©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characteristics of NIFNE in Azotobacter vinelandii Strains
IMPLICATIONS FOR THE SYNTHESIS OF THE IRON-MOLYBDENUM COFACTOR OF DINITROGENASE (*)

(Received for publication, October 20, 1994)

Jon T. Roll (1) Vinod K. Shah (2) Dennis R. Dean (3) Gary P. Roberts (1)

From the  (1)Departments of Bacteriology and (2)Biochemistry and Center for Studies of Nitrogen Fixation, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706 and the (3)Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The products of the nifN and nifE genes of Azotobacter vinelandii function as a 200-kDa alpha(2)beta(2) tetramer (NIFNE) in the synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase, the enzyme system required for biological nitrogen fixation. NIFNE was purified using a modification of the published protocol. Immunoblot analysis of anoxic native gels indicated that distinct forms of NIFNE accumulate in strains deficient in either NIFB (DeltanifB::kan DeltanifDK) or NIFH (DeltanifHDK). During the purification of NIFNE from the DeltanifHDK mutant, its mobility in these gels changed, becoming similar to that of NIFNE from the DeltanifB::kan DeltanifDK mutant. While NIFB activity initially co-purified with the NIFNE activity from the DeltanifHDK mutant, further purification of NIFNE activity resulted in the loss of the co-purifying NIFB activity; this loss correlated with the change in NIFNE mobility on native gels. These results suggest that the form of NIFNE accumulated in the DeltanifHDK mutant is associated with NIFB activity in crude extract but loses this association during NIFNE purification. Addition of the purified metabolic product of NIFB, termed NifB-co, to either NIFNE purified from the DeltanifHDK strain or to the NIFNE in crude extract of the DeltanifB::kan DeltanifDK strain caused a change in the mobility of NIFNE on anoxic native gels to that of the form accumulated in a DeltanifHDK mutant. These results support a model where both NifB-co and dinitrogenase reductase participate in FeMo-co synthesis through NIFNE, which serves as a scaffold for this process.


INTRODUCTION

Biological nitrogen fixation is the process by which atmospheric nitrogen is reduced to ammonium. This process is carried out by the nitrogenase enzyme system, which is composed of two separate protein complexes(1, 2) . Dinitrogenase reductase (component II, iron protein, NIFH), a 65-kDa alpha(2) dimer of the nifH gene product, serves as a specific electron donor to the other protein complex, dinitrogenase. Dinitrogenase (component I, molybdenum-iron protein, NIFKD) is a 240-kDa alpha(2)beta(2) tetramer of the nifK and nifD gene products. For catalytic activity, dinitrogenase requires a unique cofactor, FeMo-co, (^1)which contains iron, molybdenum, acid-labile sulfur, and homocitrate. FeMo-co is thought to provide the site of substrate reduction(3, 4, 5) . FeMo-co has been isolated and can be used to activate dinitrogenase lacking FeMo-co (apodinitrogenase)(6) . Surprisingly, functional FeMo-co can be synthesized in the absence of dinitrogenase in vivo and in vitro(7) , indicating that it is synthesized elsewhere and inserted into the apo form of dinitrogenase.

To date, the products of six nif genes have been strongly implicated in the in vivo synthesis of FeMo-co: nifN, nifE, nifB, nifH, nifQ, and nifV. The purification and characterization of a number of molecules involved in FeMo-co synthesis has been made possible by the development of an in vitro FeMo-co synthesis system, which depends upon the above mentioned gene products as well as MgATP and MoO(4)(8) . The products of nifN and nifE function as a 200-kDa alpha(2)beta(2) tetramer (NIFNE) that has been purified, partially characterized, and found to contain what appears to be a single 4Fe-4S center(9) .

The role of NIFB in FeMo-co synthesis is undefined, but recently a small molecule containing iron and sulfur, thought to be a FeMo-co precursor produced by the activity of NIFB, termed NifB-co, has been isolated(10) .

Dinitrogenase reductase (NIFH), in addition to its roles in the maturation of apodinitrogenase (11, 12, 13) and donation of electrons to dinitrogenase(2, 14) , also plays an unknown role in FeMo-co synthesis (15, 16, 17, 18) .

NIFQ apparently plays a role in molybdenum processing, possibly in the formation of molybdenum-sulfur compounds required during FeMo-co synthesis(19, 20) . NIFV is probably a homocitrate synthase(21) , and homocitrate is an organic constituent of FeMo-co(21, 22) . Recently, the absence of other gene products, namely NIFW, NIFZ, and NIFM, has been shown to have some effect on FeMo-co synthesis(23, 24) .

Although the exact role of NIFNE in FeMo-co synthesis is unknown, it is thought to serve as a scaffold on which FeMo-co is built for the following reasons: first, the genes nifN and nifE exhibit striking similarity to nifK and nifD, respectively(25, 26) , and the cysteine residue which serves as the FeMo-co ligand in NIFKD is also conserved in NIFNE(25) . Second, both proteins function as alpha(2)beta(2) tetramers; and functional FeMo-co can be synthesized in the absence of dinitrogenase, suggesting another protein might serve as a scaffold(7) .

The purpose of this work is to further our understanding of the process of FeMo-co synthesis by examination of electrophoretic mobility in anoxic native gels of NIFNE accumulated in mutants blocked at different steps in FeMo-co synthesis. The rationale of this approach is that if NIFNE does function as a scaffold for FeMo-co synthesis, different FeMo-co precursors might accumulate on NIFNE in mutants blocked in different steps in FeMo-co biosynthesis.


EXPERIMENTAL PROCEDURES

Strains and Growth Conditions

Klebsiella pneumoniae strain UN1217 (nifN4536) has been described (27) and was grown as previously described(10) . Azotobacter vinelandii strains CA12 (DeltanifHDK)(28) , DJ (wild type)(29) , and DJ35 (25) have been described. DJ677 (DeltanifB::kan DeltanifDK) was constructed by transforming strain DJ33 (30) with a hybrid plasmid (pDB218) which carries both an insertion and deletion in the nifB gene. pDB218 was constructed by replacing a SphI restriction fragment contained within the nifB-coding sequence (31) with a Km^R gene cassette isolated from pUC-KISS(32) . The parental plasmid used in the construction of pDB218 was pDB160 and it contains a 1.7-kb SalI restriction fragment from the A. vinelandii genome cloned into the SalI restriction enzyme site of pUC7. The insertion and deletion of the appropriate nifB region in strain DJ677 was accomplished by selecting transformants that exhibited Km^R and Amp^S as described previously(30) .

A. vinelandii strains CA12 and DJ were grown by inoculating 220 ml of modified Burk's medium (33) + 5 mM NH(4)Cl with 1 ml of a cell stock frozen at -80 °C in 20% glycerol. This culture was grown overnight at 30 °C in a baffled flask with vigorous shaking. It was then divided between two 20-liter carboys containing 20 liters of modified Burk's medium + 2.8 mM NH(4)Cl. These carboys were placed in a 32 °C water bath and sparged vigorously with filter-sterilized air until 5.5 h after exhaustion of ammonium, as determined by Nessler's reagent. Cells were harvested by concentration using a Pellicon 0.45-µm tangential flow cell concentrator (Millipore) followed by centrifugation. Cell pellets were frozen in liquid nitrogen and stored at -80 °C until use. Cells were broken anoxically by osmotic shock as described(34) . All crude extracts were made 10% in glycerol and stored as 5-ml aliquots in anoxic vials at -80 °C.

A. vinelandii strain DJ677 was grown as above except that the carboys had 30 ml of 15.3% NH(4)Cl added to assure excess ammonium. Three hundred eighty liters of modified Burk's medium in a 400-liter fermentor was inoculated with 1 liter of 22% ammonium acetate (Fisher) and the 20-liter overnight culture. The cells were harvested at 5.5 h after exhaustion of ammonium with a Sharples continuous flow centrifuge.

Purification of NIFNE

NIFNE was purified according to Paustian et al.(9) with the following modifications. The DE52 column was washed with 1 liter of 0.11 M NaCl, and NIFNE was eluted with 0.2 M NaCl (NIFNE from wild-type was eluted with 0.16 M NaCl). The volume of the Reactive Red 120 column used in the second step was 315 cm^3. The fraction from the Q-Sepharose column containing NIFNE was diluted in an equal volume of buffer containing 1.5 M (NH(4))(2)SO(4) (Life Technologies, Inc.), 0.1 M MOPS (Sigma) pH 7.4, 1.7 mM sodium dithionite (DTH) (Fluka), 0.5 µg/ml leupeptin (U. S. Biochemical Corp.), and 0.2 mM phenylmethylsulfonyl flouride (Sigma), and applied to a 7.5 mm times 7.5 cm Beckman Spherogel TSK phenyl hydrophobic interaction column previously reduced and equilibrated in starting buffer using a System Gold Beckman high performance liquid chromatograph. NIFNE was eluted with a decreasing (NH(4))(2)SO(4) gradient from 0.75 M to 0 M. Buffers also contained 10% glycerol, MOPS, DTH, phenylmethylsulfonyl flouride, and leupeptin as described above. NIFNE eluted at approximately 0.32 M (NH(4))(2)SO(4). Active fractions were stored at -80 °C.

Assay for NIFNE and NIFB Activities

NIFNE activity was determined by addition of 1-50 µl of the fraction in question to a degassed, reduced 9-ml stoppered serum vial containing 100 µl of 25 mM MOPS, pH 7.4, 1.7 mM DTH, 20 µl of 5 mM homocitrate acid (Sigma) that had been converted from its lactone form at pH 11, 10 µl of 1 mM sodium molybdate (Merck), 200 µl of an ATP-generating system prepared as described (10) , and 200 µl of DJ35 (DeltanifE) crude extract prepared as previously described(10) . To provide NIFB activity in excess, 40 µl of Sarkosyl eluate of K. pneumoniae UN1217 (nifN) membranes, prepared as described elsewhere(10) , were added. This fraction is highly enriched for a small iron- and sulfur-containing molecule thought to be the product of NIFB, and therefore termed NifB-co(10) . Vials were incubated at 30 °C for 30 min to allow FeMo-co synthesis and insertion into apodinitrogenase. An additional 800 µl of the ATP-generating system were then added, the vials brought to 1 atm, and a 30-min acetylene reduction assay was performed as previously described(8) . Unlike the prior NIFNE activity assay(9) , the NIFNE activity assay in this report replaces K. pneumoniae with A. vinelandii extract as a source of all required protein factors for FeMo-co synthesis except for NIFNE. This avoids any possibility of interference by FeMo-co synthesis inhibitors associated with K. pneumoniae extract(35) .

In the previous report(9) , the activity of NIFNE was given as a specific activity in nmoles of ethylene formed per min per mg of protein in NIFNE-containing fractions. We feel this nomenclature should be revised for the following reasons. First, the formation of ethylene in the assay reflects dinitrogenase activity and is therefore only an indirect measure of NIFNE activity. Secondly, the assay that is used to measure NIFNE activity involves a 30 min incubation prior to the acetylene reduction assay. During this time, it is believed that FeMo-co synthesis has gone to completion (8) and the FeMo-co synthesis activity of NIFNE is exhausted. Because FeMo-co synthesis has gone to completion, NIFNE activity should not be expressed as a function of time. This aspect of NIFNE activity is currently being investigated in detail.

In this study, NIFNE activity is expressed as the nanomoles of ethylene produced per min by dinitrogenase formed per mg protein in the NIFNE-containing fraction. This nomenclature is distinctly different than the previous nomenclature in that it avoids both the implication of NIFNE directly producing ethylene, and the implication of NIFNE activity being measured as a rate. Until we determine the exact nature of NIFNE function and the in vitro FeMo-co synthesis assay and also determine an efficient way in which to separately analyze FeMo-co synthesis and insertion, a direct analysis of NIFNE's specific activity cannot be performed.

NIFB activity was determined by the previously described method(10) .

Anoxic Native Gel Electrophoresis

Proteins were separated at 4 °C on anoxic native gels with 7-10% acrylamide (total acrylamide, w/v; monomer:cross-linker ratio of 37.5:1) and 0-20% sucrose gradients under a 4% stacking gel, both of which were in 400 mM Tris-HCl, pH 9.0. The electrophoresis buffer was 66 mM Tris base (Sigma) and 88 mM glycine (Mallinkrodt), pH 8.6, that had been sparged with N(2). DTH (1.7 mM final concentration) was added to the electrophoresis buffer. Gels were electrophoresed for 1 h at 122 V, then samples were loaded and electrophoresed for 15 h at 100 V. Treatment of crude extracts and purified proteins with experimental and control solutions prior to electrophoresis was done by addition of the protein solutions to anoxic 5-ml vials. The experimental solutions were then added to the vials which were then incubated at 30 °C with shaking for 10-30 min. After treatment, the vials were placed on ice and samples were immediately loaded onto the gel. Where required as a control, NifB-co was oxygen-treated by placing 100 µl of NifB-co in a vial containing air and vortexed gently. The vial was incubated at room temperature for 15 min and then made anoxic by evacuation and flushing with argon. DTH was then added to the NifB-co to 1.7 mM. Gels shown are representative of two or more separate experiments.

SDS-Polyacrylamide Gel Electrophoresis (PAGE)

SDS-PAGE of NIFNE-containing fractions was done with a 2.8% stacking gel and a 12% resolving gel electrophoresed at 60 V until the bromphenol blue had entered the resolving gel, then electrophoresed at 120 V until the dye front reached the bottom of the gel. Low molecular weight markers (Bio-Rad) were used.

Immunoblotting

Immunoblotting was performed as described by Blake et al.(36) with modifications by Brandner et al.(37) . After electrophoresis, gels were equilibrated in transfer buffer for 15 min and transferred at 4 °C to Immobilon P (Millipore) at 0.7 A for 2 h.

Dot Blot Quantitation of NIFNE in Crude Extracts

The relative levels of NIFNE protein in crude extracts were quantitated using an antibody capture assay described by Harlow and Lane(38) .

Protein Assay

Protein concentrations were determined by the method of Minamide and Bamburg (39) except that bound Coomassie Blue was eluted into 700 µl instead of 1 ml.

Production of Antibody

Antibody was produced by the Animal Care Unit of the University of Wisconsin Medical School using a Center for Health Sciences-Animal Care and Use Committee approved protocol for standard rabbit polyclonal antibody production.


RESULTS

Modified Purification Scheme for NIFNE

The modified purification scheme for NIFNE is described under ``Experimental Procedures.'' The new protocol decreases the number of manipulations with a similar yield of protein (Table 1). The resulting protein is >90% pure as judged by scanning densitometry of a Coomassie Blue-stained SDS-acrylamide gel (Fig. 1) and has an activity of >8000 nmol of ethylene produced per min by dinitrogenase formed per mg of protein in the NIFNE-containing fraction.




Figure 1: Purification of NIFNE. Coomassie Blue-stained SDS-polyacrylamide (12%) gel separation of proteins in NIFNE-containing fractions during the purification of NIFNE from DJ677 and purified NIFNE from CA12 and wild type. Lane 1, DJ677 crude extract; lane 2, DJ677 NIFNE-containing fraction from a DE52 cellulose column; lane 3, DJ677 NIFNE-containing fraction from a Reactive Red column; lane 4, DJ677 NIFNE-containing fraction from a TSK phenyl column; lane 5, CA12 NIFNE-containing fraction from a TSK phenyl column; lane 6, wild-type NIFNE-containing fraction from a TSK phenyl column. Molecular mass markers are (in kilodaltons): rabbit muscle phosphorylase B (97.4), bovine serum albumin (66.2), hen egg white ovalbumin (45.0), bovine carbonic anhydrase (31.0), and soybean trypsin inhibitor (21.5).



Because of the structural similarities between NIFNE and dinitrogenase, the latter protein can be a major contaminant during the purification of NIFNE. Therefore, strains deleted for nifK and nifD, the genes encoding dinitrogenase, simplify the purification. When NIFNE accumulated in DJ54 (DeltanifH) extracts was directly compared with NIFNE accumulated in CA12 (DeltanifHDK) extracts, there was no detectable difference in anoxic native gel mobility nor activity (data not shown). Similarly, when NIFNE accumulated in extracts from UW45 (nifB) and DJ677 (DeltanifB::kan DeltanifDK) were compared, no differences were observed. Therefore the absence of dinitrogenase does not appear to alter these properties of NIFNE in either of these strains.

Modified Functional Assay for NIFNE

The assay for functional NIFNE was modified from that described previously (9) for reasons explained under ``Experimental Procedures.'' Differences in activities of NIFNE between this report and the previous one may reflect the use of A. vinelandii crude extracts in place of K. pneumoniae crude extracts. For this reason, direct comparison of activities of NIFNE has not been made between the data sets.

The Predominant Forms of NIFNE Accumulated in CA12 and DJ677 Are Different Based on Mobility upon Anoxic Native Gel Electrophoresis

Immunoblot analysis was used to determine the mobility of NIFNE in crude extracts upon anoxic native gel electrophoresis. This allowed the examination of the forms of NIFNE that accumulated in strains either proficient (DJ, wild-type) or deficient (CA12, DeltanifHDK; and DJ677, DeltanifB::kan DeltanifDK) in FeMo-co synthesis. As shown in Fig. 2, the accumulated NIFNE species from DJ677 (lane 2) and CA12 (lane 3) have distinct mobilities. The NIFNE from the crude extracts of CA12 migrates as a sharp band (band B) while that in crude extracts of DJ677 migrates as a diffuse band of lower mobility (band A). Although difficult to distinguish due to the relatively lower amounts in the crude extracts, NIFNE accumulated in wild type seems to contain at least some of the species that predominate in CA12 and DJ677, in addition to other species (lane 1). It is clear that the forms of NIFNE that predominate in CA12 and DJ677 are distinct, and possible biological interpretation of these results will be discussed below.


Figure 2: Immunoblot analysis of NIFNE. Western blot of a 7-10% anoxic native gel was developed with antibody to NIFNE. Lane 1, wild-type extract; lane 2, DJ677 extract; lane 3, CA12 extract; lanes 4-9, DJ677 extract preincubated with increasing amounts of NifB-co; lane 10, CA12 NIFNE-containing fraction from a DE52 cellulose column; lane 11, CA12 NIFNE-containing fraction from a Reactive Red column; lane 12, CA12 NIFNE-containing fraction from a TSK phenyl column; lanes 13 and 14, CA12 NIFNE-containing fractions from a TSK phenyl column preincubated with increasing amounts of NifB-co; lane 15, DJ677 NIFNE-containing fraction from a TSK phenyl column; lane 16, DJ677 NIFNE-containing fraction from a TSK phenyl column preincubated with NifB-co; lane 17, wild-type NIFNE-containing fraction from a TSK phenyl column; lane 18, wild-type NIFNE-containing fraction from a TSK phenyl column preincubated with NifB-co; lane 19, wild-type extract pre-incubated with NifB-co.



The Native Electrophoretic Mobility of NIFNE from CA12 Changes during Purification

When anoxic native gels were used to examine the NIFNE from these backgrounds at different stages during purification, it was observed that the mobility of the NIFNE from CA12 (DeltanifHDK) changed during purification. In crude extracts, the NIFNE from CA12 ran as the relatively faster migrating sharp band (Fig. 2, lane 3), but changed during the purification (Fig. 2, lanes 10-12) to a form that comigrates with the form of NIFNE purified from DJ677 (DeltanifB::kan DeltanifDK) and also with the NIFNE found in the crude extract of DJ677 (Fig. 2, lanes 15 and 2, respectively). The basis for the changes in the migration pattern is unknown, but this observation indicates that the form of NIFNE purified from CA12 is not the same as found in crude extracts of that strain. As discussed below, it is our hypothesis that the NIFNE from CA12 is associated with NifB-co in crude extracts, and loses this association during its purification. This change in migration upon purification of the NIFNE is not observed in the case of NIFNE from DJ677. We currently do not know the reason for the repeated observation that partially purified NIFNE from CA12 displays a mobility different from the above species after the DE52 column (Fig. 2, lane 11).

Addition of NifB-co to NIFNE from DJ677 Changes the Mobility to That of the NIFNE Accumulated in CA12

Because NIFB is known to be essential for FeMo-co synthesis and NIFN and NIFB proteins are actually fused in Clostridium pasteurianum, (^2)NifB-co is a candidate for interaction with NIFNE. The recent purification of NifB-co, an iron-sulfur compound that is the apparent metabolic product of the NIFB protein(10) , allowed us to test for a possible interaction between NifB-co and NIFNE as assayed by NIFNE's anoxic native gel mobility. We reasoned that, if the NIFNE accumulated in extracts of a NIFB mutant (DJ677) is allowed to interact with NifB-co, the mobility may change, possibly to that of NIFNE accumulated in extracts of a dinitrogenase reductase mutant (CA12). Furthermore, if the accumulated form of NIFNE from CA12 is already associated with NifB-co (NIFB and NifB-co activity is present in this strain) and that during purification NifB-co was stripped from the NIFNE, then the purified NIFNE might return to the original form following the addition of NifB-co.

To test the first of these possibilities, a partially pure source of NifB-co was added to crude extracts of DJ677 (DeltanifB::kan DeltanifDK). Addition of increasing amounts of NifB-co to crude extracts from DJ677 changed the form of NIFNE from the slower, diffuse species (Fig. 2, band A) to the faster migrating, sharper species (Fig. 2, band B, lanes 4-9). Identical results were obtained with NifB-co that had been further purified. Addition of NifB-co or Sarkosyl elute to crude extracts of wild type (DJ) caused a similar effect (Fig. 2, lane 1 versus 19) suggesting much of the accumulated form of NIFNE in wild-type can also interact with NifB-co. Incubation of NIFNE with the detergent that solubilizes NifB-co leads to a loss of NIFNE cross-reacting material from the described bands (Fig. 2, lane 2 versus 4-9). This effect is exacerbated when low levels of protein (lane 1 versus 19) or highly purified protein (lane 12 versus 13-14, 15 versus 16 and 17 versus 18) is assayed (see below). Oxygen treatment of NifB-co prior to preincubation, which destroys its activity in a FeMo-co synthesis assay, eliminated the appearance of the faster migrating, sharp band (band B) (data not shown), suggesting that the effect is specific to functional NifB-co. A solvent-only control showed no effect on the NIFNE unless allowed to incubate for an extended period of time (30 min) after which the NIFNE was no longer detected by immunoblot analysis of the native gels (data not shown). As determined by examination of immunoblots of native gels, NIFNE from CA12 (NIFNE associated with NifB-co) is relatively stable to the same treatment. No mobility shifts of NIFNE were seen with the addition of up to 1000-fold molar excess of ferrous ammonium sulfate to DJ677 (DeltanifB::kan DeltanifDK) extract, with the addition of isolated FeMo-co to DJ677 extract, nor with the addition of dinitrogenase reductase to CA12 (DeltanifHDK) extract (data not shown).

To test the second of these predictions, NifB-co was added to the NIFNE purified from CA12 (DeltanifHDK). If this species has lost the associated NifB-co, the model predicts that the original mobility might be regained upon the addition of NifB-co. Fig. 2shows that, when NifB-co is added, the NIFNE purified from CA12 is converted from the predominantly slowly migrating diffuse band (Fig. 2, lane 12, band A) to the form which migrates more rapidly as a sharp band (Fig. 2, lanes 13 and 14, band B). Addition of NifB-co to NIFNE purified from DJ677 (DeltanifB::kan DeltanifDK) and wild type also exhibited the same effect (Fig. 2, lanes 15 versus 16 and 17 versus 18, respectively). These results indicate that functional NifB-co associates with NIFNE, and that the forms of NIFNE accumulated in different genetic backgrounds can be converted from one to another by the addition or loss of this factor. Thus, it appears that the form of NIFNE accumulated in CA12 is associated with NifB-co, and that during purification NifB-co is stripped from NIFNE. The results also suggest that the NIFNE accumulated in DJ677 (DeltanifB::kan DeltanifDK) is not associated with NifB-co but can associate with NifB-co in vitro. The association of NIFNE and NifB-co activities is addressed below.

NIFNE and NIFB Activities from CA12 Initially Copurify and Separation of the Two Activities Corresponds to the Changes Seen in Mobility of NIFNE on Native Gels

If the accumulated form of NIFNE from CA12 (DeltanifHDK) is associated with NifB-co, it should be able to provide both NIFNE and NIFB activity in the in vitro FeMo-co synthesis assay. We reasoned that NIFB activity might co-purify with that of NIFNE early in the purification, prior to the dissociation suggested by the change in NIFNE mobility on anoxic native gels. For this test, NIFNE and NIFB activities from CA12 were monitored through the purification. On the initial DE52 column, 60% of the total NIFB activity coelutes with the peak of NIFNE activity (Table 2). However, during subsequent steps in the purification of NIFNE, the NIFB activity was lost from the fractions containing NIFNE (data not shown). When the mobility of NIFNE throughout the purification protocol was monitored by anoxic native gel electrophoresis, the loss of comigrating NIFB activity (data not shown) correlated with the disappearance of the faster migrating (sharper) species of NIFNE (Fig. 2, lanes 10-12).



As a necessary control that the NIFB activity was associated with NIFNE and not co-purifying coincidentally, the first step of the NIFNE purification protocol was followed using an extract of a DeltanifE mutant (DJ35). This strain has comparable NIFB activity (approximately 50% of CA12), but lacks the NIFNE complex. In this strain, where the NIFNE complex is absent, only 9% of the NIFB activity was found to elute in the fraction that would contain NIFNE if present (Table 2). The simplest explanation for this difference in the elution of NIFB activity is that NifB-co and NIFNE are physically associated.

The Activities of NIFNE Purified from DJ, DJ677, and CA12 Are Similar

The activities were determined using the in vitro FeMo-co synthesis assay and are given in Table 3, column 3. While no more than a 2-fold difference in activity was observed among the forms of NIFNE purified from the different strains, changes during the purification, as noted above, make unambiguous interpretation of this result difficult. The similarities, however, could be due to either alteration of the protein during the purification, or due to the inability of the in vitro assay to detect any differences.



More NIFNE Accumulates in Strains Defective in FeMo-co Synthesis

The varying amount of NIFNE protein apparent in Fig. 2(lanes 1-3) is confirmed in Table 3(column 1). As determined by antibody capture quantitation, the amount of protein accumulated in wild-type (DJ) is approximately 5-fold less than the amount accumulated in CA12 (DeltanifHDK) and approximately 15-fold less than the amount accumulated in DJ677 (DeltanifB::kan DeltanifDK). These results suggest that mutants defective in FeMo-co synthesis either synthesize more NIFNE or that NIFNE accumulated in those mutants is relatively more stable than NIFNE accumulated in wild type. This fact, in combination with the need to avoid dinitrogenase contamination, accounts for the difficulty in obtaining more highly purified NIFNE from wild type.

When the in vitro FeMo-co synthesis activities of NIFNE in crude extracts of CA12 (DeltanifHDK) and DJ677 (DeltanifB::kan DeltanifDK) are normalized to the amounts of NIFNE protein accumulated in those extracts, the activities are again comparable (Table 3, column 2). This suggests that the similarity in activities is not due to the changes observed during purification. It is not possible to quantitate the activity of NIFNE in wild-type crude extracts due to the overwhelming amount of nitrogenase activity caused by the fully functional nitrogenase system in these extracts.


DISCUSSION

The general role of NIFNE in FeMo-co synthesis has been thought to be that of a scaffold protein upon which FeMo-co is built (25, 40) both because of sequence identity when compared to dinitrogenase (25) and because dinitrogenase is not required for the synthesis of FeMo-co(7) . This report provides the first biochemical evidence for that function.

Our current working model is that construction of FeMo-co occurs on NIFNE in steps, shown diagrammatically in Fig. 3. It is based on the assumption that when FeMo-co synthesis is blocked by a mutation affecting a critical step in the process, the accumulated NIFNE is expected to harbor the form of immature FeMo-co that is the substrate of the mutationally absent gene product. Because a nifH mutant (lacking dinitrogenase reductase) accumulates a ``NifB-co-charged'' version of NIFNE, it is likely that NIFNE interacts with NifB-co prior to any interaction with dinitrogenase reductase. Because the form of NIFNE accumulated in a NIFB mutant lacks molybdenum(9) , and is dependent upon homocitrate in the current in vitro FeMo-co synthesis assay, it appears that molybdenum and homocitrate are incorporated subsequent to NIFNE's interaction with NifB-co. Although the model suggests that dinitrogen-ase reductase functions directly after NIFNE's association with NifB-co, it is possible that intermediate steps occur in vivo which may not cause detectable changes in anoxic native gel mobility.


Figure 3: A model for the role of NIFNE in FeMo-co synthesis. Large circles and squares represent the dinitrogenase analogous alpha and beta subunits of NIFNE, respectively. Small circles represent different forms of maturing FeMo-co.



The number of steps needed to build the form of NIFNE that interacts with NifB-co (NIFNE accumulated in DJ677(DeltanifB::kan DeltanifDK)) is unknown, but it does appear that this form contains a minimum of 4Fe arranged in what is believed to be a single 4Fe-4S center(9) . Rather than being a FeMo-co precursor, however, this 4Fe-4S center may be in a region of NIFNE analogous to dinitrogenase's P-cluster and could conceivably be involved in electron transfer to a FeMo-co precursor. Molecular modeling of NIFNE based on the structure of dinitrogenase and sequence alignment is consistent with NIFNE containing a 4Fe-4S cluster in each analogous P-site. (^3)The number of steps subsequent to NifB-co's interaction with NIFNE is also unknown. Indeed, it is not yet clear whether or not FeMo-co synthesis is completed on NIFNE or on another protein.

Anoxic native gel electrophoresis described here revealed that different NIFNE complexes accumulate in different genetic backgrounds of A. vinelandii. Although the causes of mobility differences in anoxic native gels are unknown, these differences are reminiscent of the differences between the active form of dinitrogenase and that lacking FeMo-co (apodinitrogenase) (13, 41) in which the acquisition of a small molecule containing molybdenum, iron, and sulfur (FeMo-co) induces a large change in mobility on native gels. These results also suggest that, in mutants blocked in FeMo-co synthesis, these mutants predominantly accumulate a single form of NIFNE, which is likely to be the normal substrate of the mutationally altered gene product.

The observation that the original mobility of NIFNE purified from CA12 (DeltanifHDK) could be regained upon incubation with NifB-co, the product of NIFB, suggests that (i) NifB-co and NIFNE directly contact each other before FeMo-co is mature and (ii) the interaction of NifB-co with NIFNE is prior to the interaction of dinitrogenase reductase with NIFNE during FeMo-co synthesis. Addition of NifB-co to crude extracts of DJ677 (DeltanifB::kan DeltanifDK) revealed the same mobility change. The change in mobility of the NIFNE from CA12 during purification is therefore likely to be due to the loss of the associated NifB-co from the NIFNE. This association of NifB-co with NIFNE is not stable enough to survive purification. Disruption of this complex does not, however, render the NIFNE inactive in the in vitro assay. Moreover, once stripped of the NifB-co, NIFNE and NifB-co can again associate, suggesting the association is a transient one. The in vitro association of NifB-co with NIFNE does not require the addition of any nucleotides. Efforts toward analyzing the metal content of isolated NIFNE containing NifB-co are underway and should allow some characterization of the NifB-co molecule.

The purified NIFNE has an activity of 10,000-18,000 nmol of ethylene produced by dinitrogenase formed per mg of NIFNE added to the assay. Thus, approximately 5-9 mol of apodinitrogenase are activated per mol of NIFNE added, assuming a maximum specific activity of dinitrogenase of 2000 nmol ethylene formed per min per mg of dinitrogenase. This result can be explained only if NIFNE is functioning catalytically in the in vitro assay. This also suggests that our ability to measure a reliable specific activity of NIFNE is dependent upon whatever other molecules are necessary for this turnover and may become limiting in vitro. We expect that further investigation of NIFNE function and a purified FeMo-co synthesis system will help resolve these questions.

Finally, we report that wild type accumulates significantly less NIFNE than do mutants blocked in FeMo-co synthesis. While this feature might be due to the effects of differential transcriptional or post-transcriptional regulation, there is no known mechanism whereby synthesis or translation of nif transcripts is independently regulated. If protein stability is responsible for these differences, the result suggests the surprising possibility that NIFNE accumulated in mutants unable to complete synthesis of FeMo-co is more stable that the NIFNE accumulated in wild type.

The precise function of NIFNE in FeMo-co synthesis both in vitro and in vivo remains unknown and could include one or any combination of the following. (i) NIFNE is simply the scaffold on which FeMo-co is synthesized. (ii) NIFNE associates with a small molecule, possibly containing iron and sulfur, which it then donates to the synthesis process. (iii) NIFNE functions as a catalyst during FeMo-co synthesis. While the data presented here do not address the above possibilities directly, they do support the scaffold model and provide an approach for further analysis.

In conclusion, the results of this study are that (i) distinct forms of NIFNE accumulate in different genetic backgrounds, (ii) NifB-co directly interacts with NIFNE, (iii) dinitrogenase reductase performs a necessary step in FeMo-co synthesis subsequent to the interaction between NifB-co and NIFNE, and (iv) NIFNE is apparently a scaffold on which FeMo-co is synthesized.


FOOTNOTES

*
This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, National Science Foundation Grant MCB-9317059 and Hatch Project 36634 (to G. P. R.) and by National Institutes of Health Grant GM35332 to Paul W. Ludden. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: FeMo-co, iron-molybdenum cofactor; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; DTH, dithionite.

(^2)
J. S. Chen, personal communication.

(^3)
S. Muchmore, personal communication.


ACKNOWLEDGEMENTS

We thank Paul W. Ludden, Douglas P. Lies, and Mary J. Homer for valuable discussion.


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