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• W2077875467 abstract "NIFH (the nifH gene product) has several functions in the nitrogenase enzyme system. In addition to reducing dinitrogenase during nitrogenase turnover, NIFH functions in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), and in the processing of α2β2 apodinitrogenase 1 (a catalytically inactive form of dinitrogenase 1 that lacks the FeMo-co) to the FeMo-co-activatable α2β2γ2 form. The molybdenum-independent nitrogenase 2 (vnf-encoded) has a distinct dinitrogenase reductase protein, VNFH. We investigated the ability of VNFH to function in the in vitro biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. VNFH can replace NIFH in both the biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. These results suggest that the dinitrogenase reductase proteins do not specify the heterometal incorporated into the cofactors of the respective nitrogenase enzymes. The specificity for the incorporation of molybdenum into FeMo-co was also examined using the in vitro FeMo-co synthesis assay system. NIFH (the nifH gene product) has several functions in the nitrogenase enzyme system. In addition to reducing dinitrogenase during nitrogenase turnover, NIFH functions in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), and in the processing of α2β2 apodinitrogenase 1 (a catalytically inactive form of dinitrogenase 1 that lacks the FeMo-co) to the FeMo-co-activatable α2β2γ2 form. The molybdenum-independent nitrogenase 2 (vnf-encoded) has a distinct dinitrogenase reductase protein, VNFH. We investigated the ability of VNFH to function in the in vitro biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. VNFH can replace NIFH in both the biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. These results suggest that the dinitrogenase reductase proteins do not specify the heterometal incorporated into the cofactors of the respective nitrogenase enzymes. The specificity for the incorporation of molybdenum into FeMo-co was also examined using the in vitro FeMo-co synthesis assay system. The reduction of atmospheric N2 to ammonium by biological systems is catalyzed by the nitrogenase enzymes. The aerobeAzotobacter vinelandii harbors three genetically distinct nitrogenase enzymes that are regulated by the metal content of the growth medium, among other factors (1Bishop P.E. Joerger R.D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1990; 41: 109-125Crossref Scopus (95) Google Scholar, 2Eady R.R. Adv. Inorg. Chem. 1991; 36: 77-102Crossref Scopus (59) Google Scholar, 3Luque F. Pau R.N. Mol. Gen. Genet. 1991; 227: 481-487Crossref PubMed Scopus (37) Google Scholar). Nitrogenases 1, 2, and 3 are encoded by the nif, vnf, and anfgenes, respectively. nif-encoded nitrogenase 1 is a molybdenum-containing enzyme that is expressed in the presence of molybdenum. Expression of the vnf-encoded nitrogenase 2, a vanadium-containing enzyme, requires medium that is depleted in molybdenum and that contains vanadium. The anf-encoded nitrogenase 3 is expressed in medium deficient in both metals. All three nitrogenases are two-component metalloenzymes comprised of dinitrogenase and dinitrogenase reductase (1Bishop P.E. Joerger R.D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1990; 41: 109-125Crossref Scopus (95) Google Scholar, 2Eady R.R. Adv. Inorg. Chem. 1991; 36: 77-102Crossref Scopus (59) Google Scholar). Dinitrogenase contains the active site metal center of the enzyme, and dinitrogenase reductase functions as the obligate electron donor to dinitrogenase during enzyme turnover in a MgATP- and reductant-dependent process (4Dean D.R. Bolin J.T. Zheng L. J. Bacteriol. 1993; 175: 6737-6744Crossref PubMed Google Scholar,5Howard J.B. Rees D.C. Annu. Rev. Biochem. 1994; 63: 235-264Crossref PubMed Scopus (167) Google Scholar). The active site of dinitrogenase 1, the iron-molybdenum cofactor (FeMo-co), 1The abbreviations and designative terms used are: FeMo-co, the iron-molybdenum cofactor of dinitrogenase 1; FeV-co, the iron-vanadium cofactor of dinitrogenase 2; NIFH, thenifH-encoded dinitrogenase reductase (iron protein) component of nitrogenase 1; VNFH, the vnf-encoded dinitrogenase reductase (iron protein) of nitrogenase 2; apodinitrogenase 1, the FeMo-co-deficient form of dinitrogenase 1; apodinitrogenase 2, the FeV-co-deficient form of dinitrogenase 2; NifB-co, the metabolic product of the NIFB protein which serves as an iron and sulfur donor to FeMo-co and is thought to donate iron and sulfur to FeV-co as well; γ protein, a chaperone-insertase required for insertion of FeMo-co into apodinitrogenase 1; ANFH, anfhgene product; DTH, dithionite; PAGE, polyacrylamide gel electrophoresis. is composed of molybdenum, iron, sulfur, and homocitrate ((R)-2-hydroxyl-1,2,4-butanetricarboxylic acid) in a 1:7:9:1 ratio (6Shah V.K. Brill W.J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3249-3253Crossref PubMed Scopus (477) Google Scholar, 7Chan M.C. Kim J. Rees D.C. Science. 1993; 260: 792-794Crossref PubMed Scopus (491) Google Scholar, 8Kim J. Rees D.C. Nature. 1992; 257: 553-560Google Scholar, 9Hoover T.R. Imperial J. Ludden P.W. Shah V.K. Biochemistry. 1989; 28: 2768-2771Crossref PubMed Scopus (97) Google Scholar). The biosynthesis of FeMo-co involves the products of at least six nif genes, including nifQ,nifB, nifV, nifE, nifN, andnifH (9Hoover T.R. Imperial J. Ludden P.W. Shah V.K. Biochemistry. 1989; 28: 2768-2771Crossref PubMed Scopus (97) Google Scholar, 10Filler W.A. Kemp R.N. Ng J.C. Hawkes T.R. Dixon R.A. Smith B.E. Eur. J. Biochem. 1986; 160: 371-377Crossref PubMed Scopus (47) Google Scholar, 11Robinson A.C. Dean D.R. Burgess B.K. J. Biol. Chem. 1987; 262: 14327-14332Abstract Full Text PDF PubMed Google Scholar, 12Shah V.K. Imperial J. Ugalde R.A. Ludden P.W. Brill W.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1636-1640Crossref PubMed Scopus (59) Google Scholar). The nifQ gene product might be involved in the formation of a molybdenum-sulfur precursor to FeMo-co (13Ugalde R.A. Imperial J. Shah V.K. Brill W.J. J. Bacteriol. 1985; 164: 1081-1087Crossref PubMed Google Scholar), and the nifV gene product encodes homocitrate synthase (9Hoover T.R. Imperial J. Ludden P.W. Shah V.K. Biochemistry. 1989; 28: 2768-2771Crossref PubMed Scopus (97) Google Scholar, 10Filler W.A. Kemp R.N. Ng J.C. Hawkes T.R. Dixon R.A. Smith B.E. Eur. J. Biochem. 1986; 160: 371-377Crossref PubMed Scopus (47) Google Scholar). 2D. R. Dean, personal communication. The product of NIFB, termed NifB-co, is an iron- and sulfur-containing precursor to FeMo-co (14Shah V.K. Allen J.R. Spangler N.J. Ludden P.W. J. Biol. Chem. 1994; 269: 1154-1158Abstract Full Text PDF PubMed Google Scholar, 15Allen R.M. Chatterjee R. Ludden P.W. Shah V.K. J. Biol. Chem. 1995; 270: 26890-26896Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Based on the amino acid sequence identity of NIFEN to NIFDK (the structural polypeptides of dinitrogenase 1), and the fact that NIFEN has been shown to bind NifB-co (15Allen R.M. Chatterjee R. Ludden P.W. Shah V.K. J. Biol. Chem. 1995; 270: 26890-26896Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 16Brigle K.E. Weiss M.C. Newton W.E. Dean D.R. J. Bacteriol. 1987; 169: 1547-1553Crossref PubMed Google Scholar, 17Roll J.T. Shah V.K. Dean D.R. Roberts G.P. J. Biol. Chem. 1995; 270: 4432-4437Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), NIFEN has been proposed to be a scaffold for FeMo-co assembly; however, the precise function of NIFEN in FeMo-co biosynthesis is unknown, as is the function of NIFH (dinitrogenase reductase 1). In addition to being necessary for the biosynthesis of FeMo-co, the gene products of both nifV and nifB are required for the biosynthesis of the iron-vanadium cofactor (FeV-co) of dinitrogenase 2 and the putative iron-only cofactor (FeFe-co) of dinitrogenase 3 (18Kennedy C. Dean D. Mol. Gen. Genet. 1992; 231: 494-498Crossref PubMed Scopus (96) Google Scholar, 19Joerger R.D. Premakumar R. Bishop P.E. J. Bacteriol. 1986; 168: 673-682Crossref PubMed Google Scholar, 20Smith B.E. Eady R.R. Lowe D.J. Gormal C. Biochem. J. 1988; 250: 299-302Crossref PubMed Scopus (74) Google Scholar, 21Davis R. Lehman L. Petrovich R. Shah V.K. Roberts G.P. Ludden P.W. J. Bacteriol. 1996; 178: 1445-1450Crossref PubMed Google Scholar). Thus, homocitrate is presumed to be present as a component of FeV-co and FeFe-co, and NifB-co is believed to serve as the iron and sulfur donor to all three cofactors. Homologs ofnifE and nifN have been identified in thevnf but not in the anf system (22Wolfinger E.D. Bishop P.E. J. Bacteriol. 1991; 173: 7565-7572Crossref PubMed Google Scholar); homologs ofnifH exist in both molybdenum-independent systems (23Joerger R.D. Loveless T.M. Pau R.N. Mitchenall L.A. Simon B.H. Bishop P.E. J. Bacteriol. 1990; 172: 3400-3408Crossref PubMed Google Scholar, 24Joerger R.D. Jacobson M.R. Premakumar R. Wolfinger E.D. Bishop P.E. J. Bacteriol. 1989; 171: 1075-1086Crossref PubMed Google Scholar). Additional gene products required for FeV-co and FeFe-co biosynthesis have not been identified. An in vitro system for the synthesis of FeMo-co that requires at least molybdate, homocitrate, an ATP-regenerating mixture, a source of reductant, NifB-co, NIFEN, and NIFH has been described (12Shah V.K. Imperial J. Ugalde R.A. Ludden P.W. Brill W.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1636-1640Crossref PubMed Scopus (59) Google Scholar,14Shah V.K. Allen J.R. Spangler N.J. Ludden P.W. J. Biol. Chem. 1994; 269: 1154-1158Abstract Full Text PDF PubMed Google Scholar, 25Chatterjee R. Allen R.M. Shah V.K. Ludden P.W. J. Bacteriol. 1994; 176: 2747-2750Crossref PubMed Google Scholar, 26Allen R.M. Chatterjee R. Ludden P.W. Shah V.K. J. Biol. Chem. 1996; 271: 4256-4260Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The in vitro FeMo-co synthesis system utilizes molybdenum with high specificity as addition of 100-fold excess tungstate (a competitive inhibitor of the molybdenum transport system in Klebsiella pneumoniae) or vanadate do not significantly inhibit FeMo-co synthesis (12Shah V.K. Imperial J. Ugalde R.A. Ludden P.W. Brill W.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1636-1640Crossref PubMed Scopus (59) Google Scholar). The replacement of molybdenum with vanadium or iron in the cofactor during in vitro synthesis has not been achieved. The preferential incorporation of molybdenum into FeMo-co suggests that a component(s) involved in FeMo-co biosynthesis might exclusively select for molybdenum. The presence of a dinitrogenase reductase associated with each nitrogen fixation system makes that protein a likely candidate for specifying the heterometal incorporated into the respective cofactors of the nitrogenase enzymes. NIFH has multiple roles in the nitrogenase 1 enzyme system. In addition to MgATP-dependent electron transfer to dinitrogenase during substrate reduction, NIFH is required for the biosynthesis of FeMo-co (10Filler W.A. Kemp R.N. Ng J.C. Hawkes T.R. Dixon R.A. Smith B.E. Eur. J. Biochem. 1986; 160: 371-377Crossref PubMed Scopus (47) Google Scholar, 11Robinson A.C. Dean D.R. Burgess B.K. J. Biol. Chem. 1987; 262: 14327-14332Abstract Full Text PDF PubMed Google Scholar) and for the maturation of apodinitrogenase 1 (a catalytically inactive form of dinitrogenase 1 that lacks FeMo-co) to its FeMo-co-activatable form (27Allen R.M. Homer M.J. Chatterjee R. Ludden P.W. Roberts G.P. Shah V.K. J. Biol. Chem. 1993; 268: 23670-23674Abstract Full Text PDF PubMed Google Scholar, 28Homer M.J. Dean D.R. Roberts G.P. J. Biol. Chem. 1995; 270: 24745-24752Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In the latter process, NIFH is required for the association of the γ protein (a non-nif-encoded protein) (28Homer M.J. Dean D.R. Roberts G.P. J. Biol. Chem. 1995; 270: 24745-24752Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) with α2β2 apodinitrogenase 1 to form the FeMo-co-activatable α2β2γ2hexamer (27Allen R.M. Homer M.J. Chatterjee R. Ludden P.W. Roberts G.P. Shah V.K. J. Biol. Chem. 1993; 268: 23670-23674Abstract Full Text PDF PubMed Google Scholar). Some altered forms of NIFH that are unable to function as a reductant for nitrogenase-dependent substrate reduction are fully functional in FeMo-co biosynthesis and in the maturation of apodinitrogenase 1 (29Shah V.K. Davis L.C. Gordon J.C. Orme-Johnson W.H. Brill W.J. Biochim. Biophys. Acta. 1973; 292: 246-255Crossref PubMed Scopus (49) Google Scholar, 30Gavini N. Burgess B.K. J. Biol. Chem. 1992; 267: 21179-21186Abstract Full Text PDF PubMed Google Scholar, 31Wolle D. Dean D.R. Howard J.B. Science. 1992; 258: 992-995Crossref PubMed Scopus (67) Google Scholar), indicating that the characteristics of NIFH that enable it to function in nitrogenase turnover are not necessary for its role in the formation of active dinitrogenase. In vivo studies by Joerger et al. (23Joerger R.D. Loveless T.M. Pau R.N. Mitchenall L.A. Simon B.H. Bishop P.E. J. Bacteriol. 1990; 172: 3400-3408Crossref PubMed Google Scholar) and Gollanet al. (32Gollan U. Schneider K. Müller A. Schüddekopf K. Klipp W. Eur. J. Biochem. 1993; 215: 25-35Crossref PubMed Scopus (43) Google Scholar) suggest that NIFH supports FeV-co synthesis and that ANFH (the anfh gene product) supports FeMo-co synthesis. We utilized the in vitro FeMo-co synthesis assay system to definitively determine whether VNFH would function in FeMo-co biosynthesis; the ability of VNFH to replace NIFH in the formation of the FeMo-co-activatable α2β2γ2 form of apodinitrogenase 1 was also examined. Studies on the specificity of the incorporation of molybdenum into FeMo-co are discussed. DEAE-cellulose was a Whatman DE52 product. Sephacryl S-100 and the Mono Q anion exchange column were from Pharmacia Biotech Inc. The fast protein liquid chromatography instrument was from LKB. Sodium dithionite (DTH) was purchased from Fluka Chemicals. Sodium metavanadate (NaVO3, 99.995% purity), Tris base, and glycine were Fisher products. Acrylamide/bisacrylamide solution was obtained from Bio-Rad. All reagents used for A. vinelandii growth medium were of analytical grade or higher purity. Tetrathiomolybdate ((NH4)2MoS4) was a gift from D. Coucouvanis, and [K2(H2O)5][(VO2)2(R,S-homocitrate)2]·H2O was a gift from W. Armstrong (33Wright D.W. Chang R.T. Mandal S.K. Armstrong W.H. Orme- Johnson W.H. J. Bio-inorg. Chem. 1996; 1: 143-151Crossref Scopus (39) Google Scholar). All other chemicals were from Sigma. A. vinelandii strains DJ1030 (ΔnifHΔnifB) (28Homer M.J. Dean D.R. Roberts G.P. J. Biol. Chem. 1995; 270: 24745-24752Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), CA12 (ΔnifHDK) (34Chisnell J.R. Premakumar R. Bishop P.E. J. Bacteriol. 1988; 170: 27-33Crossref PubMed Google Scholar), UW45 (nifB [minus0]) (35Bishop P.E. Premakumar R. Dean D.R. Jacobson M.R. Chisnell J.R. Rizzo T.M. Kopzcynski J. Science. 1985; 232: 92-94Crossref Scopus (79) Google Scholar), and CA117.30 (ΔnifDKB) (36Chatterjee R. Allen R.M. Ludden P.W. Shah V.K. J. Biol. Chem. 1996; 271: 6819-6826Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) have been described. All vessels used in preparing media and for cell culture were rinsed thoroughly in 4 n HCl and then in deionized water. Cultures (15 liters) of strain DJ1030 were grown in 20-liter polycarbonate carboys with vigorous aeration at 30 °C on Burk's medium that lacked sodium molybdate and contained 10 μm NaVO3 (for derepression of thevnf system) and 40 μg of nitrogen/ml as ammonium acetate. The cultures were monitored for depletion of ammonium, following which they were derepressed for 4.5 h. The cells were concentrated using a Pellicon cassette system equipped with a filtration membrane (0.45 μm, Millipore Corp., Bedford, MA) and were centrifuged. The cell pellets were frozen in liquid N2 and stored at −80 °C. Strain DJ1030 was grown on Burk's medium containing 1 mmsodium molybdate in place of NaVO3 for derepression of thenif system. Strain UW45 was grown and derepressed on tungsten-containing medium (molybdenum free) as described previously (12Shah V.K. Imperial J. Ugalde R.A. Ludden P.W. Brill W.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1636-1640Crossref PubMed Scopus (59) Google Scholar). Strain CA117.30 was grown in 250-ml cultures on Burk's medium containing 10 μm NaVO3; cells were concentrated by centrifugation, and derepression was initiated (for 4 h) by suspending the cell pellets in nitrogen-free Burk's medium. Cells were harvested by centrifugation and frozen as described above. Cell-free extracts were prepared by the osmotic shock method (6Shah V.K. Brill W.J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3249-3253Crossref PubMed Scopus (477) Google Scholar). All buffers were sparged with purified N2 (and degassed on a gassing manifold where appropriate) for 10–30 min, and DTH was added to a final concentration of 1.7 mm. All buffers used in column chromatography contained 0.2 mm phenylmethylsulfonyl fluoride and 0.5 μg/ml leupeptin. Buffers used in fast protein liquid chromatography were filtered through a 0.45-μm filter. Tris-HCl was at pH 7.4 unless stated otherwise. All column chromatography steps except for fast protein liquid chromatography were performed at 4 °C. VNFH was purified from extract of strain DJ1030 (ΔnifHΔnifB, vnf-derepressed) with modifications to the method described by Hales et al. (37Hales B.J. Langosch D.J. Case E.E. J. Biol. Chem. 1986; 261: 15301-15306Abstract Full Text PDF PubMed Google Scholar). One hundred fifty ml of cell-free extract (from 50 g of cell paste) were applied to a 2.5 × 17-cm DEAE-cellulose column that had been equilibrated in buffer containing 0.1 m NaCl in 0.025 m Tris-HCl, pH 7.4. Following application of the extract, the column was washed with 2 bed volumes of buffer containing 0.125 m NaCl in 0.025 m Tris-HCl; VNFH was eluted using 0.22 m NaCl in 0.025 m Tris-HCl. The DEAE-cellulose fraction was concentrated by ultrafiltration using a XM100-A membrane, and the retentate (4 ml) was applied to a 2.5 × 78-cm Sephacryl S-100 column that had been equilibrated with 0.05m NaCl in 0.025 m Tris-HCl. The column was developed with the same buffer, and the VNFH-containing fractions that exhibited the highest activity were concentrated by ultrafiltration (described above) and purified further on a Mono Q anion exchange column used in conjunction with a fast protein liquid chromatography system. Two ml (6.8 mg of protein) of the VNFH-containing retentate from the ultrafiltration cell was applied onto the Mono Q column that had been equilibrated with 0.15 m NaCl in 0.025m Tris-HCl. The column was washed with 1 bed volume of the equilibration buffer, following which VNFH was eluted using a 20-ml increasing linear gradient from 0.15 to 0.4 m NaCl (in 0.025 m Tris-HCl, pH 7.4). VNFH eluted with 0.32m NaCl in 0.025 m Tris-HCl. Active fractions were stored in 9-ml, serum-stoppered vials at −80 °C. The ability of VNFH to transfer electrons to dinitrogenase 1 was tested using the acetylene reduction assay for nitrogenase activity, and the results were consistent with the published results. VNFH was equally effective as NIFH in transferring electrons to dinitrogenase 1, consistent with the results of Chisnell et al. (34Chisnell J.R. Premakumar R. Bishop P.E. J. Bacteriol. 1988; 170: 27-33Crossref PubMed Google Scholar). FeMo-co was prepared in N-methylformamide as described previously (6Shah V.K. Brill W.J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3249-3253Crossref PubMed Scopus (477) Google Scholar). The reactions were performed in 9-ml, serum-stoppered vials that were repeatedly evacuated, flushed with argon, and rinsed with 0.3 ml of 0.025 m Tris-HCl containing 1.7 mm DTH. The following components were added to the vials in the order indicated: 100 μl of 0.025 mTris-HCl; 200 μl of an ATP-regenerating mixture (containing 3.6 mm ATP, 6.3 mm MgCl2, 51 mm phosphocreatine, 20 units/ml creatine phosphokinase, and 6.3 mm DTH); 200 μl (3.8 mg protein) of extract of strain DJ1030 (ΔnifHΔnifB,nif-derepressed) as a source of α2β2 apodinitrogenase 1 and the γ protein; and 10–50 μl (0.1 mg of protein) of the appropriate dinitrogenase reductase. The vials were incubated for 10 min at room temperature to allow the formation of α2β2γ2 apodinitrogenase 1. One hundred μl of anoxic 50% glycerol were added to the reactions to be analyzed by native PAGE, and these vials were placed on ice. Ten μl of a solution containing an excess of FeMoco were added to the remaining vials, which were incubated for 10 min at room temperature during which α2β2γ2apodinitrogenase 1 was activated by FeMo-co to form dinitrogenase 1. Fifty nmol of (NH4)2MoS4 (prepared in N-methylformamide containing 1.7 mm DTH) were added to the vials to prevent further FeMo-co insertion into apodinitrogenase 1. Activity of the newly reconstituted dinitrogenase 1 was monitored by the C2H2 reduction assay for nitrogenase (12Shah V.K. Imperial J. Ugalde R.A. Ludden P.W. Brill W.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1636-1640Crossref PubMed Scopus (59) Google Scholar). (NH4)2MoS4 was excluded in certain control reactions, and 0.1 mg of the appropriate dinitrogenase reductase (that used in the insertion phase of the assay) was added in place of 0.1 mg of NIFH normally added during the C2H2 reduction phase of the assay (12Shah V.K. Imperial J. Ugalde R.A. Ludden P.W. Brill W.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1636-1640Crossref PubMed Scopus (59) Google Scholar). Nine-ml serum vials were repeatedly evacuated, flushed with argon, and rinsed with buffer containing 1.7 mm DTH. Components were added to the vials in the following order: 100 μl of 0.025 m Tris-HCl, 10 μl of 1 mm Na2MoO4, 20 μl of 5 mm homocitrate (that had been treated with base to cleave the lactone, pH 8.0), and 200 μl of the ATP-regenerating mixture (defined above). The vials were incubated at room temperature for 10–20 min. Two hundred μl of extract (∼3.8 mg protein) of either DJ1030 (ΔnifHΔnifB,nif-derepressed) or CA12 (ΔnifHDK,nif-derepressed), 25 μl of a solution containing NifB-co, and 10–50 μl (0.1 mg protein) of the appropriate dinitrogenase reductase were added to the vials. The vials were incubated at 30 °C for 30–90 min. Following this incubation, 100 μl of anoxic 50% glycerol were added to the reactions to be analyzed by anoxic native PAGE, and these vials were placed on ice. Five nmol of (NH4)2MoS4 (prepared as described above) were added to the remaining vials to prevent further FeMo-co synthesis during the subsequent C2H2 reduction phase of the assay. The activity of the newly formed dinitrogenase 1 was monitored by the C2H2 reduction assay. (NH4)2MoS4 was excluded from certain reactions to which 0.1 mg of the appropriate dinitrogenase reductase (that used in the synthesis phase of the assay) was added in place of 0.1 mg of NIFH normally added during the C2H2 reduction phase of the assay. Proteins were resolved on anoxic native gels with a 7–14% acrylamide (37.1% acrylamide, 1% bisacrylamide) and 0–20% sucrose gradient. The electrophoresis buffer was N2-sparged, 65 mm Tris-glycine (pH 8.5) containing 1.7 mm DTH. Gels were pre-electrophoresed for at least 60 min at 120 V for initial reduction, and proteins were electrophoresed for 1920 V-h (at 120 V) at 4 °C. One hundred μl of the reaction mixtures were applied onto the gel. Polyclonal antibodies to NIFH and the γ protein were raised in rabbits (the anti-γ protein antibodies were prepared and made available by Drs. Mary Homer and Gary Roberts). Immunoblotting and developing procedures have been described (38Brandner J.P. McEwan A.G. Kaplan S. Donahue T. J. Bacteriol. 1989; 171: 360-368Crossref PubMed Google Scholar). The native gels were equilibrated in transfer buffer for at least 15 min prior to blotting. Protein concentrations of cell-free extracts and purified proteins were measured using the bicinchoninic acid method (39Smith P.K. Krohn R.I. Hermanson A.K. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar). The association of the γ protein with α2β2apodinitrogenase 1 to form the α2β2γ2 FeMo-co-activable species requires the presence of NIFH and nucleotide (27Allen R.M. Homer M.J. Chatterjee R. Ludden P.W. Roberts G.P. Shah V.K. J. Biol. Chem. 1993; 268: 23670-23674Abstract Full Text PDF PubMed Google Scholar, 28Homer M.J. Dean D.R. Roberts G.P. J. Biol. Chem. 1995; 270: 24745-24752Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), as diagrammed in Reaction 1.α2β2→MgATPNIFHγ α2β2γ2 →FeMo­co α2β2 +γ2Apodinitrogenase1 Apodinitrogenase1Dinitrogenase1*(catalytically active)REACTION1The FeMo-co insertion assay and anoxic native PAGE were employed to test whether VNFH might replace NIFH in the maturation of apodinitrogenase 1. The results in Table I show that treatment of extract containing α2β2 apodinitrogenase 1 and the γ protein with equivalent levels of purified NIFH or VNFH resulted in similar levels of activity in the FeMo-co insertion assay, indicating that VNFH is as effective as NIFH in the conversion of α2β2 apodinitrogenase 1 to the α2β2γ2 form. Nucleotide is necessary for the VNFH-dependent maturation process as is maturation supported by NIFH (27Allen R.M. Homer M.J. Chatterjee R. Ludden P.W. Roberts G.P. Shah V.K. J. Biol. Chem. 1993; 268: 23670-23674Abstract Full Text PDF PubMed Google Scholar). Control reactions in which (NH4)2MoS4 was not added to quench further FeMo-co insertion and which contained VNFH in both insertion and C2H2 reduction phases of the assay exhibited similar levels of activity as reactions to which NIFH was added (following (NH4)2MoS4addition) during the C2H2 reduction phase. Thus, the activities reported in Table I for reactions that contained VNFH in the insertion phase alone were not a result of NIFH functioning to attach the γ protein to α2β2apodinitrogenase 1 during the C2H2 reduction phase of the assay. NIFH from another organism (Rhodospirillum rubrum) also supported activity in the FeMo-co insertion assay (Table I).Table IAbility of VNFH to function in the FeMo-co insertion assay and in in vitro FeMo-co synthesisDinitrogenase reductase1-aAssays contained 0.1 mg of the appropriate dinitrogenase reductase protein and 3.8 mg of extract of strain DJ1030 (ΔnifHΔnifB, nif-derepressed) as a source of α2β2 apodinitrogenase and γ protein.FeMo-co insertion1-bFeMo-co insertion assays were performed as described under “Experimental Procedures”; activities are expressed as nanomoles of C2H4 formed/min/assay.FeMo-co synthesis1-cFeMo-co synthesis assays were performed as described under “Experimental Procedures”; activities are expressed as nanomoles of C2H4 formed/min/assay.−MgATP+MgATPC2 H4 formed/min/assayNone0.30.40.03NIFH0.514.629.8VNFH0.414.58.2NIFH (R. rubrum)1-dNIFH was purified from R. rubrum as described in Ludden and Burris (42).0.413.7ND1-eNot determined.1-a Assays contained 0.1 mg of the appropriate dinitrogenase reductase protein and 3.8 mg of extract of strain DJ1030 (ΔnifHΔnifB, nif-derepressed) as a source of α2β2 apodinitrogenase and γ protein.1-b FeMo-co insertion assays were performed as described under “Experimental Procedures”; activities are expressed as nanomoles of C2H4 formed/min/assay.1-c FeMo-co synthesis assays were performed as described under “Experimental Procedures”; activities are expressed as nanomoles of C2H4 formed/min/assay.1-d NIFH was purified from R. rubrum as described in Ludden and Burris (42Ludden P.W. Burris R.H. Biochem. J. 1978; 175: 251-259Crossref PubMed Scopus (52) Google Scholar).1-e Not determined. Open table in a new tab To confirm the results of the FeMo-co insertion assays, we employed anoxic, native PAGE to monitor the association of the γ protein with α2β2 apodinitrogenase 1 in extracts of strain DJ1030 (ΔnifHΔnifB,nif-derepressed) in the presence of nucleotide and the different dinitrogenase reductase proteins. Fig. 1, an immunoblot of an anoxic, native gel (developed with antibody to the γ protein), illustrates that VNFH functions in the association of the γ protein with α2β2 apodinitrogenase 1 (Fig. 1, lane 3). These results are consistent with the activities observed in the FeMo-co insertion assays testing the different dinitrogenase reductase proteins (Table II).Table IISpecificity of the incorporation of molybdenum into FeMo-coExtract of strain2-aTwo hundred μl (3.8 mg of protein) of extract of strain UW45 (tungsten-grown) were used in the FeMo-co synthesis assays as a source of all nif-encoded proteins. Two hundred μl (3.6 mg of protein) of extract of strain CA117.30 (vnf-derepressed) was used as a source ofvnf-encoded proteins for the synthesis of FeV-co.Metal added2-bMolybdenum was included the assays in the form of Na2MoO4; vanadium was added to the assays as NaVO3, V2O5, VCl3, VOPO4, or [K2(H2O)5][(VO2)2(R,S-homocitrate)2]·H2O. Iron was included in the assays as FeNO3 or FeCl3.Cofactor synthesis2-cActivities are expressed as nanomoles of C2H4 formed/min/assay.nmol C2 H4 formed/min/assayUW45 (nifB −, tungsten-grown)None0.1As aboveMolybdenum18.3As aboveVanadium0.06As aboveIron0.08CA117.30 (ΔnifDKBvnf-derepressed)Molybdenum0.02As aboveVanadium0.022-a Two hundred μl (3.8 mg of protein) of extract of strain UW45 (tungsten-grown) were used in the FeMo-co synthesis assays as a source of all nif-encoded proteins. Two hundred μl (3.6 mg of protein) of extract of strain CA117.30 (vnf-derepressed) was used as a source ofvnf-encoded proteins for the synthesis of FeV-co.2-b Molybdenum was included the assays in the form of Na2MoO4; vanadium was added to the assays as NaVO3, V2O5, VCl3, VOPO4, or [K2(H2O)5][(VO2)2(R,S-homocitrate)2]·H2O. Iron was included in the assays as FeNO3 or FeCl3.2-c Activities are expressed as nanomoles of C2H4 formed/min/assay. Open table in a new tab The high degree of amino acid sequence identity between NIFH and VNFH (91%) (1Bishop P.E. Joerger R.D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1990; 41: 109-125Crossref Scopus (95) Google Scholar) is consistent with the effectiveness of VNFH in both substrate reduction (when complemented with dinitrogenase 1) and in the maturation of apodinitrogenase 1. The domain(s) of NIFH required for both the above functions are quite likely highly conserved in VNFH. At present, the role(s) of the dinitrogenase reductase protein in the maturation of apodinitrogenase 1 remains under investigation. VNFH was tested in the in vitro FeMo-co synthesis assay in place of NIFH (Table I). VNFH typically exhibited 25–30% of the FeMo-co synthesis activity (in our fixed time assay) observed with an equivalent level of NIFH, despite exhibiting similar levels of activity in the C2H2 reduction assay. Addition of increasing levels of VNFH and increasing the time allowed for in vitro FeMo-co synthesis did not result in a linear increase in activity (data not shown). The limiting step(s) in the assay is not the maturation of apodinitrogenase 1, because VNFH functions as effectively as NIFH in the maturation process (discussed above). The reasons for the lower level of FeMo-co synthesis observed with VNFH are not known. It is possible that VNFH is unable or slow to dissociate from a nif protein(s) with which it interacts during the course of FeMo-co synthesis, thus limiting further turnover of the protein(s) involved. Homer et al. (28Homer M.J. Dean D.R. Roberts G.P. J. Biol. Chem. 1995; 270: 24745-24752Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) demonstrated that the γ protein dimer (present in extracts of A. vinelandii strains unable to synthesize FeMo-co) monomerized upon associating with FeMo-co, and thus it was possible to employ the monomerization of the γ protein (detected by anoxic native PAGE) as an alternate assay for the completion of FeMo-co synthesis. Thus, FeMo-co synthesized in vitro in reaction mixtures containing an extract of strain CA12 (ΔnifHDK, nif-derepressed) would accumulate on the γ protein (resulting in the monomerization of the γ protein dimer) due to the absence of apodinitrogenase 1 in extracts of this strain. Fig. 2 is an immunoblot (developed with antibody to the γ protein) of an anoxic native gel that demonstrates the results of this study. When dinitrogenase reductase is excluded from the in vitro FeMo-co synthesis reaction, the γ dimer and a slow migrating species of γ that is uncharacterized (indicated by X on Fig. 2) are observed (Fig. 2, lane 1); the dimeric form of the γ protein is observed in extracts of strains that are impaired in FeMo-co biosynthesis (33Wright D.W. Chang R.T. Mandal S.K. Armstrong W.H. Orme- Johnson W.H. J. Bio-inorg. Chem. 1996; 1: 143-151Crossref Scopus (39) Google Scholar). That both NIFH and VNFH support FeMo-co biosynthesis is illustrated by the monomerization of the γ protein observed as the faster migrating γ protein-FeMo-co form in reactions that included NIFH or VNFH (Fig. 2, lanes 2 and 3). Does dinitrogenase reductase specify the heterometal contained in the nitrogenase cofactors? Two lines of evidence suggest that the dinitrogenase reductases do not specify or select against the heterometal that is incorporated into the cofactors of the nitrogenase enzymes: 1) the ability of VNFH to function in in vitroFeMo-co synthesis (albeit less effectively than NIFH), and 2) the observation by Joerger et al. (23Joerger R.D. Loveless T.M. Pau R.N. Mitchenall L.A. Simon B.H. Bishop P.E. J. Bacteriol. 1990; 172: 3400-3408Crossref PubMed Google Scholar) that NIFH supported vanadium-dependent diazotrophic growth of an A. vinelandii strain containing a deletion in the vnfHgene, indicating that, in vivo, NIFH functions in FeV-co biosynthesis. Gollan et al. (32Gollan U. Schneider K. Müller A. Schüddekopf K. Klipp W. Eur. J. Biochem. 1993; 215: 25-35Crossref PubMed Scopus (43) Google Scholar) demonstrated the in vivo synthesis and incorporation of FeMo-co into the dinitrogenase 3 polypeptides of a Rhodobacter capsulatus strain containing deletions in the nifHDK genes; the synthesis of FeMo-co in the absence of a nifH gene suggests that ANFH most likely replaced NIFH in the synthesis of FeMo-co. Our results demonstrating the ability of VNFH to function in the in vitro biosynthesis of FeMo-co suggest that the dinitrogenase reductase protein quite likely does not select against the incorporation of molybdenum into FeV-co and FeFe-co. Cofactor structures of the three nitrogenases are proposed to be essentially similar with vanadium and iron atoms replacing the molybdenum atom in FeV-co and FeFe-co, respectively (2Eady R.R. Adv. Inorg. Chem. 1991; 36: 77-102Crossref Scopus (59) Google Scholar, 21Davis R. Lehman L. Petrovich R. Shah V.K. Roberts G.P. Ludden P.W. J. Bacteriol. 1996; 178: 1445-1450Crossref PubMed Google Scholar, 40Smith B.E. Eady R.R. Eur. J. Biochem. 1992; 205: 1-15Crossref PubMed Scopus (101) Google Scholar). The requirement of the nifB and nifV gene products for the biosynthesis of all three cofactors suggests that certain steps in the biosynthesis of FeMo-co are shared in the biosynthetic pathways of all three cofactors. Although FeV-co is largely uncharacterized, extended x-ray absorption fine structure studies on dinitrogenase 2 indicate that FeV-co is similar in structure to FeMo-co with the octahedral vanadium atom surrounded by 3 oxygen atoms and 3 sulfur atoms as is the molybdenum atom in FeMo-co (41Arber J.M. Dobson B.R. Eady R.R. Hasnain S.S. Garner C.D. Matsushita T. Nomura M. Smith B.E. Biochem. J. 1989; 258: 733-737Crossref PubMed Scopus (49) Google Scholar). Other similarities between FeMo-co and FeV-co include the ability to extract FeV-co intoN-methylformamide (20Smith B.E. Eady R.R. Lowe D.J. Gormal C. Biochem. J. 1988; 250: 299-302Crossref PubMed Scopus (74) Google Scholar) and its probable ligation to the dinitrogenase 2 polypeptide via the conserved cysteine and histidine residues (analogous to Cys-275 and His-442 of NIFD) that ligate FeMo-co to dinitrogenase 1 (8Kim J. Rees D.C. Nature. 1992; 257: 553-560Google Scholar, 23Joerger R.D. Loveless T.M. Pau R.N. Mitchenall L.A. Simon B.H. Bishop P.E. J. Bacteriol. 1990; 172: 3400-3408Crossref PubMed Google Scholar). To determine whether the FeMo-co synthesis system would utilize vanadium and iron in the synthesis of FeV-co and FeFe-co, respectively, we tested various vanadium- and iron-containing compounds in place of molybdenum in the in vitro FeMo-co synthesis assay. Extract of A. vinelandii strain UW45 (nifB −, tungsten-grown) was used as a source of all the nif-encoded proteins necessary for the synthesis of FeMo-co. Active dinitrogenase 1 was formed only when molybdenum (in the form of Na2MoO4) was included in the in vitro reactions (Table II). Molybdenum added to in vitro FeMo-co synthesis reactions in the form of (NH4)2MoO2S2, K2MoO3S, and MoS2 also supportedin vitro FeMo-co synthesis (data not shown). Vanadium added in the form of NaVO3, V2O5, VCl3,VOPO4, or [K2(H2O)5][(VO2)2(R,S-homocitrate)2]·H2O did not produce active dinitrogenase 1. Similar results were obtained when iron (in the form of FeCl3 and Fe(II)NO3) was included in the assay. Several possibilities might account for these results. The FeMo-co synthesis machinery might indeed discriminate against vanadium and iron; however, in vivostudies demonstrating the ability of NIFEN and NIFH to support vanadium-dependent diazotrophy suggest that certainnif proteins required for FeMo-co biosynthesis do function in FeV-co biosynthesis in vivo (22Wolfinger E.D. Bishop P.E. J. Bacteriol. 1991; 173: 7565-7572Crossref PubMed Google Scholar, 23Joerger R.D. Loveless T.M. Pau R.N. Mitchenall L.A. Simon B.H. Bishop P.E. J. Bacteriol. 1990; 172: 3400-3408Crossref PubMed Google Scholar). Vanadium and iron might not be in their correct oxidation states or precursor forms necessary for incorporation into the cofactor under the in vitro assay conditions. We employed cell-free extracts of strain CA117.30 (ΔnifDKB) that was derepressed on vanadium to determine whether FeV-co could be synthesized under conditions similar to those used to synthesize FeMo-co in vitro. When extract of CA117.30 (ΔnifDKB, vnf-derepressed) was used as a source of vnf-encoded proteins in in vitroreactions containing vanadium (in the form of NaVO3, V2O5, VCl3, VOPO4, or K2(H2O)5][(VO2)2(R,S-homocitrate)2]·H2O), homocitrate, ATP (in the form of an ATP-regenerating mixture), and NifB-co, formation of active dinitrogenase 2 was not observed (Table II). Varying the nucleotides included in the reactions, the pH of the reaction mixture, and addition of partially purified apodinitrogenase 1 and NIFEN (in case of limiting levels of apodinitrogenase 2 and VNFEN in the vnf-derepressed extracts) to certain reactions also produced negative results. Clearly, the in vitro conditions under which FeMo-co is synthesized are inadequate for the synthesis of FeV-co. As discussed above, the conversion of vanadium to the form required for its incorporation into FeV-co might not occur in vitro; alternatively, intermediates in the FeV-co biosynthetic pathway might be unstable under our cell-breakage and assay conditions. These observations suggest that steps and precursors unique to the synthesis of FeV-co quite likely exist. The identification of additional vnf genes and the characterization of phenotypes of strains carrying lesions in vnf genes might enable the elucidation of steps involved in the biosynthesis of FeV-co. We thank Gary Roberts and Priya Rangaraj for helpful discussions and for critically reading this manuscript. We thank Mary Homer and Gary Roberts for making anti-γ protein antibodies available to us. Sandra Grunwald is gratefully acknowledged for providing purified R. rubrum NIFH." @default.
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• W2077875467 title "In Vitro Synthesis of the Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase" @default.
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