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J. Biol. Chem., Vol. 275, Issue 23, 17631-17638, June 9, 2000

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Identification of an Fe protein Residue (Glu¹⁴⁶) of Azotobacter vinelandii Nitrogenase That Is Specifically Involved in FeMo Cofactor Insertion^*

Markus W. Ribbe, Evan H. Bursey, and Barbara K. Burgess

From the Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697

Received for publication, December 9, 1999, and in revised form, February 15, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The Fe protein of nitrogenase has three separate functions. Much is known about the regions of the protein that are critical to its function as an electron donor to the MoFe protein, but almost nothing is known about the regions of the protein that are critical to its functions in either FeMo cofactor biosynthesis or FeMo cofactor insertion. Using computer modeling and information obtained from Fe protein mutants that were made decades ago by chemical mutagenesis, we targeted a surface residue Glu¹⁴⁶ as potentially being involved in FeMo cofactor biosynthesis and/or insertion. The Azotobacter vinelandii strain expressing an E146D Fe protein variant grows at ~50% of the wild type rate. The purified E146D Fe protein is fully functional as an electron donor to the MoFe protein, but the MoFe protein synthesized by that strain is partially (~50%) FeMo cofactor-deficient. The E146D Fe protein is fully functional in an in vitro FeMo cofactor biosynthesis assay, and the strain expressing this protein accumulates "free" FeMo cofactor. Assays that compared the ability of wild type and E146D Fe proteins to participate in FeMo cofactor insertion demonstrate, however, that the mutant is severely altered in this last reaction. This is the first known mutation that only influences the insertion reaction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Nitrogenase is composed of two separately purified proteins (for recent reviews see Refs. 1-6). The iron protein (Fe protein) is a 60,000 M_r dimer of two identical subunits encoded by the nifH gene. The two subunits are bridged by a single [4Fe-4S] cluster, and each of the subunits has a binding site for MgATP. The molybdenum-iron protein (MoFe protein) is a 230,000 M_{r 22} tetramer with the  and  subunits encoded by the nifD and nifK genes, respectively. The MoFe protein contains two different types of metal clusters, the [8Fe-7S] P-clusters and the [Mo-7Fe-9S-homocitrate] clusters that are designated FeMo cofactor. Substrate reduction by the enzyme requires both component proteins with the Fe protein serving as a specific electron donor to the MoFe protein. To carry out this function in nitrogenase catalysis, the reduced Fe protein first binds two molecules of MgATP and then undergoes a global conformational change before forming a very specific complex with the MoFe protein. Electrons can then be transferred from the Fe protein to the P-clusters of the MoFe protein in a reaction that is somehow coupled to MgATP hydrolysis. Subsequently, the oxidized Fe protein dissociates from the MoFe protein, the Fe protein is rereduced, MgADP dissociates, and the cycle is repeated.

In addition to its role in dinitrogen reduction, the Fe protein has at least two other functions in the cell. One of them is in the initial biosynthesis of FeMo cofactor (for reviews see Refs. 5 and 7-13). Although the complete pathway for FeMo cofactor biosynthesis is not yet established, it has been known for some time that the FeMo cofactor is synthesized separately from the MoFe protein polypeptides (14-16) and that its synthesis requires the combined action of the nif Q, B, N, E, X, and V genes (5, 7-13, 17). In 1986, surprising reports appeared that mutants of Klebsiella pneumoniae and Azotobacter vinelandii that did not synthesize the nifH polypeptide also did not synthesize FeMo cofactor (15, 16). Based on these and other experiments (18-21), it is now accepted that the nifH gene product is required for the initial biosynthesis of FeMo cofactor. Its role in this process, however, has not been established, nor is it known what features of the Fe protein are required for its participation in FeMo cofactor biosynthesis.

A third function of the Fe protein involves the insertion of preformed FeMo cofactor into a FeMo cofactor-deficient form of the MoFe protein (for reviews see Refs. 5 and 7-13). This final maturation of the MoFe protein also appears to occur in a series of steps. First, a P-cluster containing but FeMo cofactor-deficient MoFe protein is synthesized that has the FeMo cofactor site somehow inaccessible to FeMo cofactor insertion (18-21). This form of the protein accumulates in strains that have deletions in the nifH gene. Next, in a reaction that requires both the Fe protein and MgATP, the FeMo cofactor-deficient MoFe protein is converted to another form with the FeMo cofactor site accessible for FeMo cofactor insertion (20, 22). This reaction appears to require at least one additional protein, nifY in K. pneumoniae or gamma in A. vinelandii, and the role of the Fe protein/MgATP may be to facilitate the association of these proteins with the FeMo cofactor-deficient MoFe protein (23-27). If the Fe protein is available in vivo, as it is in nif B, N, or E minus strains, then this step has already occurred, and the FeMo cofactor-deficient MoFe protein that accumulates can be directly activated with isolated FeMo cofactor (20). Again, the exact role that the Fe protein plays in the conversion of the FeMo cofactor-deficient protein, from a form with the FeMo cofactor site inaccessible to one that can be directly activated with FeMo cofactor, is not known, nor is it known what features of the Fe protein are required for this reaction.

In recent years a large number of site-directed mutant variants of the Fe protein have been constructed to examine which features of the Fe protein are required for its first function, electron transport to nitrogenase (1-6). In some cases, the strains carrying those mutations have also been examined with respect to whether or not the Fe proteins could carry out the other two Fe protein functions (28-33). These and other studies (34) have led to the conclusions that to participate in FeMo cofactor biosynthesis and insertion, the Fe protein may have to bind MgATP, but it does not need to undergo the MgATP-induced conformational change, it does not need to form the normal complex with the MoFe protein, it does not need to transfer electrons to any protein, and it does not need to hydrolyze MgATP, at least using the mechanism that is used during nitrogenase catalysis.¹ Because these mutations were designed to study the mechanism of nitrogenase catalysis, all were defective in normal nitrogenase turnover (28-33). As a result they could not be used to identify regions of the Fe protein that might be uniquely required for the other two functions of the protein.

Prior to the availability of site-directed mutagenesis, a large number of mutants of A. vinelandii and K. pneumoniae were created by chemical mutagenesis followed by selection for their inability to grow under dinitrogen-fixing conditions. Subsequently, several of these mutations were mapped to the nifH gene, and some were sequenced to identify their nifH genotype (28, 35-38). Very recently, the structure of an Fe protein/MoFe protein complex from A. vinelandii became available (39), and we used that structure to search for those nifH mutations that were not located in any known region of importance for normal nitrogenase catalysis. Here we report that this information was used to construct a site-directed variant of the Fe protein, E146D, that is defective in its ability to participate in FeMo cofactor insertion but that participates normally in nitrogenase turnover and FeMo cofactor biosynthesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Unless otherwise noted, all chemicals and reagents were obtained from Fisher, Baxter Scientific, or Sigma.

Construction and Expression of Variant A. vinelandii Strains-- A fragment of the A. vinelandii chromosome containing the entire nifH and a portion of the nifD genes was cloned into the bacteriophage M13mp18 (28). Site-directed mutagenesis was performed using the Mutagene mutagenesis kit, version 2, from Bio-Rad. The oligonucleotides used for mutagenesis were purchased from the Integrated DNA Technologies (Coralville, IA). They were both 32 bases long and were complementary to the region surrounding the Glu¹⁴⁶ codon. One oligonucleotide was degenerate at the Glu¹⁴⁶ codon, allowing for the production of several Fe protein mutants at this position, including the glutamate to lysine mutation. The other oligonucleotide was specific for the glutamate to aspartate mutation. After mutagenesis, bacteriophage containing mutated nifH genes were selected through DNA sequencing using the Sequenase II sequencing kit (U. S. Biochemical Corp.). Two mutated genes were selected, corresponding to E146K and E146D mutations. In each case double-stranded DNA was isolated from these phage using a Qiagen kit (Qiagen, Chatsworth, CA). This DNA was then transformed back into two A. vinelandii strains, the wild type Trans strain and the nifH strain DJ54, using a published method (40). Here the chromosomal copy of the gene is replaced with the mutated gene through homologous recombination, allowing production of variant Fe protein in its native background, under the control of its native promoter. The recombination of the mutated nifH gene containing the Glu to Lys mutation into the bacterial chromosome of the Trans strain resulted in a strain that was not able to grow under dinitrogen-fixing conditions. The recombination of the gene containing the Glu to Asp mutation into the bacterial chromosome of the DJ54 strain resulted in a strain that grew slowly under dinitrogen-fixing conditions. All subsequent work was done on this DJ54-derived strain.

Cell Growth and Protein Purification-- A. vinelandii strains expressing wild type and E146D Fe proteins were grown in 180-liter batches in a 200-liter New Bruswick fermentor under N₂-fixing conditions on Burke's minimal media. The growth rate was measured by cell density at 436 nm using a Spectronic 20 Genesys (Spectronic Instruments, Rochester, NY). The cells were harvested at the end of the exponential phase using a flow through centrifugal harvester (Cepa, Germany). Published methods were used for the purification of wild type and E146D Fe proteins (41), wild type MoFe protein (42), and the MoFe protein synthesized by the strain expressing the E146D Fe protein (21). A. vinelandii strains E146K nifH and DJ54 (nifH) were grown in 10-liter batches in a 12-liter New Brunswick fermentor on Burke's minimal medium supplemented with 2 mM ammonium acetate. After the consumption of the ammonia, the cells were derepressed for 3 h followed by harvesting using a Sorvall RC5C centrifuge (10,000 rpm, 30 min, 4 °C). The cell paste was washed with 50 mM Tris-HCl, pH 8.0, and kept on dry ice until needed. Cell-free extracts were prepared from these strains by passing bacterial suspensions (10 g of wet weight in 20 ml of 50 mM Tris-HCl, pH 8.0) three times through a French pressure cell and centrifuging them (10,000 rpm, 30 min, 4 °C) under anaerobic conditions.

Protein Characterization and Spectroscopy-- All spectroscopic samples were prepared in a Vacuum Atmospheres dry box with an oxygen level of less than 1 ppm. With the exception of the all ferrous Fe protein, all reduced Fe protein, and MoFe protein samples that were characterized spectroscopically were 2 mM in Na₂S₂O₄. Ti(III)citrate and the Ti(III)citrate reduced all ferrous Fe protein samples were prepared following published methods (43, 44). For visible range absorption experiments reduced samples were prepared in anaerobic cuvettes that were previously blanked. Spectra were recorded on an HP 8452A Diode Array spectrophotometer. Visible region CD samples were prepared in the same manner, but in that case the Fe protein samples were first oxidized to the [4Fe-4S]²⁺ state by passage over a specially prepared column as described elsewhere (29). Spectra were recorded on a Jasco spectrometer with a scan speed of 50 nm/min. Traces shown are the accumulation of five scans. All perpendicular and parallel mode EPR spectra were recorded on dithionite or Ti(III)citrate reduced samples using a Bruker ESP 300 E_z spectrophotometer, interfaced with an Oxford Instruments ESR-9002 liquid helium continuous flow cryostat.

All nitrogenase activity assays (8.7-ml vials) were carried out as described previously (42). The products H₂ and C₂H₄ were analyzed as published elsewhere (28). Ammonium was determined by a high performance liquid chromatography fluorescence method (45). Molybdenum (46) and iron (47) were determined as published elsewhere. The iron chelation assays were performed as described elsewhere (44) using a Fe protein concentration of 0.8 mg/ml.

FeMo Cofactor Biosynthesis and Insertion Assays-- Experiments that monitored the insertion of FeMo cofactor into the FeMo cofactor-deficient MoFe protein synthesized by A. vinelandii nifH strain DJ54 were all done in the presence of excess isolated FeMo cofactor in N-methyl formamide (NMF),² which was isolated as described elsewhere (42). The anaerobic mixture (argon gas) contained, in a 0.35-ml total volume, 25 mM Tris-HCl, pH 7.4, 1.7 mM ATP, 3.4 mM MgCl₂, 20 mM creatine phosphate, 17 units of creatine phosphokinase, 20 mM Na₂S₂O₄, 4 mg of DJ54 cell-free extract protein, and 45 µg of purified wild type or E146D Fe protein. The reactions were started by injecting 2 µl of concentrated FeMo cofactor in NMF. Following incubation at 30 °C for the times indicated in the text, the insertion was stopped by addition of 40 nmol of (NH₄)₂MoS₄. Subsequently, the enzyme activity was measured by C₂H₄ evolution. Those enzyme activity assays, containing 0.1 ml of the insertion mixture, were carried out as described previously (42). The assays designed to test for the presence of "free" or uninserted FeMo cofactor in E146D nifH cell-free extracts contained, in a 0.35-ml total volume, 25 mM Tris-HCl, pH 7.4, 20 mM Na₂S₂O₄, and 0.15 mg of purified cofactor-deficient MoFe protein from nifB strain DJ1143 (48). The insertion was started by the addition of isolated FeMo cofactor in NMF or E146D nifH cell-free extracts. The samples were incubated at 30 °C for 30 min, and subsequently, the enzyme activity of 0.1 ml of the insertion mixture was determined as described previously (42).

Assays designed to test the ability of the E146D Fe protein to participate in FeMo cofactor biosynthesis in vitro contained, in a 0.35-ml total volume, 25 mM Tris-HCl, pH 7.4, 0.3 mM homocitrate, 30 µM sodium molybdate, 3.4 mM ATP, 6.8 mM MgCl₂, 40 mM creatine phosphate, 34 units of creatine phosphokinase, 20 mM Na₂S₂O₄, and 8 mg of DJ54 cell-free extracts, and the reactions were started by addition of 120 µg of purified wild type or E146D Fe protein. The samples were incubated at 30 °C for 30 min, stopped by adding 40 nmol (NH₄)₂MoS₄ (34, 49), and the enzyme activity was determined as described previously (42). Homocitrate lactone was converted to the free acid as described elsewhere (50).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The Glu¹⁴⁶ Region of the Fe Protein-- As discussed in the Introduction, the Fe protein of nitrogenase has at least three separate functions in the cell: electron donation to the MoFe protein to support N₂ reduction; participation in the initial biosynthesis of FeMo cofactor; and insertion of preformed FeMo cofactor in a FeMo cofactor-deficient MoFe protein. How the Fe protein participates in the last two reactions and what features of the Fe protein are required for those two reactions is not known. In this study we used computer modeling with the recently published structure of an Fe protein/MoFe protein complex (39) to try to locate conserved Fe protein residues that might be specifically involved only in one or both of the last two reactions. To do this we relied on information concerning the nifH genotypes of strains of K. pneumoniae or A. vinelandii that had been produced decades ago using chemical mutagenesis and that had Nif phenotypes (35-38). The idea was to look for residues that were not close to the [4Fe-4S] cluster or any of the regions of the Fe protein that were known to be important for MgATP binding, the MgATP-induced conformational change or for complex formation with the MoFe protein. In this way Glu¹⁴⁶ was selected as a target for mutagenesis.

Fig. 1 shows the region around Glu¹⁴⁶ in the Fe protein sequence. The residue is completely conserved in 57 known Fe proteins sequences, it is located in a highly conserved region of the protein, it is on the surface of the protein and part of  sheet number 6, and it is not close to any regions known to be required for Fe protein participation in nitrogenase turnover (Fig. 1). Fig. 2 shows the position of the residue in the A. vinelandii complex structure relative to the [4Fe-4S] cluster, the nucleotide-binding region, the dimer interface, and the MoFe protein interaction surface. It is difficult to see from this figure why a mutation at this position would affect the ability of the protein to serve as an electron donor for the MoFe protein. Nonetheless one of the chemically generated nifH mutations that led to a Nif phenotype was later shown to be the substitution of Glu by Lys at the homologous position, 147 in K. pneumoniae (36). Fig. 3A is a close up of Glu¹⁴⁶ showing again that this hydrophilic residue is on the surface of the protein and is salt-bridged to another surface residue, Arg³. Fig. 3B is a model of the nonconservative replacement by Lys, a mutation that introduces a positive charge at that position and that would by necessity break the salt bridge and cause a rearrangement of Arg³.³ The chemically generated E147K K. pneumoniae NifH that led to a Nif phenotype was never purified, but it was reported that it accumulated to lower levels in the cell than the wild type protein. This might be due to destabilization of the NH₂ terminus caused by breaking the salt bridge to Arg³. We constructed the homologous mutation E146K nifH in A. vinelandii and confirmed both the Nif phenotype and the fact that it accumulated to lower levels in the cell making purification difficult (Fig. 4). In addition, because all three functions of the Fe protein are dependent on Fe protein concentration, it is difficult to determine to what extent the Nif phenotype is due to a specific critical role played by Glu¹⁴⁶ rather than being due to simply having less Fe protein present in the cell.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Comparison of selected Fe protein sequences in the region of Glu146. The position of residue 146, which is located in -sheet 6, is indicated in red. Residues involved in binding to the MoFe protein and to nucleotide are indicated in green and blue, respectively. Residues implicated in the dimer interaction within the Fe protein are indicated in brown. Asterisks represent residues that are identical in 57 aligned nifH sequences (57).

View larger version (69K): [in this window] [in a new window]   Fig. 2.   The position of Glu146 as seen in the Fe-MoFe-MgADP AlF4 complex structure. One-half of the MoFe protein is shown, with the  and  subunits colored in red and blue, respectively. The two Fe protein subunits are shown in green and yellow. The position of Glu146 is indicated relative to the metal clusters and the bound MgADP AlF4 molecules. This figure depicts the structure published by Schindelin et al. (39). Figs. 2 and 3 were created using the computer programs Molscript (58) and Raster3D (59).

View larger version (90K): [in this window] [in a new window]   Fig. 3.   A close-up view of residue 146. The left side of A shows a ball and stick representation of Glu146 (wild type) and some surrounding amino acids. The interaction between Glu146 and Arg3 is indicated by a dashed line between the terminal oxygen and nitrogen atoms. The right side of each panel shows a space filling model of the Fe protein surface with residues 146 and 3 shown as ball and stick representations. B and C show the hypothetical structures of the E146K and E146D mutants, respectively, in the region of the mutation. The structures used in these last two panels were generated by homology modeling the mutant sequences to existing Fe protein structures and subsequently energy minimizing the mutant structure using GROMOS96. This process is completely automated and is available at the SWISS-MODEL web site (60-62).

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Coomassie-stained SDS-polyacrylamide gel electrophoresis (A) and Western blot using Fe protein specific antibody (B) of cell-free extracts from wild type and Glu146 nifH mutant strains of A. vinelandii. Lane 1, purified wild type Fe protein (5 µg); lane 2, extract of wild type; lane 3, extract of E146K nifH; lane 4, extract of E146D nifH; lane 5, extract of nifH A. vinelandii strain DJ54 (40 µg each).

To test the importance of Glu¹⁴⁶ per se we constructed a conservative mutation in which Glu was substituted by Asp leaving the negative charge at this position but decreasing the extent to which the residue protrudes into the solvent (Fig. 3C). In the absence of an x-ray structure, it is not known whether or not Arg³ rearranges to retain the salt bridge. Fig. 4 shows however, that unlike the situation with E146K, the E146D and wild type Fe proteins do accumulate to the same levels in the cell, a fact that was also confirmed by purification yields (see below). Fig. 5 is a growth curve showing that nonetheless this conservative mutation leads to a slow growth phenotype under dinitrogen-fixing conditions with doubling time of 4 h versus 2 h for the strains expressing E146D and wild type Fe proteins, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   N2-fixing growth of wild type and E146D nifH of A. vinelandii. Both strains were grown in 10-liter batches in a 12-liter New Brunswick fermentor in Burke's minimal medium. In both cases the inocula was 200 ml, A436 = 1.6. Growth for the wild type () and E146D nifH A. vinelandii strain () was monitored by measuring the absorbance at 436 nm.

The Slow Growth Phenotype Is Not Due to Defects in the Ability of the E146D Fe Protein to Serve as an Electron Donor to the MoFe Protein-- To examine the ability of the E146D Fe protein to serve as an electron donor, the protein was purified and characterized. The protein was easily purified using the wild type procedure, and the final yields of protein were the same as those normally obtained from a wild type preparation (42). Fig. 6 compares the EPR spectra in the g = 2 region exhibited by the wild type and E146D Fe proteins. These spectra are both qualitatively and quantitatively indistinguishable from each other, leading to the conclusion that the E146D Fe protein has a normal complement of [4Fe-4S] cluster and that the cluster can be reduced by dithionite to the [4Fe-4S]¹⁺ level.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   EPR spectra of purified wild type (A, C, and E) and E146D Fe protein (B, D, and F) without nucleotide (A and B) and in the presence of MgATP (C and D) or MgADP (E and F). A final concentration of 6.25 mM ADP or ATP and 13 mM MgCl2 was used in the measurements containing nucleotide. All protein concentrations were 15 mg/ml, and the solutions contained 2 mM sodium dithionite. The spectra were recorded at 13 K using a microwave power of 20 mW, a microwave frequency of 9.43 GHz, and a gain of 5 × 104.

Recent studies have shown that wild type Fe protein can be further reduced to an unprecedented all ferrous [4Fe-4S]⁰ oxidation state in vitro (43, 51) and have suggested that this state might be utilized in vivo (44, 52). If that were the case, then a mutant Fe protein that is unable to achieve the 0 oxidation level should exhibit a slow growth phenotype. The all ferrous Fe protein exhibits a very distinctive UV-visible spectrum (43) that is observed for both the wild type and E146D Fe proteins following reduction by Ti(III) citrate to the all ferrous level (data not shown). Another distinctive feature of the all ferrous Fe protein is that it is S = 4 and exhibits a g = 16 EPR signal in the parallel mode (43). Fig. 7 compares the parallel mode EPR spectra exhibited by the wild type and E146D Fe proteins following reduction by Ti(III) citrate to the all ferrous level. Again these spectra are indistinguishable from each other, leading to the conclusion that the E146D Fe protein can be reduced by two electrons to the all ferrous level.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Parallel mode none EPR spectra of purified wild type (A) and E146D (B) Fe proteins in their Ti(III)citrate reduced all ferrous oxidation state. Samples were prepared as described under "Experimental Procedures". All protein concentrations were 8 mg/ml. The spectra were measured at a microwave power of 50 mW, a gain of 5 × 104, a microwave frequencies of 9.41 GHz, and a temperature of 4 K.

Following reduction of the [4Fe-4S]²⁺ cluster, the next step in the nitrogenase reaction involves the Fe protein binding MgATP and undergoing a global conformational change (1, 2, 4). This reaction can be easily monitored using a chelation assay because in the absence of MgATP (or the presence of MgADP) the [4Fe-4S]¹⁺ or [4Fe-4S]⁰ clusters are resistant to chelation by bathophenanthroline disulfonate, whereas in the presence of MgATP iron is rapidly removed from the protein (53, 54). Many mutant Fe proteins have been described that are either blocked in this reaction or show altered chelation kinetics (e.g.1, 4, 29, 41). Fig. 8 compares the behavior of the wild type and E146D Fe proteins in a standard chelation assay. Again the results are indistinguishable, leading to the conclusion that the E146D Fe protein is not defective in its ability to bind MgATP and undergo the conformational change. The altered MgATP conformation has also been examined by EPR spectroscopy (Fig. 6), and again there are no differences observed between the wild type and E146D Fe proteins in these reactions.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Iron chelation of the [4Fe-4S]+ cluster of wild type (A) and E146D (B) Fe proteins. The formation of the complex between the iron chelator bathophenanthroline disulfonate and the iron from the [4Fe-4S]+ clusters of the Fe proteins was measured at 535 nm in the presence of either MgATP, MgATP, and MoFe protein or MgADP. A final concentration of 2 mM ADP or ATP and 4 mM MgCl2 was used. The MoFe and Fe protein concentrations were 1.3 and 0.8 mg/ml, respectively. Curves obtained in the presence of MgATP were fitted to a single exponential over 50 s giving the observed rate constants of 0.043 and 0.044 s1 for the wild type and E146D Fe proteins, respectively. A total of 3.9 and 3.8 atoms Fe per molecule of wild type and E146D Fe protein, respectively, was determined from the extinction coefficient for the phenanthroline-Fe2+ complex in the presence of MgATP (535 nm = 22,140 cm1 M1).

Once the Fe protein is in the MgATP conformation, it is positioned to form a complex with the MoFe protein. When that occurs, the MoFe protein covers up the [4Fe-4S] cluster of the Fe protein, protecting it from chelation (39, 53). Fig. 8 shows again that there are no observed differences in the behavior of the wild type and E146D Fe proteins in this chelation protection assay. Thus the slow growth phenotype cannot be attributed to an inability of E146D Fe protein to bind normally to the MoFe protein, an observation that is consistent with the location of the residue (Fig. 2).

Following the formation of a productive complex, an electron is transferred from the Fe protein to the MoFe protein in a reaction coupled to MgATP hydrolysis. This is followed by complex dissociation, and the Fe protein is left in a conformation with MgADP bound (1). Figs. 6 and 9 compare the MgADP conformations of the wild type and E146D Fe proteins using EPR and CD spectroscopies, respectively. Again the spectra are indistinguishable. Following repeated cycles of electron transfer, the MoFe protein accumulates enough electrons to reduce substrates. Table I shows that the specific activities of the E146D and wild type Fe proteins are within experimental error of each other not only in the H₂ evolution and C₂H₂ reduction assays but also in the N₂ fixation assay. Thus the slow growth phenotype of the strain expressing E146D Fe protein cannot be explained by its inability to serve normally as an electron donor to the MoFe protein.

View larger version (25K): [in this window] [in a new window]   Fig. 9.   CD spectra of oxidized wild type (A) and E146D (B) Fe proteins without nucleotide and in the presence of MgADP. A final concentration of 6.25 mM ADP and 13 mM MgCl2 was used in the measurements containing nucleotide. The protein concentration was 7.5 mg/ml.

The Slow Growth Phenotype Can Be Explained by Defects in the MoFe Protein-- Having established that the slow growth phenotype was not due to a defect in the first function of the Fe protein, we went on to examine whether or not the E146D Fe protein might be defective in FeMo cofactor biosynthesis and/or insertion. The MoFe protein synthesized by that strain was therefore purified and characterized. Once the pure protein was available, it was immediately obvious that the MoFe protein synthesized by the strain expressing E146D Fe protein was lighter in color than wild type MoFe protein, indicating that it did not have a full complement of metal clusters. Fig. 10 compares the EPR signals exhibited by wild type MoFe protein and MoFe protein from E146D nifH when the proteins are in the presence of excess dithionite. This well characterized S = 3/2 EPR signal arises from the FeMo cofactor center of the MoFe protein (1, 7). Spin integration shows that the MoFe protein synthesized by the E146D nifH strain accumulates only 54% as much FeMo cofactor as the wild type.

View larger version (12K): [in this window] [in a new window]   Fig. 10.   EPR spectra of dithionite reduced wild type (A) and E146D nifH MoFe protein (B). All protein concentrations were 5 mg/ml. The spectra were measured at 10 K using a microwave power of 50 mW, a microwave frequency of 9.43 GHz, and a gain of 5 × 104.

This lower amount of FeMo cofactor is also consistent with the observed metal content of 0.83 ± 0.09 Mo and 18.17 ± 0.26 Fe atoms per molecule of E146D nifH MoFe protein compared with 1.86 ± 0.02 Mo and 30.88 ± 3.03 Fe atoms per molecule for wild type MoFe protein. The observed ratio of ~1 Mo:23 Fe atoms for the E146D nifH MoFe protein versus 1 Mo:15 Fe atoms for the wild type MoFe protein is consistent with having two P clusters present for every one FeMo cofactor. The data cannot distinguish whether all molecules of MoFe protein are missing one FeMo cofactor or whether a statistical distribution is present with some holo protein and some totally FeMo cofactor-deficient protein. We think the latter is unlikely, however, because the FeMo cofactor-deficient MoFe protein from a NifH strain is very difficult to purify and can only be obtained in small yield (21), whereas good yields of MoFe protein were obtained from the strain expressing E146D nifH. The independent assembly of the two halves of the protein is also not unprecedented and has been demonstrated for the vanadium nitrogenase system (55).

It is known that FeMo cofactor-deficient MoFe proteins synthesized by NifB and NifH strains are not the same (20). In this case we expect that the MoFe protein molecules that are missing one FeMo cofactor should be of the NifH and not the NifB type. This is confirmed by Fig. 11, which shows that the MoFe protein synthesized by the E146D nifH strain is an ₂₂ tetramer, like the FeMo cofactor-deficient MoFe protein from a NifH strain (21) and not an ₂₂₂ hexamer like the FeMo cofactor-deficient MoFe protein from NifB strains (27).

View larger version (40K): [in this window] [in a new window]   Fig. 11.   SDS-polyacrylamide gel electrophoresis of purified wild type and E146D nifH MoFe protein. Lane 1, protein standard (20 µg); lane 2, purified wild type MoFe protein (4 µg); lane 3, purified E146D nifH MoFe protein (4 µg). A molecular weight of 23,000-26,000 was reported for the monomeric gamma protein from SDS-polyacrylamide gel electrophoresis (26). The molecular weights of the native wild type and of the E146D nifH MoFe protein appeared to be identical based on the criteria of the elution profile of a gel filtration Ultrogel AcA34 (ICF, France) column.

As expected, this lower FeMo cofactor content correlates with lower MoFe protein specific activity such that the MoFe protein synthesized by the E146D nifH strain is ~55% as active as the wild type MoFe protein not only for H₂ evolution and C₂H₂ reduction but also for N₂ fixation (Table II). This reduction in activity further correlates with the growth rate of the strain (Fig. 5), explaining the slow growth phenotype.

Evidence That the E146D Fe Protein Can Function Normally in FeMo Cofactor Biosynthesis-- As discussed above, the MoFe protein synthesized by the E146D nifH strain accumulates only about half as much FeMo cofactor as the protein from wild type cells leading to a slow growth phenotype. One possibility that would explain that result would be that the E146D Fe protein was defective in its ability to function in the initial biosynthesis of FeMo cofactor such that FeMo cofactor biosynthesis could not keep up with protein synthesis/assembly. Table III shows, however, that we were unable to distinguish E146D Fe protein and wild type Fe protein in an in vitro biosynthesis assay, making this possibility seem unlikely.

In addition, it has been known for many years that in the absence of the MoFe protein polypeptides in vivo small amounts of FeMo cofactor accumulate on some other protein (14-16). We reasoned that if the E146D Fe protein were not defective in the initial biosynthesis of FeMo cofactor, then the cell-free extracts might contain small amounts of FeMo cofactor that had not been inserted into the MoFe protein. To test this we added purified FeMo cofactor-deficient MoFe protein from a NifB strain to try to capture this free cofactor. It is known that the insertion of FeMo cofactor into that protein does not require the Fe protein (27, 48), and therefore the presence of the E146D Fe protein in the cell-free extracts should not interfere. As shown in Table IV, the inactive NifB FeMo cofactor-deficient MoFe protein could be activated either by addition of isolated FeMo cofactor in NMF or by free FeMo cofactor contained within the E146D nifH, cell-free extracts. Thus, free FeMo cofactor does accumulate in the E146D strain leading to the conclusion that the E146D Fe protein is not defective in the initial biosynthesis of FeMo cofactor.

The E146D Fe Protein Is Defective in FeMo Cofactor Insertion-- Strains that do not synthesize the Fe protein accumulate a FeMo cofactor-deficient form of the MoFe protein that can be activated in vitro, in cell-free extracts, by addition of isolated FeMo cofactor in NMF (15, 18-21). Previous studies have established that this "insertion" reaction also requires the Fe protein and MgATP and at least one additional protein, which has been designated gamma in A. vinelandii (18-27). One role of the Fe protein and MgATP appears to be to promote the association of gamma with the FeMo cofactor-deficient MoFe protein in such a way that the protein is converted from a form with the FeMo cofactor site buried to one with the FeMo cofactor site accessible for FeMo cofactor insertion (26). Fig. 12 is a time course for the insertion reaction where either wild type Fe protein or E146D Fe protein were added to cell-free extracts from nifH strain DJ54 along with MgATP and isolated FeMo cofactor. The results show that E146D Fe protein is obviously defective in the insertion of isolated FeMo cofactor into the FeMo cofactor-deficient MoFe protein. The shape of the insertion curve indicates that the E146D Fe protein is specifically defective in the initial phase of the reaction. After this barrier is overcome, the insertion can carry on at a higher rate. Previous studies would suggest that this early phase of the reaction may involve the Fe protein mediated association of the FeMo cofactor-deficient MoFe protein with gamma (26).

View larger version (22K): [in this window] [in a new window]   Fig. 12.   Time course of FeMo cofactor insertion by purified wild type () and E146D () Fe proteins into FeMo cofactor-deficient MoFe protein present in the extracts of nifH strain DJ54. As previously published (18, 19), the insertion required the addition of Fe protein and MgATP. Excess isolated FeMo cofactor in NMF was inserted into the FeMo cofactor-deficient MoFe protein of nifH strain DJ54 using extracts. The insertion assays were performed as described under "Experimental Procedures" and stopped at the indicated time points by adding (NH4)2MoS4 (34, 50). After the insertion, the enzyme activity was determined by measuring C2H4 evolution using the standard activity assay.

Conclusion-- The Fe protein of nitrogenase has three functions in the cell: 1) electron donation to the MoFe protein; 2) the initial biosynthesis of FeMo cofactor; and 3) the insertion of preformed FeMo cofactor into the FeMo cofactor-deficient MoFe proteins synthesized by nifH strains. In this study we have used sequence and structural information to identify a residue that might be involved in only the second or third functions. The above data establish that the E146D Fe protein is competent in electron transfer and the initial biosynthesis of FeMo cofactor but is specifically blocked for the first phase of the insertion reaction. Thus, the cells expressing the E146D Fe protein accumulate uninserted FeMo cofactor and partially FeMo cofactor-deficient MoFe protein. This is the first mutant that has been described that is only defective in this reaction. Glu¹⁴⁶ is a negatively charged surface residue. When it is converted to the smaller aspartate, the protein is partially active in the insertion reaction, whereas when it is converted to a positively charged lysine, a Nif phenotype is obtained. When combined with the information that the initial phase of the insertion reaction involves Fe protein/MgATP/gamma-mediated modification of the FeMo cofactor-deficient MoFe protein (26), the data suggest that Glu¹⁴⁶ is likely to be critical for specific protein-protein interactions. Future studies will be directed toward elucidating the details of those reactions.

	ACKNOWLEDGEMENTS

We acknowledge Professor Dennis Dean of Virginia Polytechnic Institute and State University for kindly providing nifB A. vinelandii strain DJ1143 and Dr. Hayley Angove for providing purified FeMo cofactor-deficient MoFe protein from DJ1143 and isolated FeMo cofactor.

	FOOTNOTES

^* This work was supported by National Institute of Health Grant GM-43144 (to B. K. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 949-824-4297; Fax: 949-824-8551; E-mail: bburgess@uci.edu.

¹ FeMo cofactor biosynthesis and insertion both appear to require MgATP hydrolysis and the binding of MgATP to the Fe protein. Many Fe protein variants that do not hydrolyze MgATP when bound to the MoFe protein still carry out FeMo cofactor biosynthesis and insertion. At present the possibility that these same mutants will hydrolyze MgATP when bound to another protein cannot be eliminated.

³ We have previously demonstrated by x-ray crystallography that breaking a salt bridge between surface Asp¹⁵ and Lys⁸⁴ of another [Fe-S] protein, by construction of a D15N mutation, caused Lys⁸⁴ to rearrange substantially and find a new partner (56).

	ABBREVIATIONS

The abbreviation used is: NMF, N-methyl formamide.

	REFERENCES

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