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Regulation of Dinitrogenase Reductase ADP-ribosyltransferase and Dinitrogenase Reductase-activating Glycohydrolase by a Redox-dependent Conformational Change of Nitrogenase Fe Protein

2000; Elsevier BV; Volume: 275; Issue: 5 Linguagem: Inglês

10.1074/jbc.275.5.3493

1083-351X

Cale M. Halbleib, Yaoping Zhang, Paul W. Ludden,

Microbial Fuel Cells and Bioremediation

The nitrogenase-regulating enzymes dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG), from Rhodospirillum rubrum, were shown to be sensitive to the redox status of the [Fe4S4]1+/2+ cluster of nitrogenase Fe protein from R. rubrum or Azotobacter vinelandii. DRAG had <2% activity with oxidized R. rubrum Fe protein relative to activity with reduced Fe protein. The activity of DRAG with oxygen-denatured Fe protein or a low molecular weight substrate,N α-dansyl-N ω-(1,N6-etheno-ADP-ribosyl)-arginine methyl ester, was independent of redox potential. The redox midpoint potential of DRAG activation of Fe protein was −430 mVversus standard hydrogen electrode, coinciding with the midpoint potential of the [Fe4S4] cluster from R. rubrum Fe protein. DRAT was found to have a specificity opposite that of DRAG, exhibiting low (<20%) activity with 87% reduced R. rubrum Fe protein relative to activity with fully oxidized Fe protein. A mutant of R. rubrum in which the rate of oxidation of Fe protein was substantially decreased had a markedly slower rate of ADP-ribosylation in vivo in response to 10 mm NH4Cl or darkness stimulus. It is concluded that the redox state of Fe protein plays a significant role in regulation of the activities of DRAT and DRAG in vivo. The nitrogenase-regulating enzymes dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG), from Rhodospirillum rubrum, were shown to be sensitive to the redox status of the [Fe4S4]1+/2+ cluster of nitrogenase Fe protein from R. rubrum or Azotobacter vinelandii. DRAG had <2% activity with oxidized R. rubrum Fe protein relative to activity with reduced Fe protein. The activity of DRAG with oxygen-denatured Fe protein or a low molecular weight substrate,N α-dansyl-N ω-(1,N6-etheno-ADP-ribosyl)-arginine methyl ester, was independent of redox potential. The redox midpoint potential of DRAG activation of Fe protein was −430 mVversus standard hydrogen electrode, coinciding with the midpoint potential of the [Fe4S4] cluster from R. rubrum Fe protein. DRAT was found to have a specificity opposite that of DRAG, exhibiting low ( 95% active. DRAG activity was determined by a modification of the nitrogenase-coupled DRAG assay (29.Saari L.L. Triplett E.W. Ludden P.W. J. Biol. Chem. 1984; 259: 15502-15508Abstract Full Text PDF PubMed Google Scholar), in which the removal of ADP-ribose was differentiated from the acetylene reduction reaction. The standard demodification step was conducted in an anaerobic, stoppered vial containing 5 mm ATP, 25 mm phosphocreatine, 50 μg of creatine phosphokinase (Sigma), 25 mm MgCl2, 0.5 mmMnCl2, 10 mmNa2S2O4, 20–40 μg of ADP-ribosylated Fe protein, 350 ng of DRAG, and 50 mm MOPS, pH 7.8, in a 500-μl total volume under nitrogen headspace. Na2S2O4 or ATP were excluded from some assays. After a 10-min incubation at 30 °C, 2 mmADP-ribose (Sigma) was added to inhibit further DRAG activity. The acetylene reduction step was initiated by the addition of any components excluded from the demodification step, along with 75 μg of MoFe protein and 10% acetylene in the headspace. After a 10-min incubation at 30 °C, the reaction was stopped by the addition of 5% trichloroacetic acid. The headspace was analyzed for C2H4 content by gas chromatography. Reactions were conducted in 50-μl volumes in microcentrifuge tubes inside anaerobic, N2-filled vials. Inside the vials, but outside the reaction tubes, 0.5 ml of 100 mm Na2S2O4 was present to act as an oxygen scavenger (26.Lowery R.G. Saari L.L. Ludden P.W. J. Bacteriol. 1986; 166: 513-518Crossref PubMed Google Scholar). Component concentrations were identical to those in the decoupled DRAG assays described above, with the following exceptions: creatine phosphokinase (2.5 μg), ADP-ribosylated Fe protein (35 μg), and DRAG (200 ng). The reactions were incubated 10 min at 30 °C and then were stopped by the addition of 50 μl of SDS sample buffer. A sample (20 μl) of each stopped assay was loaded onto a 10% acrylamide (0.6% bis-acrylamide) SDS minigel. After electrophoresis, each gel was stained with Coomassie Blue R-250 stain (Sigma). The relative amounts of the Fe protein upper band (ADP-ribosylated) and lower band (unmodified) were quantitated by ImageQuant software (Molecular Dynamics). A standard correction was made in each assay for a contaminant of creatine phosphokinase, which comigrated with the upper band of Fe protein. Note that only one subunit of the Fe protein homodimer becomes ADP-ribosylated, and thus a 1:1 ratio of upper to lower band represents completely modified (inactive) Fe protein dimer. A continuous assay for DRAG cleavage of the N-glycosidic bond of ε-ADPR-DAME has been described (45.Pope M.R. Saari L.L. Ludden P.W. Anal. Biochem. 1987; 160: 68-77Crossref PubMed Scopus (5) Google Scholar). Assays were performed in 5-mm (outer diameter) × 3.5-cm sealed quartz tubes. Reaction mixtures contained 50 mm MOPS, pH 7.8, 1 mmMnCl2, 50 μm ε-ADPR-DAME, and 150 ng of purified DRAG in a total volume of 250 μl. Reactions were conducted anaerobically in the presence or absence of 0.5 mmNa2S2O4. The reactions were irradiated with 304 nm UV light, and the fluorescence emission at 405 nm was monitored. Reactions were initiated by the addition of DRAG. After 3.5 h, 1 μg of phosphodiesterase (Sigma) and 0.5 mm MgCl2 were added to completely cleave all ADP-ribosylarginine bonds. Finally, each reaction was exposed to air for 20 min, to react away dithionite, before obtaining the final fluorescence intensities. The extent of DRAG activity toward ADP-ribosylated Fe protein was determined in reactions poised at varied redox potentials. In an anaerobic glove box, DRAG decoupled assays (500 μl total volume) were set up, as described above, containing 1 mm benzyl viologen (Sigma). High concentrations of indigo carmine-oxidized ADP-ribosylated Fe protein (100–300 μg/reaction) were required in these assays. Reactions were poised by the addition of Na2S2O4, and the redox potentials were measured by direct potentiometry, using a saturated Ag/AgCl2 electrode (E m°′ = +199 mV versus standard hydrogen electrode (SHE)) as a reference. Reactions were initiated by the addition of 150 ng of DRAG. After a 10-min incubation, the redox potentials were again measured, and the reactions were stopped by the addition of 2 mmADP-ribose. Because benzyl viologen inhibits nitrogenase reactions at concentrations greater than 0.1 mm, portions of the stopped assays (50 μl) were diluted 10-fold into acetylene reduction reactions. The acetylene reduction assays contained 5 mmATP, 25 mm phosphocreatine, 50 μg of creatine phosphokinase, 10 mm MgCl2, 2 mmADP-ribose, 10 mmNa2S2O4, 75 μg of MoFe protein, and 50 mm MOPS, pH 7.8, under a headspace of 10% acetylene in nitrogen. The reactions were incubated for 10 min at 30 °C and then were stopped by the addition of 5% trichloroacetic acid. The headspace was analyzed for C2H4 content by gas chromatography. The midpoint potentials for modified and unmodified R. rubrum Fe protein were determined by dye-mediated, visible spectrum-monitored redox titration. Two solutions in matched glass cuvettes were prepared, containing 50 μm Fe protein, 50 mm MOPS, pH 7.8, 3 mm MgATP, and 15 μm methyl viologen (MV) (Sigma). The spectral differences between the cuvettes were recorded as 2-μl aliquots of 2 mmNa2S2O4 were added to one cuvette. The redox potential of the titrated solution was determined from the intensity of the absorption at 608 nm due to reduced MV. The fraction of Fe protein in the reduced ([Fe4S4]1+) state was determined from the change in the characteristic Fe protein absorbance at 420 nm (46.Shah V.K. Brill W.J. Biochim. Biophys. Acta. 1973; 305: 445-454Crossref PubMed Scopus (105) Google Scholar, 47.Ljones T. Biochim. Biophys. Acta. 1973; 321: 103-113Crossref PubMed Scopus (28) Google Scholar). An assay for DRAT activity by 32P-NAD radiolabeling of Fe protein has been described (28.Lowery R.G. Ludden P.W. J. Biol. Chem. 1988; 263: 16714-16719Abstract Full Text PDF PubMed Google Scholar). The standard reaction mix contains 50 mm MOPS, pH 7.8, 1 mm ADP, 5 mmMgCl2, 0.25 mm [α-32P]NAD (20 μCi/μmol), 80 μg of indigo carmine-oxidized, unmodified Fe protein, and DRAT in a volume of 50 μl. To this mix were added 10 μm MV and 4 μg of carbon monoxide dehydrogenase (CODH) from R. rubrum. To produce reducing conditions, the headspace of the reaction vial was filled with CO, which was oxidized to CO2 by CODH with a concomitant reduction of MV. Assay mixtures were incubated for 20 min at 30 °C and then were stopped with 5% trichloroacetic acid. Precipitated protein was collected on nitrocellulose filters, and the amount of [α-32P]ADP-ribose incorporated into Fe protein was determined by a Packard Tri-Carb 2100TR liquid scintillation counter. R. rubrum strains UR2 (wild-type) and UR145 (nifD::kan (48.Ludden P.W. Lehman L. Roberts G.P. J. Bacteriol. 1989; 171: 5210-5211Crossref PubMed Google Scholar)) were grown on supplemented malate-ammonium medium (40.Fitzmaurice W.P. Saari L.L. Lowery R.G. Ludden P.W. Roberts G.P. Mol. Gen. Genet. 1989; 218: 340-347Crossref PubMed Scopus (76) Google Scholar). Each strain was inoculated into malate-glutamate medium (33.Kanemoto R.H. Ludden P.W. J. Bacteriol. 1984; 158: 713-720Crossref PubMed Google Scholar) at a 65-fold dilution. After illuminated, anaerobic growth for 2 days, for derepression of Fe protein, the cells were treated with darkness or 10 mm NH4Cl, as described (33.Kanemoto R.H. Ludden P.W. J. Bacteriol. 1984; 158: 713-720Crossref PubMed Google Scholar). Proteins were extracted quickly by a trichloroacetic acid precipitation method (49.Zhang Y. Burris R.H. Ludden P.W. Roberts G.P. J. Bacteriol. 1993; 175: 6781-6788Crossref PubMed Google Scholar, 50.Zhang Y. Burris R.H. Ludden P.W. Roberts G.P. J. Bacteriol. 1994; 176: 5780-5787Crossref PubMed Google Scholar). Fe protein subunits were separated by electrophoresis on a 10% acrylamide (0.6% bis-acrylamide) SDS minigel. Proteins from SDS-polyacrylamide gel electrophoresis were transferred onto nitrocellulose and then were immunoblotted with polyclonal antibody against A. vinelandii Fe protein and were visualized with ECL Western blotting reagents (Amersham Pharmacia Biotech). The protein bands on the x-ray film were quantitated by ImageQuant software. The activation of ADP-ribosylated Fe protein by DRAG was assayed with oxidized Fe protein or reduced Fe protein by the “decoupled DRAG assay” described under “Materials and Methods.” In both cases, Fe protein was first oxidized as described under “Materials and Methods.” To obtain reduced Fe protein, sodium dithionite was added to the incubation mixture. In the second phase of the decoupled DRAG assay, the extent of activation of Fe protein was determined from acetylene reduction assays conducted in the presence of MoFe protein, excess dithionite, and 10% acetylene in the headspace. In these decoupled assays for DRAG activity, with indigo carmine-oxidized Fe protein from R. rubrum as the substrate, activity in the absence of Na2S2O4was found to be negligible in comparison to activity with the reductant added (Table I). This result was validated by demonstration of the efficacy of the assay system. In the decoupled assays, the addition of 2 mm ADP-ribose completely inhibited DRAG activity but only slightly inhibited the acetylene reduction reaction of Fe protein and MoFe protein (data not shown). The addition of this specific inhibitor stopped DRAG activation of Fe protein, effectively separating the DRAG-dependent activation of Fe protein from the Fe protein-dependent acetylene reduction phase of the assay and thus allowing analysis of the DRAG reaction in isolation. The decoupled assay system was demonstrated to be a linear assay for DRAG. The dependence of the DRAG reaction on the presence of MgATP, first reported by Saari et al. (31.Saari L.L. Pope M.R. Murrell S.A. Ludden P.W. J. Biol. Chem. 1986; 261: 4973-4977Abstract Full Text PDF PubMed Google Scholar), was confirmed (Table I). Also, the dependence on reductant was shown to exist regardless of the divalent cation (Mn2+ or Fe2+) present in the assay (data not shown).Table IActivity of DRAG with oxidized and reduced R. rubrum Fe proteinReaction conditions during incubation with DRAGRedox state of Fe protein added to incubationRedox state of Fe protein during incubationnmol C2H4produced min−1 mg Fe protein−1during C2H2 reduction phase of assayaDecoupled assays for DRAG activity were performed, activating Fe protein with DRAG under the following conditions: 10 min at 30 °C, followed by a Fe protein-dependent acetylene reduction phase with 2 mm ADP-ribose (to inhibit DRAG activity), MoFe protein, 10 mmNa2S2O4, and 10% acetylene in the headspace.Complete (350 ng DRAG, 10 mmNa2S2O4, 5 mmMgATP)OxbFe protein was oxidized by indigo carmine affixed to a Dowex chloride anion exchange column.Red848 ± 2CompleteRedcReduced Fe protein was passed over a 5-ml G-25 Sephadex column to remove excess dithionite.Red760 ± 20Minus DRAGOxbFe protein was oxidized by indigo carmine affixed to a Dowex chloride anion exchange column.Red10.2 ± 0.5Minus DRAGRedcReduced Fe protein was passed over a 5-ml G-25 Sephadex column to remove excess dithionite.Red4.7 ± 0.5Minus Na2S2O4OxbFe protein was oxidized by indigo carmine affixed to a Dowex chloride anion exchange column.Ox23 ± 1Minus Na2S2O4RedcReduced Fe protein was passed over a 5-ml G-25 Sephadex column to remove excess dithionite.ReddFe protein was subject to oxidation following removal of Na2S2O4.163 ± 6Minus MgATPePhosphocreatine and creatine phosphokinase were also excluded.OxbFe protein was oxidized by indigo carmine affixed to a Dowex chloride anion exchange column.Red14 ± 1a Decoupled assays for DRAG activity were performed, activating Fe protein with DRAG under the following conditions: 10 min at 30 °C, followed by a Fe protein-dependent acetylene reduction phase with 2 mm ADP-ribose (to inhibit DRAG activity), MoFe protein, 10 mmNa2S2O4, and 10% acetylene in the headspace.b Fe protein was oxidized by indigo carmine affixed to a Dowex chloride anion exchange column.c Reduced Fe protein was passed over a 5-ml G-25 Sephadex column to remove excess dithionite.d Fe protein was subject to oxidation following removal of Na2S2O4.e Phosphocreatine and creatine phosphokinase were also excluded. Open table in a new tab The stimulation of DRAG activity by reductant was correlated to a change in Fe protein subunit composition, indicative of the removal of ADP-ribose from Fe protein (51.Preston G.G. Ludden P.W. Biochem. J. 1982; 205: 489-494Crossref PubMed Scopus (21) Google Scholar, 52.Pope M.R. Murrell S.A. Ludden P.W. Biochemistry. 1985; 24: 2374-2380Crossref PubMed Scopu