The conversion of dinitrogen to ammonium by biological systems is catalyzed by nitrogenase. Nitrogenase is composed of two oxygen-labile metalloproteins: dinitrogenase (also called MoFe protein or component I) and dinitrogenase reductase (also called NIFH, Fe protein, or component II; (1) and (2) ). Dinitrogenase is an tetramer of the nifD and nifK gene products, and it contains two pairs of unique metal clusters, known as the iron-molybdenum cofactor (FeMo-co; ()(3) and (4) ) and the P-cluster(4, 5) . Dinitrogenase is specifically reduced by dinitrogenase reductase. Dinitrogenase reductase, which contains a single FeS cluster, is an dimer of the nifH gene product (6) . The electrons transferred to dinitrogenase are ultimately channeled to FeMo-co, the site of substrate reduction (see (7) for a concise review).

FeMo-co is composed of molybdenum, iron, sulfur, and homocitrate ((R)-2-hydroxyl-1,2,4-butane tricarboxylic acid) in a ratio of 1:7:9:1(4, 8) . The products of at least six nitrogen fixation (nif) genes, including nifQ, nifV, nifB, nifH, nifN, and nifE, are required for the biosynthesis of FeMo-co(8, 9, 10, 11) . Interestingly, the genes that encode dinitrogenase (nifD and nifK) are not required for FeMo-co biosynthesis, suggesting that FeMo-co is assembled elsewhere in the cell and is then inserted into FeMo-co-deficient dinitrogenase (apodinitrogenase; Refs. 12 and 13). The high degree of sequence similarity between the nifN and nifK sequences and the nifE and nifD sequences suggests that NIFNE might serve as a scaffold for FeMo-co biosynthesis(14) . This hypothesis is supported by the recent observation that the mobility of NIFNE on native (non-denaturing) gels changes specifically upon the addition of NifB-co, a likely FeMo-co precursor (described below; (15) ). An in vitro FeMo-co synthesis system that requires an ATP-regenerating system, molybdate, homocitrate, and at least NIFB, NIFNE, and NIFH has been described(11) . Although use of the in vitro system has yielded significant information concerning FeMo-co biosynthesis, the nature of the iron and sulfur donor(s) for the biosynthesis of FeMo-co remains unknown.

NifB-co is one potential source of iron and sulfur for FeMo-co biosynthesis. In the course of attempting to purify the NIFB protein from Klebsiella pneumoniae, Shah et al.(16) isolated and purified the apparent product of NIFB as a detergent-solubilized, small molecule termed NifB-cofactor (NifB-co). Solutions of NifB-co exhibit certain characteristics that are similar to solutions of purified FeMo-co, including color, stability in N-methylformamide, and oxygen lability. The requirement for NIFB in the in vitro FeMo-co synthesis assay is satisfied by the addition of NifB-co, and the amount of FeMo-co synthesized in vitro is proportional to the amount of NifB-co added to the system in which all other components are present in excess. The stoichiometric requirement of NifB-co is consistent with the hypothesis that NifB-co is an iron-sulfur precursor of FeMo-co. Because a functional nifB gene is also required for the molybdenum-independent nitrogen fixation systems(17) , it has been proposed that NifB-co is the basic iron-sulfur cluster for the synthesis of FeMo-co, the vanadium-containing cofactor (FeV-co) of the vnf-encoded nitrogenase, and the iron-only cofactor of the anf-encoded nitrogenase(16) .

In vitro activation of apodinitrogenase by FeMo-co apparently requires the presence of a protein designated as gamma(18) . Recent studies show that addition of purified FeMo-co to crude extracts and partially purified fractions containing gamma results in a shift in the electrophoretic mobility of gamma on native gels. The mobility change correlates with the incorporation of iron into gamma(19) . In addition, crude extracts that contain this faster migrating form of gamma (with associated FeMo-co) are capable of activating apodinitrogenase in vitro(19) .

To date there has been no direct evidence for the incorporation of iron and sulfur from NifB-co into FeMo-co or for the flow of iron and sulfur from NifB-co through the NIFNE and gamma proteins. This report directly demonstrates that iron and sulfur from NifB-co can become associated with NIFNE and gamma, and ultimately become incorporated into apodinitrogenase as FeMo-co.

EXPERIMENTAL PROCEDURES

Materials

Azobacter vinelandii Strains and Growth Conditions

Strain UW45 was grown and derepressed in tungsten-containing medium (molybdenum-free) as described previously(11) . When necessary, small molecules (i.e. homocitrate) were removed from the cell extracts by Sephadex G-25 column chromatography.

K. pneumoniae Strain and Growth Conditions

For sulfur donation studies, a starter culture of strain UN1217 was grown aerobically on a rotary shaker at 30 °C for 20-24 h in 275 ml of medium (minimal medium as described previously; (16) ) containing 15.6 mM ammonium acetate (filter-sterilized). One ml of this culture (stationary phase) was transferred to 250 ml of fresh medium containing 0.2 mM sulfur added as NaSO. Approximately 15 h later, this starter culture was used to inoculate a larger culture that would be derepressed. A 5-liter Pyrex carboy containing 4.5 liter of the medium plus 3 ml of a 22% ammonium acetate solution (approximately 1.9 mM final concentration) and 28.7 ml of a 10 mM NaSO solution (approximately 0.063 mM sulfur final concentration) was inoculated with 105 ml of the starter culture. The large culture was grown at 30 °C and sparged vigorously with compressed air (filter-sterilized). The culture was spot tested for ammonium using Nessler's reagent. Thirty min after exhaustion of the ammonium (which occurred approximately 4 h post inoculation), derepression of the Nif proteins was initiated by switching the gas from air to 95% N + 5% CO and by adding 4.4 ml of a sterile 10% L-serine solution. At this time, approximately 4 mCi of NaSO (specific activity 561-572 mCi/mmol) were added to the culture. Six hours after the start of derepression, the cells were harvested (approximately 5 g, wet weight) by centrifugation and stored at -80 °C. Approximately 60% of the radiolabel added to the medium was incorporated into the cells.

Growth and derepression of strain UN1217 in the presence of FeCl were similar to that described above, with the following modifications. The starter cultures were not supplemented with any iron. The medium in the 5-liter carboy was supplemented with FeCl to a final concentration of approximately 10 µM. At the start of derepression, 2 mCi of FeCl (specific activity 26.7-29.2 mCi/mg) were added. Fe-Labeled ferric citrate was prepared by diluting the FeCl into 1 ml of distilled HO containing 5 mg of sodium citrate (pH approximately 7). The solution was heated and the entire volume was added to the carboy. Approximately 50-70% of the radiolabel added to the medium was incorporated into the cells.

UN1217 Cell Breakage and Preparation of Crude NifB-co Samples

Approximately 4.5 ml of the cell suspension were transferred to 10-ml polycarbonate centrifuge tubes (16 76 mm) that contained 9.5 g of 0.1-0.2 mm glass beads. The beads had been equilibrated overnight with anoxic 0.1 M Tris-HCl buffer (pH 7.4). The tubes were degassed, and cell breakage was initiated by vortexing (standard bench-top vortexer on high setting) each tube for 1 min. Each tube was placed on ice for at least 2 min following vortexing. Seven vortex/ice cycles were used for K. pneumoniae cell breakage. N-Lauroylsarcosine was added to a final concentration of 2% (from a 20% anoxic stock solution in 0.1 M Tris-HCl (pH 7.4)). After 10 min at room temperature, the tubes were centrifuged at 27,200 g for 10 min. The temperature during centrifugation was maintained at approximately 20 °C to avoid precipitation of the detergent. The supernatants containing NifB-co were anoxically transferred and combined. To ensure that all of the NifB-co was collected, 2 ml of 0.1 M Tris-HCl (pH 7.4) and 0.2 ml of 20% N-lauroylsarcosine stock solution were added to each tube immediately after the supernatant was removed. The tubes were degassed, vortexed for 1 min, and centrifuged as described above. The supernatants were anoxically transferred and combined (but kept separately from the first supernatant). Both supernatants were assayed for NifB-co activity using the in vitro FeMo-co synthesis assay (described below). The supernatants were stored at -20 °C.

NifB-co Purification

In Vitro FeMo-co Synthesis Assay

In control experiments, various components of the complete reaction mixture were excluded as indicated under ``Results and Discussion.'' Where indicated, apodinitrogenase in 200 µl of the appropriate extract was activated by incubation with an excess of purified, unlabeled FeMo-co for 10 min before performing the in vitro FeMo-co synthesis reaction with the labeled NifB-co.

Activation of Apodinitrogenase by FeMo-co (FeMo-co Insertion Assay)

Anoxic Native Gel Electrophoresis

Immunoblots

Visualization of Radioactivity

Acid-labile Sulfide Determination

RESULTS AND DISCUSSION

Identification of an Iron and Sulfur Donor for FeMo-co Synthesis

To investigate the role of NifB-co as an iron-sulfur donor for FeMo-co biosynthesis, NifB-co was independently labeled in vivo with Fe or S. The labeled cofactor was purified as described under ``Experimental Procedures.'' The Fe- and S-labeled NifB-co fractions contained an average of 2,200,000 and 300,000 cpm/ml, respectively. The average activities of the Fe and S-labeled NifB-co fractions were 770 and 400 nmol of CH reduced/min by dinitrogenase formed/ml of the NifB-co-containing fraction, respectively.

The in vitro FeMo-co synthesis system together with anoxic, native gel electrophoresis was employed to monitor the incorporation of iron and/or sulfur from NifB-co into the FeMo-co of dinitrogenase. A complete reaction mixture that included all of the components known to be required for FeMo-co biosynthesis was used to monitor donation of iron and sulfur from NifB-co to FeMo-co. The complete reaction mixtures contained molybdenum, homocitrate, an ATP-regenerating system, and cell-free extract from strain UW45 (nifB), which served as a source of NIFNE, dinitrogenase reductase, apodinitrogenase, and any other unidentified factors required for in vitro FeMo-co biosynthesis. Purified Fe- or S-labeled NifB-co was added to complete the reaction mixture. A number of control reactions (in which iron and sulfur from NifB-co were not expected to be incorporated into dinitrogenase) were performed to demonstrate the specificity of incorporation of radiolabel from active NifB-co into dinitrogenase. Homocitrate, molybdenum, and MgATP were omitted from some in vitro FeMo-co synthesis reaction mixtures to prevent FeMo-co synthesis. In other control reaction mixtures, the oxygen-labile, labeled NifB-co was inactivated by exposure to air prior to addition to the reactions. In other control assays, all of the apodinitrogenase present in the UW45 extracts was activated in vitro with unlabeled, purified FeMo-co prior to the addition of labeled NifB-co to the complete reaction mixture. In this system, all of the available FeMo-co binding sites on the apodinitrogenase should be occupied by unlabeled FeMo-co, and therefore iron and sulfur from labeled NifB-co were not expected to be associated with dinitrogenase.

To investigate the incorporation of Fe and S from NifB-co into FeMo-co, the proteins in the various in vitro FeMo-co synthesis reaction mixtures were separated on anoxic native gels. The position to which dinitrogenase migrated was determined by immunoblot analysis (data not shown). The data in Fig. 1A revealed that incorporation of Fe from labeled NifB-co into dinitrogenase required the presence of all of the components known to be required for in vitro FeMo-co synthesis. A prominently labeled band that comigrated with dinitrogenase was only detected in the lanes to which the complete FeMo-co synthesis reaction mixture (plus apodinitrogenase) was applied (Fig. 1A, lanes 7 and 8). The species that migrated slightly faster than dinitrogenase has been identified as NIFNE and is discussed in detail below. At least five lines of evidence suggested that in the complete in vitro FeMo-co synthesis reaction mixture, iron from NifB-co was specifically incorporated into the FeMo-co of dinitrogenase. (i) Only very low levels of iron were associated with dinitrogenase when FeMo-co synthesis was prevented in the reaction mixtures by omission of MgATP, molybdenum, or homocitrate (each of which is a known requirement for in vitro FeMo-co synthesis; see Fig. 1A, lanes 2, 6, and 9, respectively). (ii) The absence of a band that co-migrated with dinitrogenase in samples where the air-inactivated Fe-NifB-co was utilized demonstrated that active NifB-co was required for incorporation of the Fe into FeMo-co of dinitrogenase (Fig. 1A, lane 5). (iii) Activation of apodinitrogenase with unlabeled FeMo-co prior to synthesizing FeMo-co using the Fe-labeled NifB-co resulted in almost no association of Fe with dinitrogenase (Fig. 1A, lane 3). (iv) No radiolabel was detected at the dinitrogenase position in the lane containing only free Fe-NifB-co (Fig. 1A, lane 4). (v) Labeled dinitrogenase was not observed when the complete reaction mixture was oxidized following the FeMo-co synthesis reaction, but prior to being applied to the gel (data not shown). Note that the CH reduction activities of the various assays (Fig. 1A) are consistent with the conclusion that holodinitrogenase was only formed in the complete system (by FeMo-co synthesis using Fe-NifB-co; Fig. 1A, lanes 7 and 8) and in the FeMo-co activated sample (by activation of apodinitrogenase with unlabeled FeMo-co; Fig. 1A, lane 3). Similar results were obtained when S-labeled NifB-co was used in the various in vitro FeMo-co synthesis reaction mixtures (Fig. 1B, compare lanes 1, 2, and 4 (control experiments) with lane 3 (complete system)). The differences in the band intensities in reactions that used S-labeled NifB-co compared to those that used Fe-labeled NifB-co (Fig. 1, compare A and B) are most likely because the cpm/ml of the S-labeled NifB-co was only 14% that of the Fe-labeled NifB-co. Together these data show that iron and sulfur from NifB-co were only incorporated into dinitrogenase under conditions where FeMo-co was synthesized and demonstrate that iron and sulfur from NifB-co were specifically incorporated into the FeMo-co of dinitrogenase. These data provide direct biochemical identification of an iron and sulfur precursor of FeMo-co.

Figure 1: Phosphorimage of a native, anoxic gel of various in vitro FeMo-co synthesis reactions that included Fe-labeled-NifB-co (panel A) and S-labeled-NifB-co (panel B). Arrows indicate the position of dinitrogenase and NIFNE as determined by immunoblot analysis. Synthesis reactions used UW45 (nifB) extract except where indicated. A, lane 1, purified NIFNE protein (no cell-free extract); lane 2, minus MgATP reaction; lane 3, apodinitrogenase initially activated with purified, unlabeled FeMo-co; lane 4, Fe-labeled-NifB-co only; lane 5, Fe-NifB-co air-inactivated prior to addition to complete in vitro synthesis reaction mixture; lane 6, minus MoO reaction; lane 7, complete in vitro synthesis reaction mixture (50 µl applied); lane 8, complete in vitro synthesis reaction mixture (100 µl applied); lane 9, minus homocitrate reaction. B, lane 1, S-labeled-NifB-co; lane 2, minus homocitrate reaction; lane 3, complete in vitro synthesis reaction mixture; lane 4, minus MoO and homocitrate reaction; lane 5, minus apodinitrogenase reaction (CA11.1 (nifHDK vnfDGK1::spc)). Where appropriate, the CH reduction activities of the assays (nmol of CH formed/[minassay]) are reported. Table below the figures indicate components present (+) in the reaction mixture.

The lack of Fe associated with dinitrogenase in the sample in which apodinitrogenase was initially activated with purified, unlabeled FeMo-co suggested that, once bound to the protein, there was not a significant amount of turnover of FeMo-co in this system. The absence of a labeled dinitrogenase band indicated that the synthesized Fe-FeMo-co did not displace the FeMo-co that originally activated the apodinitrogenase.

The failure of NifB-co to form a complex with apodinitrogenase was also tested by investigating its possible ability to inhibit the insertion of FeMo-co into apodinitrogenase. As shown in Table 1, preincubation of apodinitrogenase with an excess of NifB-co did not result in any detectable inhibition of FeMo-co insertion, consistent with the labeling results in Fig. 1.

Accurate quantitation of the number of iron and sulfur atoms donated by NifB-co for FeMo-co synthesis remains to be accomplished. However, based on previous activity and iron analysis studies, NifB-co has been predicted to donate all of the iron for FeMo-co biosynthesis(16) .

In the complete in vitro FeMo-co synthesis reaction mixture (Fig. 1A, lanes 7 and 8), the iron from NifB-co was obviously associated with more than one protein. The slowest migrating species is currently unidentified (Fig. 1A, lane 8). However, the species was also present in extracts of wild type cells grown on NH (data not shown) and was likely binding iron from denatured NifB-co because it was also observed in samples containing oxidized NifB-co (Fig. 1A, lane 5) and, in fact, became more prominent as the labeled NifB-co samples lost activity, most likely due to oxygen inactivation. The slowest migrating species was not detected when S-labeled NifB-co was used (Fig. 1B).

Iron and Sulfur from NifB-co Associate with NIFNE

The labeled species that migrated slightly faster than dinitrogenase (Fig. 1A, lanes 7 and 8) was identified as NIFNE by immunoblot analysis (data not shown) and by use of purified NIFNE protein (Fig. 1A, lane 1). Purified NIFNE with associated Fe from NifB-co (Fig. 1A, lane 1) comigrated with the labeled species observed in a number of the in vitro FeMo-co synthesis reaction mixtures including: (i) reaction mixtures from which MgATP (Fig. 1A, lane 2), molybdenum (Fig. 1A, lane 6), homocitrate (Fig. 1A, lane 9), or dinitrogenase reductase (data not shown) were excluded, (ii) complete in vitro FeMo-co synthesis reaction mixtures in which all of the apodinitrogenase was activated with unlabeled FeMo-co prior to the addition of the Fe-NifB-co (Fig. 1A, lane 3), and (iii) the complete reaction mixture (Fig. 1A, lanes 7 and 8). Active NifB-co was required for this association, as determined by the absence of this species when air-inactivated Fe-NifB-co was used in the FeMo-co synthesis reactions (Fig. 1A, lane 5). These data suggest that in the absence of molybdenum, homocitrate, MgATP, or dinitrogenase reductase (each of which is a requirement for FeMo-co synthesis) NifB-co is associated with NIFNE. These results are consistent with a model where these compounds are all necessary for later steps of FeMo-co synthesis on NIFNE and suggest that FeMo-co might be completed on NIFNE. The Fe-NifB-co data presented here definitively demonstrate that the previously observed shift in the mobility of NIFNE on native gels in the presence of NifB-co was due to the association of iron and sulfur from NifB-co with NIFNE.

It is currently unclear why less Fe from NifB-co was reproducibly associated with NIFNE when homocitrate was excluded from the reaction mix compared to when MgATP or molybdenum were excluded (Fig. 1A, compare lane 9 with lanes 2 and 6). At least two hypotheses might explain this observation. Although the activity assay showed no CH reduction activity (which suggests that no FeMo-co was synthesized), an organic acid other than homocitrate might have been present in the reaction mixture, and thus a low amount of an aberrant form of FeMo-co (containing an organic acid other than homocitrate) might have been synthesized and proceeded along the biosynthetic pathway (i.e. been passed to another protein). The ability of other organic acids to substitute for homocitrate in vitro has been demonstrated(27) . Alternatively, it is possible that in this reaction system (that lacked homocitrate) the iron-sulfur precursor was converted to a form (possibly with added molybdenum, but not the finished FeMo-co) that had a lesser affinity for NIFNE than did the iron-sulfur precursor.

Binding of Fe and S from NifB-co to NIFNE was also observed in reactions that utilized cell extracts of strains with mutations in nifDK (apodinitrogenase mutants; CA11.1 (nifHDK vnfDGK1::spc), which produces both NIFNE and NifB-co (Fig. 2, lane 2); DJ677 (nifB::kan nifKD), which produces NIFNE (Fig. 1B, lane 5)). Interestingly, the observed amount of Fe from NifB-co associated with NIFNE in extracts of strain CA11.1 in the presence and absence of added dinitrogenase reductase suggests that the NIFNE-NifB-co complex is a physiologically relevant precursor along the FeMo-co biosynthetic pathway. Significantly more Fe (from NifB-co) was associated with NIFNE from CA11.1 (nifHDK vnfDGK1::spc) extracts when dinitrogenase reductase was added in addition to all of the other requirements for in vitro FeMo-co synthesis (Fig. 2, compare lanes 2 and 3). Extracts of strain CA11.1 derepressed for the Nif proteins exhibited high levels of NIFB and NIFNE activities as monitored by the FeMo-co synthesis assay. Immunoblot analysis of a native gel containing crude extract from this strain showed that the NIFNE present migrated at the position for NIFNE with bound NifB-co (data not shown; (15) ). We hypothesize that the majority of NIFNE in lane 3 (Fig. 2) had bound NifB-co (unlabeled, of in vivo origin), and therefore little association of the added Fe-NifB-co with NIFNE was observed in the absence of a complete in vitro FeMo-co synthesis reaction system. When dinitrogenase reductase was added to complete the reaction mixture containing CA11.1 extract, molybdenum, homocitrate, and MgATP, all of the components known to be required for FeMo-co biosynthesis were present and therefore FeMo-co was synthesized. Thus, the unlabeled NifB-co (of in vivo origin) was apparently incorporated into FeMo-co and proceeded along the biosynthetic pathway (discussed below). The NIFNE protein was then available to interact with the Fe-NifB-co that had been added to the system, and therefore an increase in Fe associated with NIFNE was observed upon addition of dinitrogenase reductase to the reaction mixture (Fig. 2, lane 2). These results demonstrate the relevance of the interaction of NifB-co with NIFNE. The apparent ability to chase NifB-co from NIFNE in CA11.1 extracts by addition of dinitrogenase reductase to the reaction mixture suggests that NIFNE is not only capable of binding NifB-co, but, in fact, the NIFNE-NifB-co complex appears to be a precursor of FeMo-co.

Figure 2: Phosphorimage of a native, anoxic gel of various in vitro FeMo-co synthesis reactions that included Fe-labeled-NifB-co. Arrows indicate the position of dinitrogenase and NIFNE as determined by immunoblot analysis. Extracts of strain CA11.1 (nifHDK vnfDGK1::spc) were used in the reactions applied to lanes 1 and 2. Lane 1, minus apodinitrogenase reaction; lane 2, minus apodinitrogenase and dinitrogenase reductase reaction; lane 3, complete in vitro FeMo-co synthesis reaction (UW45 (nifB) extract). Where appropriate, the CH reduction activities of the assays (nmol of CH formed/[minassay]) are reported. Table below the figure indicates components present (+) in the reaction mixture.

It is currently unclear if FeMo-co synthesis is completed on NIFNE, however there is evidence that NIFNE can bind to FeMo-co in crude extracts. When purified FeMo-co was added to extracts of strain DJ677 (nifB::kan nifKD), which lacks apodinitrogenase and is unable to synthesize NifB-co, and the mixture was applied to a Sephacryl S-200 sizing column, the NIFNE-containing fraction contained FeMo-co as determined by the ability to activate apodinitrogenase in extracts of strain UW45 (nifB; data not shown). When a similar experiment was done by adding purified FeMo-co to extracts of strain DJ678 (nifDK YENX::kan), which lacks both apodinitrogenase and NIFNE, no FeMo-co was associated with the Sephacryl S-200 column fraction that corresponded to the NIFNE-containing fraction in the DJ677 experiment (data not shown). These data suggest that NIFNE is capable of associating with the completed FeMo-co molecule and are consistent with the hypothesis that FeMo-co is completed on NIFNE. The observation that Fe from NifB-co was associated with NIFNE in extracts of strain UW45 that had been activated with purifed FeMo-co (Fig. 1A, lane 3) might suggest that NIFNE has a greater affinity for NifB-co than for FeMo-co.

Iron and Sulfur from NifB-co Associate with Gamma

FeMo-co synthesis assays were performed using extracts from strain DJ677 (nifB::kan nifDK), which lacks apodinitrogenase and NIFB. The absence of apodinitrogenase allows the accumulation of FeMo-co on other proteins to be detected. To visualize dinitrogenase and gamma on the same system (Fig. 3), samples were electrophoresed on a gel for significantly fewer V h than the gels shown in Fig. 1and Fig. 2. The fastest migrating species (marked ``unknown'' in Fig. 3) is unidentified (discussed below). The smear on the gel (that obscures Fe associated with NIFNE) is attributed to Fe from oxygen-inactivated NifB-co (see Fig. 3, lane 1). The position of gamma (with bound FeMo-co) on the gel was determined by immunoblot analysis (data not shown).

Figure 3: Phosphorimage of a native, anoxic gel of various in vitro FeMo-co synthesis reactions that included Fe-labeled-NifB-co. Arrows indicate the positions of dinitrogenase and gamma (with bound FeMo-co) as determined by immunoblot analysis. The unknown species is also indicated by an arrow. Synthesis reactions used extracts of strain DJ677 (nifB::kan nifDK), except in lane 6. Lane 1, minus apodinitrogenase with Fe-NifB-co inactivated by air prior to addition to the assay; lane 2, minus apodinitrogenase and homocitrate; lane 3, minus apodinitrogenase and MgATP; lane 4, minus apodinitrogenase with purified, unlabeled FeMo-co added; lane 5, minus apodinitrogenase (all other FeMo-co synthesis requirements present); lane 6, complete in vitro FeMo-co synthesis reaction (UW45 (nifB)). Where appropriate, the CH reduction activities of the assays (nmol of CH formed/[minassay]) are reported. Table below the figure indicates components present (+) in the reaction mixture.

The data in Fig. 3revealed that significant amounts of Fe from Fe-NifB-co accumulated on a protein that co-migrated with gamma in extracts of strain DJ677 only when all of the components required for FeMo-co biosynthesis were present (Fig. 3, lane 5). Fe was not associated with gamma when FeMo-co synthesis could not occur due to omission of the MgATP (Fig. 3, lane 3) or homocitrate (Fig. 3, lane 2) from the reaction mixture or when purified, unlabeled FeMo-co was added prior to the synthesis reaction (Fig. 3, lane 4). In addition, no label was associated with this protein when the Fe-NifB-co was inactivated by exposure to air prior to addition to the FeMo-co synthesis reaction (Fig. 3, lane 1). Because all components of the in vitro FeMo-co synthesis system were required to observe Fe-NifB-co-dependent labeling of gamma, these data support the conclusion that the iron associated with gamma was in the form of FeMo-co and definitively demonstrate that the iron previously observed to be associated with gamma (in the form of FeMo-co) was from NifB-co.

A Fe-labeled species that migrated slightly faster than gamma was observed in some reaction mixtures (Fig. 3, lanes 2, 3, and 5). The band did not correspond to free Fe-NifB-co and was dependent on active NifB-co because the species was absent in samples where air-inactivated Fe-NifB-co was utilized (Fig. 3, lane 1). The species was most prominent in samples that contained all of the components required for FeMo-co synthesis (Fig. 3, lane 5) and in samples that contained all of the components required for FeMo-co synthesis except homocitrate (Fig. 3, lane 2). Immunoblot analysis indicated that this species did not correspond to gamma or NIFNE. The possibility that this is another relevant protein involved in FeMo-co biosynthesis is currently under investigation. A similar species appears to have been observed when MoO was used to investigate incorporation of molybdenum into dinitrogenase in the presence of various homocitrate analogs (see Fig. 3of (27) ).

Model of FeMo-co Biosynthesis

A major question that remains concerns the source of iron and sulfur for NifB-co biosynthesis. Exciting results from the laboratory of Dean and colleagues indicate that the nifU and nifS gene products are likely to be involved in the mobilization of inorganic iron and sulfur for the synthesis of the nitrogenase iron-sulfur clusters (see (7) for a recent review). A system that utilized NIFS for the reconstitution of the FeS cluster of dinitrogenase reductase was recently described(29) . It will be interesting to attempt to generate NifB-co using a similar system.

Conclusions

FOOTNOTES

*

This research was supported by National Institutes of Health Grant GM35332 (to P. W. L.). 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.

§

To whom correspondence should be addressed. Tel.: 608-262-6859; Fax: 608-262-3453; ludden@biochem.wisc.edu.

()

The abbreviations used are: FeMo-co, iron-molybdenum cofactor; NifB-co, iron- and sulfur-containing precursor to FeMo-co; DTH, sodium dithionite; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid.

ACKNOWLEDGEMENTS

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