Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6082-6086, August 1989 Biochemistry Purification and characterization of the nifN and nifE gene products from vinelandii mutant UW45 (- biosynthss//iron-sulfur protein) TIMOTHY D. PAUSTIAN*t, VINOD K. SHAHtt, AND GARY P. ROBERTS*t§ Departments of *Bacteriology and tBiochemistry and tCenter for the Study of , College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706 Communicated by Robert H. Burris, May 15, 1989

ABSTRACT The niffl and -E gene products are involved in activity of the newly formed holodinitrogenase is then mea- the synthesis of the iron-molybdenum cofactor of dinitrogen- sured by an acetylene reduction assay in the presence of ase, the responsible for the reduction of dinitrogen to excess dinitrogenase reductase. ammonia. By using the in vitro iron-molybdenum cofactor The FeMo-co biosynthesis assay provides a system for the biosynthesis assay, we have followed the purification of these detection and isolation of factors and proteins necessary for gene products 450-fold to >95% purity. An overall recovery of FeMo-co synthesis and has been used to identify several 20% was obtained with the purified protein having a specific molecules involved in the process. Homocitrate, the product activity of 6900 units/mg of protein. The protein (hereafter of the NIFV protein, has been found to be essential for the referred to as NIFNE) was found to contain equimolar amounts synthesis of FeMo-co (17). Further work has verified the of the nifN and -E gene products and have a native molecular presence of homocitrate in the finished FeMo-co molecule mass of 200 ± 10 kDa, which indicates an a2.82 structure. (18). The in vitro FeMo-co synthesis assay, along with NIFNE was oxygen labile with a half-life of 1 min in air. A genetic evidence, has also revealed a necessity for dinitro- UV-visible spectrum of the dye-oxidized protein showed an genase reductase during the synthesis of FeMo-co (14, 15, absorption maximum at 425 nm that could be bleached by 18). In this paper we report the utilization ofthis assay for the reduction of NIFNE with sodium dithionite, suggesting the purification of the niJN and -E gene product complex and its presence of an Fe center in NIFNE. characterization. Biological nitrogen fixation is carried out by the bacterial enzyme nitrogenase. Nitrogenase catalyzes the reduction of MATERIALS AND METHODS dinitrogen to ammonia and is composed of two separate Materials. The DEAE-cellulose used was Whatman DE-52 protein complexes, dinitrogenase reductase (also called com- microgranular. The PL-SAX column was obtained from ponent II or iron protein) and dinitrogenase (component I or Polymer Laboratories, Amherst, MA. molybdenum iron protein) (1, 2). Dinitrogenase reductase is Buffer Preparation and Definition. All buffers were titrated a dimer of the nifH gene product with a Mr = 60,000-70,000 to correct pH at room temperature. Anaerobic buffers were (3). It is extremely sensitive to oxygen, contains a 4Fe-4S prepared as described (16). The following abbreviations for center, and is responsible for the reduction of dinitrogenase buffers are used throughout the text. MD buffer is 25 mM (2, 3). Dinitrogenase is an a232 tetramer of the nifK and -D Mops-NaOH (pH 7.4) and 1.7 mM sodium dithionite. MGD gene products and has a Mr of 220,000-240,000 (3). Dinitro- buffer is 25 Mops-NaOH (pH 7.4), 20% glycerol, and 1.7 mM genase is also a metalloenzyme containing Fe, Mo, and S in sodium dithionite. various centers on the protein (4, 5). One ofthese centers, the Bacterial Strains and Growth Conditions. Klebsiella pneu- iron-molybdenum cofactor (FeMo-co), is thought to be the moniae strain UN1217, containing the niflN4536 mutation, an for the reduction of nitrogen gas (6-8). The internal deletion of the niJN gene (19), and Azotobacter isolation and characterization of FeMo-co by Shah and Brill vinelandii strain UW45, containing a point mutation in the from purified dinitrogenase showed that the cofactor con- nifB gene (20-22), have been described. Growth and dere- tained Mo, Fe, and S in a ratio of 1:8:6 (4). Subsequent pression ofK. pneumoniae mutants have been described (11, isolation and analysis of FeMo-co by other groups has 23). suggested different stoichiometries of these elements in A. vinelandii cells were grown in modified Burk's minimal FeMo-co, but the consensus remains in the range of 1 medium (24). A. vinelandii strain UW45 was derepressed for Mo:6-8 Fe:8-10 S (3, 4, 8-10). The structure of FeMo-co the nifN and -E gene products, hereafter referred to as remains unknown. NIFNE, as follows. A single colony was inoculated into 100 The products of six nifgenes have been implicated in the ml of Burk's medium plus 28 mM ammonium acetate and synthesis ofthis cofactor in vivo, nifQ, -B, -N, -E, -H, and -V incubated overnight at 30'C. A 2.8-liter flask containing 750 (11-16). Recently, an assay for the in vitro synthesis of ml ofthe same medium was inoculated with 1 ml ofthis starter FeMo-co has been developed (16). This assay involves the culture and incubated in an identical fashion. An 8-liter mixing of extracts of two mutants having lesions in genes carboy containing 6 liters ofthe same medium was inoculated involved in different steps of FeMo-co biosynthesis. Al- with the 750 ml ofculture and incubated with sparging at 30'C though neither mutant is capable of synthesizing FeMo-co overnight. Three hundred liters of Burk's medium was pre- separately, when extracts of appropriate mutants are mixed pared and to this was added 600 ml of filter-sterilized 22% in the presence of an ATP-generating system and molybdate, ammonium acetate to a final concentration of 5.7 mM. The 6 FeMo-co is synthesized. FeMo-co thus synthesized activates liters of overnight UW45 culture was added to bring the FeMo-co-less dinitrogenase present in the extracts. The starting ODwo to 0.050-0.075. The culture was incubated at 30'C and aerated at 140 liters/min with an agitation rate of 150 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: FeMo-co, iron-molybdenum cofactor. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 6082 Downloaded by guest on September 24, 2021 Biochemistry: Paustian et al. Proc. Natl. Acad. Sci. USA 86 (1989) 6083 rpm. The presence of ammonium ion was monitored by using linear gradient from 1.0 to 0.0 M (NH4)2SO4 and 0.5-ml Nessler's reagent and after 10-14 hr, ammonium ion was fractions were collected. Pure NIFNE eluted at 0.5 M depleted. The cells were harvested 4 hr after the depletion of (NH4)2S04. ammonia using a Sharples continuous centrifuge. Final OD6w Purified NIFNE was mixed with an equal volume of of the culture was 2.8-3.2. Cell paste was frozen in liquid anaerobic 50%o glycerol in MD buffer to stabilize the activity. nitrogen and stored at -80'C. Typical yields were 950 g ofcell For storage, all fractions were quick frozen in a dry ice/ paste from a 300-liter fermenter. ethanol bath and stored at -20'C. NIFNE stored in this Cell Extract Preparation. K. pneumoniae cell extracts were manner was stable for >3 months. prepared as described except 0.1 M Mops-NaOH (pH 7.4) Oxygen Stability. Twelve hundred fifty units of NIFNE in was substituted for Tris HCI (16). A. vinelandii cell extracts 0.5 ml ofMD buffer was placed into a 9-ml stoppered vial that were prepared by osmotic shock as described (25). had been evacuated and flushed three times with argon and FeMo-co Synthesis Assay. The FeMo-co synthesis assay rinsed with 0.3 ml of MD buffer. The vial was then brought has been described (16, 17). Protein concentrations were to 0.2 atm (1 atm = 101.3 kPa) of oxygen and incubated with determined by the bicinchoninic acid method (26). shaking at 30'C. Samples were taken at 0, 1, 2, 3, 4, 5, and Purification of NIFNE. All steps of the purification were 15 min and were assayed for NIFNE activity. The control carried out under anaerobic conditions at 40C unless other- reaction was carried out in the same manner, without oxygen wise noted. Frozen A. vinelandii UW45 cell paste (240 g) was added. thawed and broken by osmotic shock into 1 liter of MD UV-Visible Scan of N1FNE. Sodium dithionite in the solu- buffer. The lysed cells were centrifuged at 16,000 x g for 50 tion was removed from 4 mg of NIFNE by using a PD10 min at 4TC. The supernatant was applied to a 5 x 25 cm column in an anaerobic glove box. The desalted enzyme (1.3 DEAE-cellulose column that had been reduced with 4 bed ml) was oxidized by mixing with methyl viologen, in its volumes of0.1 M NaCl MD buffer and equilibrated with 1 bed oxidized form, and desalted again with a PD10 column into volume of MGD buffer. After the extract was applied, the 100 mM Mops-NaOH (pH 7.5) to remove the methyl violo- column was washed with 500 ml of MGD buffer followed by gen. The oxidized NIFNE was placed in an anaerobic cuvette 500 ml of 0.11 M NaCl in MGD buffer. NIFNE was eluted and removed from the glove box, and a spectrum was from the column with 0.185 M NaCl in MGD buffer and 50-ml recorded from wavelengths of 600-200 nm with a Shimadzu fractions were collected anaerobically. UV160 scanning spectrophotometer. The spectrum of re- A 5 x 8 cm reactive red-120 agarose column was reduced duced NIFNE was obtained after the addition of dithionite. with 5 bed volumes of MD buffer. The column was further Metal Analyses. Metal analyses were carried out with an washed with 1 bed volume of 10 mM MgCl2 in MGD buffer. Applied Research Laboratories 34000 inductively coupled DEAE column fractions, containing NIFNE, were diluted plasma atomic emission spectrophotometer at the University with an equal volume of 20 mM MgCl2 in MGD buffer and of Wisconsin Soil and Plant Analysis Laboratory on a de- applied to the reactive red-120 column. The column was salted sample ofNIFNE. A small portion ofthe tested sample washed with 10 mM MgCl2 in MGD buffer followed by MGD was saved for protein assay. Contaminating metals in the buffer containing 1 mM ADP, 0.15 M NaCl, and 10 mM Mops buffer used for all metal analyses were removed by MgCl2. NIFNE was then eluted with 10 mM ATP/0.15 M passage through a Bio-Rad Chelex-100 column. NaCl in MGD buffer. Fractions containing NIFNE were Molecular Mass Determination by Gel Filtration. A 1.3 X 85 immediately applied onto a 1.5 x 8 cm Q Sepharose column cm Sephacryl S-300 column was equilibrated with 200 ml of that had been equilibrated with 0.15 M NaCl in MGD buffer 0.2 M NaCl in MGD buffer. One milliliter of NIFNE (1 mg) and NIFNE was eluted with 0.3 M NaCl in MGD buffer. was applied and the column was developed with the same The active fraction from the Q Sepharose column, about 20 buffer at a flow rate of 9 cm/hr. After 50 ml of buffer had ml, was desalted on a 2.5 x 18 cm Sephadex G-10 column eluted, 2-ml fractions were collected anaerobically and as- equilibrated with MGD buffer. To the desalted fraction, 0.83 sayed for activity. NIFNE eluted at 70 ml, appropriate for a ml of anaerobic 0.3 M ATP (pH 7.4) was added to make the protein of 200 ± 10 kDa. The column was calibrated with the fraction 10 mM in ATP. following protein standards: ferritin (450 kDa), catalase (240 Pharmacia-LKB HPLC was used for the final two steps of kDa), aldolase (158 kDa), bovine serum albumin (68 kDa), the purification. Buffers used for the HPLC were filtered chicken albumin (45 kDa), chymotrypsinogen A (21 kDa), through a 0.22-,um membrane and made anaerobic by sparg- and cytochrome c (12.5 kDa). ing with helium gas for 30 min. Sodium dithionite was then added and sparging was continued during use of the HPLC. All HPLC columns were operated at room temperature. The RESULTS desalted fraction, containing NIFNE, was applied to a 0.75 x Purification of NIFNE. Table 1 is a summary of the puri- 15 cm PL-SAX column that had been equilibrated with 50 fication protocol for NIFNE. The two polypeptides copuri- mM NaCl in MGD buffer. The column was washed with 2 bed fled through the entire procedure, suggesting that the proteins volumes of 50 mM NaCl in MGD buffer and then developed form a complex. Pure NIFNE had a specific activity of 6900 with a linear gradient of 50-500 mM NaCl in MGD buffer units/mg in the assay. However, due to the indirect coupled (volume, 130 ml). Four-milliliter fractions were collected nature of the assay, the specific activity varied slightly anaerobically and NIFNE was eluted from the column at 225 (±650). A 450-fold purification was achieved with typical mM NaCI. recoveries in the 15-25% range. One-dimensional NaDod- Active fractions from the PL-SAX column (three fractions, S04/polyacrylamide gel electrophoresis ofthe samples taken 12 ml) were precipitated by addition of 5.16 g of (NH4)2SO4 throughout the purification protocol is shown in Fig. 1. (65% saturation) under anaerobic conditions and the precip- Estimation of the purity of NIFNE by scanning densitometry itate was collected by centrifugation. The supernatant was of an overloaded Coomassie blue-250-stained gel showed the discarded and the precipitate was resuspended in 5 ml of 1.0 two polypeptides composed >95% of the protein in the M (NH4)2SO4 in MD buffer. The precipitated NIFNE fraction purified fraction. was then applied to a 0.5 x 5 cm Alkyl Superose column that When compared to standards, the apparent molecular had been equilibrated with 1.5 M (NH4)2SO4 in MD buffer. masses of the nifN and -E gene products are 47,000 Da and The column was washed with 1.5 bed volumes of the same 49,000 Da, respectively. This agrees well with the published buffer and then washed with 1 bed volume of 1 M (NH4)2SO4. molecular masses of 49,186 Da and 50,236 Da calculated for NIFNE was eluted from the column by a 7.3-ml decreasing nifN and -E gene products from the sequence of these genes Downloaded by guest on September 24, 2021 6084 Biochemistry: Paustian et al. Proc. Natl. Acad. Sci. USA 86 (1989) Table 1. Summary of the purification of NIFNE Protein, Volume, Activity,* Specific activity, Fold % Sample mg ml units units/mg purification recovery UW45 extract 12,500 1000 192,000 15 1 100 DEAE-cellulose 1,970 145 244,500 124 8 127 Reactive red/Q Sepharose 39 20 106,900 2738 178 56 PL-SAX 11 12 50,300 4620 301 26 Alkyl Superose 6 3 38,700 6900 450 20 *One unit corresponds to 1 nmol ofacetylene reduced per min at 300C. Assays performed after the reactive red-120 column included 200 1A of the flow-through fraction of the reactive red-120 column. (27, 28). The evidence that the purified proteins are the A difference spectrum is shown in the Inset to Fig. 4. The products of the nifNVE genes is the following: (i) the proteins maximum absorption difference for the oxidized minus the were purified by a functional assay based on their ability to reduced spectrum occurred at 425 nm. complement a K. pneumoniae mutant lacking the nifNE gene Metal Analysis. Metal analysis revealed the presence of products, and (ii) the sequences of the N termini of the significant amounts of Fe, Zn, and Cu associated with purified polypeptides match the predicted amino acid se- NIFNE. The Fe content was found to be 0.810 ppm (detec- quences for NIFN and -E derived from the DNA sequence. tion limit, 0.011), which corresponds to 4.6 mol of Fe per mol Surprisingly, this analysis also reveals that the terminal of NIFNE. The Zn content was 0.248 ppm (detection limit, methionine is removed from the NIFN polypeptide, whereas 0.010), which corresponds to 1.2 Zn per NIFNE. The Cu the NIFE protein is unprocessed. content was 0.126 ppm (detection limit, 0.025), which corre- Stoichiometry of Polypeptides in the NifVE Complex. Gel sponds to 0.63 Cu per NifNE complex. NIFNE was also filtration of NIFNE on a Sephacryl S-300 column indicated a tested for the presence of Cd, Cr, Ni, Li, Co, Mn, Mo, As, molecular mass of200 + 10 kDa for the protein complex (Fig. and Pb, which were all below detection limits. The molar 2). The results of the polyacrylamide gel indicate NIFNE ratio of metals per protein complex must be viewed with contains equal amounts of the NIFN and -E polypeptides. caution due to uncertainties in the measurement of the When the molecular masses ofthe nifN and -E gene products protein concentration. are taken into account, it is likely that NIFNE is an a2,82 Titration of the FeMo-co Synthesis Assay with NIFNE. A tetramer in the native complex. titration with NIFNE was performed to show that the protein Oxygen Stability. When exposed to oxygen. NIFNE de- is the limiting factor in the FeMo-co synthesis assay when at clined in activity with a half-life of 1 min (Fig. 3). Vigorous low concentrations (Fig. 5). The assay is linear at low shaking of the protein in an anaerobic environment did not concentrations ofNIFNE; however, the assay soon becomes result in the inactivation of the protein. NIFNE therefore is nonlinear. An excess of nifB gene product, homocitrate, inactivated in the presence of oxygen and, as noted below, dinitrogenase reductase, and FeMo-co-less dinitrogenase are this inactivation is likely due to the oxidation of an Fe-S present in the FeMo-co synthesis assay. The loss of linearity center on the protein. may therefore be due to limiting concentrations of other UV-Visible Spectrum of NIFNE. Purified NIFNE has a unknown factors needed in the assay (as discussed below). yellow-brown color and a UV-visible spectrum ofthe protein A Factor in FeMo-co Biosynthesis. During the purification of was recorded to characterize this absorbance. Oxidized NIFNE a loss in recovery was observed after the reactive NIFNE has a spectrum with an absorption shoulder in the red-120 step; typically a 20-30%o recovery was obtained. 390- to 430-nm range, typical of an Fe-S protein, and reduc- tion of NIFNE caused this signal to disappear 4) (29). (Fig. 10 6 LANE 1 2 3 4 5 6 7

zma 4. 10 5

10 4 0.80 FIG. 1. Purification of NIFNE. Active fractions from each pu- rification step were analyzed by discontinuous NaDodSO4/poly- Kav acrylamide gel electrophoresis followed by staining with Coomassie blue-250. The stacking gel was 2.8% polyacrylamide and the running FIG. 2. Gel filtration of NIFNE. One milligram of NIFNE was gel was 12% polyacrylamide. Lanes and 7, molecular mass standards chromatographed on a Sephacryl S-300 column at a flow rate of 9 (15 A.g); lane 2, UW45 extract (50.pg); lane 3, DEAE-cellulose fraction cm/hr. Standards used were ferritin (440 kDa), catalase (232 kDa), (50 pg); lane 4, reactive red-120/Q Sepharose fraction (14 ,±g); lane 5, aldolase (158 kDa), albumin (67 kDa), ovalbumin (45 kDa), chymo- PL-SAX fraction (6 u.g); lane 6, Alkyl Superose fraction (2 j&g). trypsinogen (25 kDa), and cytochrome c (12.5 kDa). Kav is the Standards used were phosphorylase B (93 kDa), albumin (67 kDa), partition coefficient calculated for each protein. Two independent ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin experiments were performed. The point of NIFNE elution is noted inhibitor (21.5 kDa), and lysozyme (14.5 kDa). by the circle. Downloaded by guest on September 24, 2021 Biochemistry: Paustian et al. Proc. Natl. Acad. Sci. USA 86 (1989) 6085

100

80

~60

40

20 30 gig NifNE

0I FIG. 5. Titration of the FeMo-co synthesis assay with NIFNE. 0 5 10 15 The indicated amount of NIFNE was assayed by using the standard Time (min) FeMo-co synthesis assay. All known components in the assay were provided in excess, except for NIFNE. FIG. 3. Oxygen stability ofNIFNE. NIFNE was incubated in the presence ot u. z aim ou oxygen. At niiie were removed and assayed for Fe iaiicaatimesi poinit, tpo Lion shoulder ofthe oxidized protein at 425 nm, which was absent hundred percent activity correspoinds to 16.8 nmol of acetylene in the dithionite-reduced spectrum. The protein contained 4.6 reduced per min. NIFNE activity shaken in air (A) and activity mol of Fe, 1.2 mol of Zn, and 0.63 mol of Cu per mol of shaken under an argon atmosphere (*) are shown. NIFNE. NIFNE contains some type ofoxidizable Fe center, which is probably the reason the enzyme is oxygen sensitive. When the flow-through fraction from the column was added The hypothesis that the nifN and -E gene products might to the assay of the peak fracti(an, a 2-fold stimulation was form a complex was first put forward by Roberts et al. (12) noticed. This nonbinding fracti4 on did not have NIFNE ac- on the basis of genetic evidence and the visualization of the tivity of its own nor did it replEace dinitrogenase reductase, products in two-dimensional gels of crude extracts. Brigle ATP, Mo, homocitrate, or Ut41217 extract in the assay. and coworkers (27, 28) have sequenced these genes and have Further tests showed this facto]r to be oxygen-stabile, inac- also suggested this possibility. Here we have presented tivated by boiling, and unable to pass through a YMT30 biochemical evidence that the two polypeptides do form an ultrafiltration membrane that has a molecular mass cut-offof a232 complex of 200 kDa. 30 kDa. During further purificatiion steps the factor continued The sequencing ofnijN and -E has also revealed amino acid to provide a 2-fold stimulating ac:tivity. The assays ofNIFNE sequence homology between these gene products and the after the reactive red-120 colur in included this factor. nifK and -D gene products, respectively (27, 28). Our work suggests that NIFNE and dinitrogenase have a similar a2132 DISCUS,SION subunit structure. Taken together these results may indicate an evolutionary relationship and suggest that NIFNE may The nifN and -E gene products have been purified 450-fold need to mimic dinitrogenase to carry out its function in with a 20% recovery. The add]ition of 20%o glycerol to the FeMo-co synthesis. This could include binding an unfinished purification buffers greatly en hanced the stability of the form of FeMo-co, modifying it and then passing it on either enzyme and allowed its purificEation to >95%. NIFNE was to dinitrogenase or some other protein, or serving as a found to be oxygen-labile with a half-life of 1 min in air. A scaffolding protein where FeMo-co is built. Alternatively, UV-visible spectrum of the prc)tein revealed an absorption NIFNE may need to have this similar structure to facilitate interaction between the NIFNE complex and dinitrogenase 0.600 0.10 reductase, possibly involving the reduction of NIFNE during oxidized reduced its reaction cycle. NIFNE isolated from the mutant UW45 absorbs light in the visible spectrum when in an oxidized state. This absorbance 0.00 / \ is bleached out when the protein is reduced with sodium 0.400 °° 425 600 dithionite. A difference spectrum of the oxidized minus the Wavelength() reduced protein reveals an absorption maximum of 425 nm,

c: us typical for an Fe-S protein (29). r_ Cysteine residues in proteins are known to be ligands for iron-containing prosthetic groups in many metalloproteins 0.200 (29-31). Comparison of the NifE protein sequence to the NifD protein sequence reveals four highly conserved cys- teine residues (27). Brigle et al. (27) proposed that these conserved cysteines may be involved in binding metal- 0.000 I containing centers to NIFNE, and results here support that hypothesis in establishing the presence of an iron-containing 200 300 400500 600 prosthetic group on NIFNE. Wave]length (nm) What is the function of this Fe-S center? One possibility is FIG. 4. UV-visible spectrum ofF oxidized and reduced NIFNE. that the Fe-S center is needed for the transfer ofelectrons that NIFNE was desalted on a PD10 column and then oxidized by are a part of NIFNE's function. Molybdenum in molybdate treatment with methyl viologen. (Inset) Oxidized minus reduced is in the +6 oxidation state and during the formation of spectrum for NIFNE with an absorption maximum occurring at 425 FeMo-co its valence could change. NIFNE may be involved nm. in this redox reaction and the Fe-S center may be needed to Downloaded by guest on September 24, 2021 6086 Biochemistry: Paustian et al. Proc. Natl. Acad Sci. USA 86 (1989) harbor the electrons used to act on molybdenum. A second 1. Bulen, W. A. & Lecomte, J. R. (1966) Proc. Natl. Acad. Sci. possibility is that the Fe-S center is itself the intermediate of USA 56, 979-989. FeMo-co biosynthesis, which is bound to NIFNE. 2. Hagemen, R. V. & Burris, R. H. (1978) Proc. Natl. Acad. Sci. USA 75, 2699-2702. The metal analysis also revealed the presence ofZn and Cu 3. Shah, V. K., Ugalde, R. A., Imperial, J. & Brill, W. J. (1984) in the NIFNE complex. It is unlikely that these metals were Annu. Rev. Biochem. 53, 231-257. contaminants in the buffer since all buffers used for this 4. Shah, V. K. & Brill, W. J. (1977) Proc. Natl. Acad. Sci. USA analysis had been stripped of metals by passage through a 74, 3249-3253. Chelex-100 column and, when this buffer was analyzed, all 5. Orme-Johnson, W. H. (1985) Annu. Rev. Biophys. Chem. 14, metals were below detection limits. The Zn and Cu found in 419-459. 6. Hawkes, T. R., McLean, P. A. & Smith, B. E. (1984) Biochem. the protein sample could be contaminants that are nonspe- J. 217, 317-321. cifically bound to NIFNE and have no biological function. It 7. Shah, V. K., Chisnell, J. R. & Brill, W. J. (1978) Biochem. is also possible that these metals are serving some function on Biophys. Res. Commun. 81, 232-236. NIFNE. 8. Smith, B. E., Bishop, P. E., Dixon, R. A., Eady, R. R., Filler, The source of NIFNE used for these experiments is the A. W. A., Lowe, D. J., Richards, A. J. M., Thomson, A. J., vinelandii mutant UW45. NIFNE obtained from this source Thorneley, R. N. F. & Postgate, J. R. (1985) in Nitrogen Fixation Research Progress, eds. Evans, H. J., Bottomley, may have different properties when compared to NIFNE P. J. & Newton, W. E. (Nijhoff, Boston), pp. 597-603. isolated from wild type, since the nifB mutation in UW45 9. Nelson, M. J., Levy, M. A. & Orme-Johnson, W. H. (1983) could either cause or prevent the accumulation of an inter- Proc. Natl. Acad. Sci. USA 80, 147-150. mediate on NIFNE. Metal analysis of NIFNE did not detect 10. Yang, S.-S., Pan, W.-H., Friesen, G. D., Burgess, B. K., the presence of Mo, although Mo was present in the medium Corbin, J. L., Stiefel, E. I. & Newton, W. E. (1982) J. Biol. used for the growth of UW45. The absence of Mo eliminates Chem. 257, 8042-8048. 11. Imperial, J., Ugalde, R. A., Shah, V. K. & Brill, W. J. (1984) the possibility that a Mo-containing intermediate accumu- J. Bacteriol. 158, 187-194. lates on NIFNE during derepression of UW45. However, it 12. Roberts, G. P., MacNeil, T., MacNeil, D. & Brill, W. J. (1978) is still possible that Mo could be present on NIFNE isolated J. Bacteriol. 136, 267-279. from wild type. 13. Ugalde, R. A., Imperial, J., Shah, V. K. & Brill, W. J. (1984) It is interesting that NIFNE binds tightly to reactive J. Bacteriol. 159, 888-893. red-120 agarose, a triazine dye column. Many proteins that 14. Filler, W. A., Kemp, R. M., Ng, J. C., Hawkes, T. R., Dixon, R. A. & Smith, B. E. (1986) Eur. J. Biochem. 160, 371-377. bind to triazine dye columns are known to be nucleotide- 15. Robinson, A. C., Dean, D. R. & Burgess, B. K. (1987) J. Biol. utilizing (32), and the elution of NIFNE with ATP Chem. 262, 14327-14332. suggests that the protein can at least bind this nucleotide. It 16. Shah, V. K., Imperial, J., Ugalde, R. A., Ludden, P. W. & is also known that ATP is needed for FeMo-co synthesis to Brill, W. J. (1986) Proc. Natl. Acad. Sci. USA 83, 1636-1640. take place (16). NIFNE may require ATP to carry out its 17. Hoover, T. R., Shah, V. K., Roberts, G. P. & Ludden, P. W. function in FeMo-co synthesis; however, experiments per- (1986) J. Bacteriol. 167, 999-1003. formed to demonstrate ATP hydrolysis by the NIFNE com- 18. Shah, V. K., Hoover, T. R., Imperial, J., Paustian, T. D., Roberts, G. P. & Ludden, P. W. (1988) in Nitrogen Fixation: plex have not been successful (data not shown). Hundred Years After, eds. Bothe, H., de Bruijn, F. J. & During the purification of NIFNE, a factor was isolated Newton, W. E. (Fisher, New York), pp. 115-120. that stimulated the activity ofthe assay 2-fold. This factor had 19. MacNeil, T., MacNeil, D., Roberts, G. P., Supiano, M. A. & no detectable NIFNE activity and could not replace any of Brill, W. J. (1978) J. Bacteriol. 136, 253-266. the known components of FeMo-co synthesis. Tests showed 20. Nagatani, H. H., Shah, V. K. & Brill, W. J. (1974) J. Bacteriol. it to be oxygen-stable, inactivated when boiled, and unable to 120, 697-701. pass through a 30-kDa ultrafiltration membrane. Also, this 21. Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H. & Brill, W. J. (1973) Biochim. Biophys. Acta 292, factor was not found in crude extracts of A. vinelandii that 246-255. had been grown in the presence of ammonia. These experi- 22. Bishop, P. E. & Brill, W. J. (1977) J. Bacteriol. 130, 954-956. ments suggest that the factor is a protein made under am- 23. Nieva-Gomez, D., Roberts, G. P., Klevickis, S. & Brill, W. J. monia-limiting conditions. In the present assay the factor is (1980) Proc. Natl. Acad. Sci. USA 77, 2555-2558. not absolutely essential, probably due to the presence of a 24. Strandberg, G. W. & Wilson, P. W. (1968) Can. J. Microbiol. similar factor in the K. pneumoniae UN1217 crude extract. 14, 25-31. The stimulation of the assay by the added factor can be 25. Shah, V. K., Davis, L. C. & Brill, W. J. (1972) Biochim. if the K. factor is in the Biophys. Acta 256, 498-511. explained pneumoniae limiting assay. 26. Smith, P. K., Krohn, R. I., Hermanson, A. K., Mallia, A. K., Efforts must be directed at identifying this factor, improving Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, the assay for it, and, if it is a protein, finding the gene that N. M., Olson, B. J. & Klenk, D. C. (1985) Anal. Biochem. 150, codes for it. 76-85. 27. Brigle, K. E., Weiss, M. C., Newton, W. E. & Dean, D. R. We thank Paul Ludden, Tim Hoover, Juan Imperial, and Mark (1987) J. Bacteriol. 169, 1547-1553. Madden for many helpful discussions. We also thank Scott Ensign 28. Brigle, K. E. & Dean, D. R. (1985) Proc. Natl. Acad. Sci. USA for assistance with the UV-visible scan and metal analysis and Dr. 82, 5720-5723. James Howard for generously offering to sequence the NIFN and E 29. Orme-Johnson, W. H. (1973) Annu. Rev. Biochem. 42, 159- proteins. This research was supported by the College ofAgricultural 204. and Life Sciences, University of Wisconsin, and by Public Health 30. Thompson, S., Cass, K. & Stellwagen, E. (1975) Proc. Natl. Service Grant GM-35163 from the National Institutes of Health. Acad. Sci. USA 72, 669-672. T.D.P. was supported in part by Cellular and Molecular Biology 31. Yoch, D. C. & Carithers, R. P. (1979) Microbiol. Rev. 43, Training Grant GM07215 from the National Institutes of Health. 384-421. V.K.S. was supported by Public Health Service Grant GM35332 32. Thony, B., Kaluza, K. & Hennecke, H. (1985) Mol. Gen. from the National Institutes of Health. Genet. 198, 441-448. Downloaded by guest on September 24, 2021