe protein interactions during nitrogenase catalysis.
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Steady state kinetic measurements are reported for nitrogenase from Azotobacter vinelandii (Av) and Clostridium pasteurianum (Cp) under a variety of conditions, using dithionite as reductant. The specific activities of Av1 and Cp1 are determined as functions of Av2:Av1 and Cp2:Cp1, respectively, at component protein ratios from 0.4 to 50, and conform to a simple hyperbolic rate law for the interaction of Av2 with Av1 and Cp2 with Cp1. The specific activities of Av2 and Cp2 are also measured as a function of increasing Av1 and Cp1 concentrations, producing 'MoFe inhibition' at large MoFe:Fe ratios. When the rate of product formation under MoFe inhibited conditions is re-plotted as increasing Av2:Av1 or Cp2:Cp1 ratios, sigmoidal kinetic behavior is observed, suggesting that the rate constants in the Thorneley and Lowe (T&L) model are more dependent upon the oxidation level of MoFe protein than previously suspected [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 224 (1984) 887-894], at least when applied to Av and Cp. Calculation of Hill coefficients gave values of 1.7-1.9, suggesting a highly cooperative Fe-MoFe protein interaction in both Av and Cp nitrogenase catalysis. The T&L model lacks analytical solutions [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 215 (1983) 393-404], so the ease of its application to experimental data is limited. To facilitate the study of steady state kinetic data for H(2) evolution, analytical equations are derived from a different mechanism for nitrogenase activity, similar to that of Bergersen and Turner [Biochem. J. 131 (1973) 61-75]. This alternative cooperative model assumes that two Fe proteins interact with one MoFe protein active site. The derived rate laws for this mechanism were fitted to the observed sigmoidal behavior for low Fe:MoFe ratios (<0.4), as well as to the commonly observed hyperbolic behavior for high values of Fe:MoFe for both Av and Cp. (+info)
Mechanistic interpretation of the dilution effect for Azotobacter vinelandii and Clostridium pasteurianum nitrogenase catalysis.
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Nitrogenase activity for Clostridium pasteurianum (Cp) at a Cp2:Cp1 ratio of 1.0 and Azotobacter vinelandii (Av) at Av2:Av1 protein ratios (R) of 1, 4 and 10 is determined as a function of increasing MoFe protein concentration from 0.01 to 5 microM. The rates of ethylene and hydrogen evolution for these ratios and concentrations were measured to determine the effect of extreme dilution on nitrogenase activity. The experimental results show three distinct types of kinetic behavior: (1) a finite intercept along the concentration axis (approximately 0.05 microM MoFe); (2) a non-linear increase in the rate of product formation with increasing protein concentration (approximately 0.2 microM MoFe) and (3) a limiting linear rate of product formation at high protein concentrations (>0.4 microM MoFe). The data are fitted using the following rate equation derived from a mechanism for which two Fe proteins interact cooperatively with a single half of the MoFe protein. (see equation) The equation predicts that the cubic dependence in MoFe protein gives rise to the non-linear rate of product formation (the dilution effect) at very low MoFe protein concentrations. The equation also predicts that the rate will vary linearly at high MoFe protein concentrations with increasing MoFe protein concentration. That these limiting predictions are in accord with the experimental results suggests that either two Fe proteins interact cooperatively with a single half of the MoFe protein, or that the rate constants in the Thorneley and Lowe model are more dependent upon the redox state of MoFe protein than previously suspected [R.N. Thornley and D. J. Lowe, Biochem. J. 224 (1984) 887-894]. Previous Klebsiella pneumoniae and Azotobacter chroococcum dilution results were reanalyzed using the above equation. Results from all of these nitrogenases are consistent and suggest that cooperativity is a fundamental kinetic aspect of nitrogenase catalysis. (+info)
Differential regulation of fixN-reiterated genes in Rhizobium etli by a novel fixL-fixK cascade.
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Among the complexities in the regulation of nitrogen fixation in the Rhizobiaceae are reiteration of regulatory components as well as variant roles for each component between species. For Rhizobium etli CFN42, we reported that the symbiotic plasmid (pCFN42d) contains a key regulatory gene (fixKd) and genes for a symbiotic cytochrome oxidase (fixNOQPd). Here we discuss the occurrence of reiteration of these genes (fixKf and fixNOQPf) and the finding of an unusual fixL homolog on a plasmid previously considered cryptic (pCFN42f). The structure of the deduced FixL polypeptide is suggestive of a fusion of the receiver and transmitter modules of a two-component regulatory system as described in R. leguminosarum bv. viciae VF39. Gene fusion analysis, coupled with mutation of each regulatory element, revealed that free-living expression of FixKf was dependent fully on FixL. In contrast, synthesis of FixKd was not detected under the conditions tested. The FixKf protein is needed for microaerobic expression of both fixN reiterations, whereas the FixKd protein appears to be dispensable. Interestingly, expression of the fixN reiterations exhibits a differential dependence for FixL, where transcription of fixNf was suppressed in the absence of FixL but expression of fixNd still showed significant levels. This suggests the existence of a FixL-independent mechanism for expression of the fixNd reiteration. Surprisingly, mutations in fixL, fixKd, or fixKf (either singly or in combination) did not alter symbiotic effectiveness. A mutation in fixNd (but not in fixNf) was, however, severely affected, indicating a differential role for these reiterations in nitrogen fixation. (+info)
Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413.
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Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a nif1 mutant; however, that expression did not prevent differentiation of heterocysts and expression of the nif1 nitrogenase in either the nif1 mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells. (+info)
Effect of P(II) and its homolog GlnK on reversible ADP-ribosylation of dinitrogenase reductase by heterologous expression of the Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase regulatory system in Klebsiella pneumoniae.
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Reversible ADP-ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase (DRAT-DRAG) regulatory system, has been characterized in Rhodospirillum rubrum and other nitrogen-fixing bacteria. To investigate the mechanisms for the regulation of DRAT and DRAG activities, we studied the heterologous expression of R. rubrum draTG in Klebsiella pneumoniae glnB and glnK mutants. In K. pneumoniae wild type, the regulation of both DRAT and DRAG activity appears to be comparable to that seen in R. rubrum. However, the regulation of both DRAT and DRAG activities is altered in a glnB background. Some DRAT escapes regulation and becomes active under N-limiting conditions. The regulation of DRAG activity is also altered in a glnB mutant, with DRAG being inactivated more slowly in response to NH4+ treatment than is seen in wild type, resulting in a high residual nitrogenase activity. In a glnK background, the regulation of DRAT activity is similar to that seen in wild type. However, the regulation of DRAG activity is completely abolished in the glnK mutant; DRAG remains active even after NH4+ addition, so there is no loss of nitrogenase activity. The results with this heterologous expression system have implications for DRAT-DRAG regulation in R. rubrum. (+info)
Isolation and characterization of draT mutants that have altered regulatory properties of dinitrogenase reductase ADP-ribosyltransferase in Rhodospirillum rubrum.
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In Rhodospirillum rubrum, dinitrogenase reductase ADP-ribosyltransferase (DRAT) is responsible for the ADP-ribosylation of dinitrogenase reductase in response to the addition of NH(+)(4) or removal from light, resulting in a decrease in nitrogenase activity. DRAT is itself subject to post-translational regulation; to investigate the mechanism for the regulation of DRAT activity, random PCR mutagenesis of draT (encoding DRAT) was performed and mutants with altered DRAT regulation were screened. Two mutants (with substitutions of K103E and N248D) were obtained in which DRAT showed activity under conditions where wild-type DRAT (DRAT-WT) did not. These mutants showed lower nitrogenase activity and a higher degree of ADP-ribosylation of dinitrogenase reductase under N(2)-fixing conditions than was seen in a wild-type control strain. DRAT-K103E was overexpressed and purified. DRAT-K103E displayed a much weaker affinity for an Affi-gel Blue matrix than did DRAT-WT, suggestive of a fairly striking biochemical change. However, there was no significant difference in kinetic constants, such as K(m) for NAD and V(max), between DRAT-K103E and DRAT-WT. Like DRAT-WT, DRAT-K103E also modified reduced dinitrogenase reductase poorly. The biochemical properties of these variants are rationalized with respect to their behaviour in vivo. (+info)
Transition state complexes of the Klebsiella pneumoniae nitrogenase proteins. Spectroscopic properties of aluminium fluoride-stabilized and beryllium fluoride-stabilized MgADP complexes reveal conformational differences of the Fe protein.
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Stable inactive 2 : 1 complexes of the Klebsiella pneumoniae nitrogenase components (Kp2/Kp1) were prepared with ADP or the fluorescent ADP analogue, 2'(3')-O-[N-methylanthraniloyl] ADP and AlF(4)(-) or BeF(3)(-) ions. By analogy with published crystallographic data [Schindelin et al. (1997) Nature 387, 370-376)], we suggest that the metal fluoride ions replaced phosphate at the two ATP-binding sites of the iron protein, Kp2. The beryllium (BeF(x)) and aluminium (AlF(4)(-)) containing complexes are proposed to correspond to the ATP-bound state and the hydrolytic transition states, respectively, by analogy with the equivalent complexes of myosin [Fisher et al. (1995) Biochemistry 34, 8960-8972]. (31)P NMR spectroscopy showed that during the initial stages of complex formation, MgADP bound to the complexed Kp2 in a manner similar to that reported for isolated Kp2. This process was followed by a second step that caused broadening of the (31)P NMR signals and, in the case of the AlF4- complex, slow hydrolysis of some of the excess ADP to AMP and inorganic phosphate. The purified BeFx complex contained 3.8 +/- 0.1 MgADP per mol Kp1. With the AlF(4)(-) complex, MgAMP and adenosine (from MgAMP hydrolysis) replaced part of the bound MgADP although four AlF(4)(-) ions were retained, demonstrating that full occupancy by MgADP is not required for the stability of the complex. The fluorescence emission maximum of 2'(3')-O-[N-methylanthraniloyl] ADP was blue-shifted by 6-8 nm in both metal fluoride complexes and polarization was 6-9 times that of the free analogue. The fluorescence yield of bound 2'(3')-O-[N-methylanthraniloyl] ADP was enhanced by 40% in the AlF(4)(-) complex relative to the solvent but no increase in fluorescence was observed in the BeFx complex. Resonance energy transfer from conserved tyrosine residues located in proximity to the Kp2 nucleotide-binding pocket was marked in the AlF(4)(-) complex but minimal in the BeFx fluoride complex, illustrating a clear conformational difference in the Fe protein of the two complexes. Our data indicate that complex formation during the nitrogenase catalytic cycle is a multistep process involving at least four conformational states of Kp2: similar to the free Fe protein; as initially complexed with detectable (31)P NMR; as detected in mature complexes with no detectable (31)P NMR; in the AlF(4)(-) complex in which an altered tyrosine interaction permits resonance energy transfer with 2'(3')-O-[N-methylanthraniloyl] ADP. (+info)
Purification and properties of nitrogenase from the cyanobacterium, Anabaena cylindrica.
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The nitrogenase complex was isolated from nitrogen-starved cultures of Anabaema cylindrica. Sodium dithionite, photochemically reduced ferredoxin, and NADPH were found to be effective election donors to nitro genase in crude extracts whereas hydrogen and pyruvate were not. The Km for acetylene in vivo is ten-fold higher than the Km in vitro, whereas this pattern does not hold for the non-heterocystous cyanobacterium, Plectonema boryanum. This indicates that at least one mechanism of oxygen protection in vivo involves a gas diffusion barrier presented by the heterocyst cell wall. The Mo-Fe component was purified to homogeneity. Its molecular weight (220,000), subunit composition, isoelectric point (4.8), Mo, Fe, and S2- content (2, 20 and 20 mol/mol component), and amino acid composition indicate that this component has similar properties to Mo-Fe-containing components isolated from other bacterial sources. The isolated components from A. cylindrica were found to cross-react, to varying degrees, with components isolated from Azotobacter vinelandii, Rhodospirillum rubrum, and P. boryanum. (+info)