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Originally published In Press as doi:10.1074/jbc.M605527200 on August 6, 2006

J. Biol. Chem., Vol. 281, Issue 41, 30534-30541, October 13, 2006
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Molecular Insights into Nitrogenase FeMoco Insertion

TRP-444 OF MoFe PROTEIN {alpha}-SUBUNIT LOCKS FeMoco IN ITS BINDING SITE*Formula

Yilin Hu{ddagger}, Aaron W. Fay{ddagger}, Benedikt Schmid§, Beshoie Makar{ddagger}, and Markus W. Ribbe{ddagger}1

From the {ddagger}Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900 and the §Department of Biotechnology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91052 Erlangen, Germany

Received for publication, June 8, 2006 , and in revised form, July 31, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Biosynthesis of the FeMo cofactor (FeMoco) of nitrogenase MoFe protein is arguably one of the most complex processes in metalloprotein biochemistry. Here we investigate the role of a MoFe protein residue (Trp-{alpha}444) in the final step of FeMoco assembly, which involves the insertion of FeMoco into its binding site. Mutations of this aromatic residue to small uncharged ones result in significantly decreased levels of FeMoco insertion/retention and drastically reduced activities of MoFe proteins, suggesting that Trp-{alpha}444 may lock the FeMoco tightly in its binding site through the sterically restricting effect of its bulky, aromatic side chain. Additionally, these mutations cause partial conversion of the P-cluster to a more open conformation, indicating a potential connection between FeMoco insertion and P-cluster assembly. Our results provide some of the initial molecular insights into the FeMoco insertion process and, moreover, have useful implications for the overall scheme of nitrogenase assembly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Nitrogenase is a multicomponent metallo-enzyme that catalyzes the reduction of atmospheric dinitrogen to bioavailable ammonia (for recent reviews see Refs. 1–8). The best studied, Mo-containing nitrogenase of Azotobacter vinelandii is composed of two proteins, the Fe protein and the MoFe protein. The homodimeric Fe protein (Av2)2 has one ATP binding site per subunit and a single [4Fe-4S] cluster bridged between the subunits; whereas the {alpha}2beta2 tetrameric MoFe protein (Av1) contains, in each {alpha}beta subunit pair, two complex metal clusters. One, designated the P-cluster, is an [8Fe-7S] cluster (9) that is located at the {alpha}beta-interface and coordinated by six Cys ligands. The other, designated FeMoco, is a [Mo-7Fe-9S-X]3 cluster that is situated within the {alpha}-subunit and bound to His-{alpha}442, Cys-{alpha}275 and an endogenous homocitrate ligand (10). These metal clusters are essential for nitrogenase reactivity, mediating the ATP-driven, interprotein electron transfer from the [4Fe-4S] cluster of the Fe protein to the P-cluster of the MoFe protein and finally to the FeMoco where substrate reduction takes place.

Biosynthesis of P-cluster and FeMoco has attracted considerable attention, because these clusters are biologically unique and chemically unprecedented. Both P-cluster and FeMoco are composed of smaller substructures: the P-cluster consists of two [4Fe-4S] subclusters sharing aµ6-sulfide (9); and the FeMoco comprises [Mo-3Fe-3S] and [4Fe-3S] subcubanes sharing three µ2-sulfides and a central µ6-atom (10). Nonetheless, while the P-cluster is likely assembled through fusion of its substructural [4Fe-4S] units in its target location (11–13), FeMoco is first assembled on a scaffold NifEN protein and then inserted into its binding site in the MoFe protein (14–17). Biosynthesis of FeMoco presumably starts with the production of an Fe/S core by NifB (encoded by nifB), which is then transferred to, and further processed on the NifEN complex (14–19). Previously, we identified a molybdenum-free, NifEN-bound FeMoco precursor that bears a striking resemblance to the Fe/S core of the mature FeMoco (15). Most recently, we showed that, upon MgATP hydrolysis, Fe protein inserts molybdenum and homocitrate into the FeMoco precursor while it is still bound to NifEN, resulting in the formation of a fully complemented cluster that can be subsequently inserted into its final location in the MoFe protein (16, 17). We also established that the transfer of the cluster from NifEN to MoFe protein likely occurs through direct protein-protein interactions (16, 17). These findings not only provide important insights into the biosynthesis and biomimetic chemical synthesis of FeMoco, they may also bear useful implications for the biosynthetic mechanism of other complex metal-containing clusters (20–22).

Though progress has been made toward understanding the assembly of FeMoco prior to its insertion into the FeMoco binding site, little is known about the final step of FeMoco incorporation into its target location within the MoFe protein. Our current knowledge in this regard is largely based upon the crystal structure of a {Delta}nifB MoFe protein (Av1{Delta}nifB) from a nifB-deletion strain of A. vinelandii (23). Consistent with the hypothesis that nifB encodes for a protein that is involved in the biosynthesis of FeMoco, Av1{Delta}nifB is FeMoco-deficient, yet it contains normal P-clusters (23). In vitro, Av1{Delta}nifB can be reconstituted, without additional factors, into an active holoprotein by isolated FeMoco (23, 24). Given that the FeMoco binding site is fully buried at ~10 Å below the surface of the wild-type MoFe protein (Av1wild-type), this observation indicates that Av1{Delta}nifB undergoes significant conformational rearrangements relative to its holo counterpart, a process that renders its FeMoco site open and allows the subsequent insertion of isolated FeMoco (23). Indeed, when compared with Av1wild-type, the {alpha}III domain of Av1{Delta}nifB has some major structural changes in that nearly all beta-strands are shorter toward their C termini while {alpha}-helices A, C, and D are shorter toward their N termini. These conformational alterations create a positively charged insertion funnel in Av1{Delta}nifB, notably missing in its holo counterpart, that presumably steers the negatively charged FeMoco all the way down the funnel into its final location (23). A closer look at the FeMoco binding sites of Av1wild-type and Av1{Delta}nifB (Fig. 1) reveals that, in the case of Av1{Delta}nifB, the C{alpha} of His-{alpha}442 shifts ~5 Å during the rearrangement of {alpha}III domain and joins two other residues, His-{alpha}274 and His-{alpha}451, in the formation of a striking His triad (23). Coupled to this rearrangement, residues His-{alpha}442 and Trp-{alpha}444 switch their relative positions (23). Additionally, substantial changes take place in non-liganding residues as well; particularly, the stretch from {alpha}355 to {alpha}359, with Gly-{alpha}356, Gly-{alpha}357, and Arg-{alpha}359 that normally form hydrogen bonds to FeMoco sulfurs in holo Av1 (23). These residues are likely the key players in the process of FeMoco insertion and, therefore, serve as ideal targets for us to tackle the mechanism of the final step of FeMoco assembly on a molecular basis.


Figure 1
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  		FIGURE 1.
Protein environment of (A) Av1wild-type and (B) Av1{Delta}nifB in the vicinity of the FeMoco binding area. Parts of the C{alpha} backbones of Av1wild-type and Av1{Delta}nifB are shown and colored as follows: Av1wild-type in yellow and Av1{Delta}nifB in green. FeMoco is shown in ball-and-stick presentation, and the colors of the FeMoco atoms are: oxygen in red, carbon in gray, molybdenum in orange, sulfur in green, iron in magenta, and central atom X (of unknown origin) in blue. The hypothetical position of FeMoco in Av1{Delta}nifB is shown in light gray. Dotted lines represent coordinations between FeMoco atoms or between FeMoco and protein residues. Important side-chain residues in the {alpha}-subunits are indicated in the figure. Among them, the key residues proposed to be involved in FeMoco insertion are: 1) Trp-{alpha}444 (part of the FeMoco lock); 2) His-{alpha}274, His-{alpha}442, and His-{alpha}451 (His triad residues); 3){alpha}355 through{alpha}359 (part of the lid loop from{alpha}353 through{alpha}364); and 4) Lys-{alpha}426 (anchor for the organic moiety of FeMoco, homocitrate). Programs MOLSCRIPT (25) and RASTER3D (26) were used to generate the figure.

 
In the current study we examine the role of Trp-{alpha}444 of Av1 in FeMoco insertion by combined mutational, biochemical, and spectroscopic approaches. Our data show that substitution of this large aromatic residue for small uncharged ones results in a drastically decreased level of FeMoco insertion/retention, thus suggesting that the sterically prohibiting Trp-{alpha}444 may function in the FeMoco insertion process by tightly locking the FeMoco into its binding site.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Unless noted otherwise, all chemicals and reagents were obtained from Fisher Scientific, Baxter Scientific, or Sigma.

Construction of Variant A. vinelandii Strains—Table 1 summarizes the A. vinelandii strains used in this study. The wild-type strain AvYM13Awild-type (expressing His-tagged Av1wild-type and non-tagged Av2wild-type) was constructed as described elsewhere (24). The variant strains AvYM18AW444A-nifD (expressing His-tagged Av1W444A-nifD and non-tagged Av2W444A-nifD), AvYM19AW444F-nifD (expressing His-tagged Av1W444F-nifD and non-tagged Av2W444F-nifD), AvYM20AW444G-nifD (expressing His-tagged Av1W444G-nifD and non-tagged Av2W444G-nifD) and AvYM21AW444Y-nifD (expressing His-tagged Av1W444Y-nifD and non-tagged Av2W444Y-nifD) were constructed as follows. First, plasmid pHR30 was constructed, which contained the chromosomal fragment of nifD and nifK genes of A. vinelandii. Then, a series of oligos was used to create desired site-directed mutations of the nifD gene carried on pHR30, following the procedure of the commercial GeneEditor in vitro Site-directed Mutagenesis System (Promega, Madison, WI). The oligos used for mutations were (i) W444A, 5'-CGTCAAATGCACTCCGCCGATTATTCGGGCCCC-3'; (ii) W444F, 5'-CGTCAAATGCACTCCTTCGATTATTCGGGCCCC-3'; (iii) W444G, 5'-CGTCAAATGCACTCCGGCGATTATTCGGGCCCC-3'; and (iv) W444Y, 5'-CGTCAAATGCACTCCTACGATTATTCGGGCCCC-3'. The resulting plasmids were pHR31 (W444A-nifD), pHR32 (W444F-nifD), pHR33 (W444G-nifD), and pHR34 (W444Y-nifD). Finally, pHR31, pHR32, pHR33, and pHR34 were transformed into AvYM13A using a previously described method (27, 28), resulting in variant strains AvYM18AW444A-nifD, AvYM19AW444F-nifD, AvYM20AW444G-nifD, and AvYM21AW444Y-nifD with site-directed mutations of nifD gene on the chromosomal DNA.


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  		TABLE 1
Designations of strains and proteins of A. vinelandii in this work

 
Cell Growth and Protein Purification—All A. vinelandii strains were grown in 180-liter batches in a 200-liter New Brunswick fermentor (New Brunswick Scientific, Edison, NJ) in Burke's minimal medium supplemented with 2 mM ammonium acetate. The growth rate was measured by cell density at 436 nm using a Spectronic 20 Genesys (Spectronic Instruments, Westbury, NY). After the consumption of ammonia, the cells were de-repressed for 3 h followed by harvesting using a flow-through centrifugal harvester (Cepa, Lahr/Schwarzwald, Germany). The cell paste was washed with 50 mM Tris-HCl (pH 8.0). Published methods were used for the purification of all Av2 proteins (29) and His-tagged Av1 proteins (12, 28).

EPR Spectroscopy—All EPR samples were prepared in a Vacuum Atmospheres dry box (Hawthorne, CA) with an oxygen level of less than 4 ppm. Unless noted otherwise, all samples were in 25 mM Tris-HCl, pH 8.0, 10% glycerol and 2 mM Na2S2O4. Av1 protein samples were oxidized by incubation with excess indigo disulfonate (IDS) for 30 min. Subsequently, IDS was removed by a single passage over an anion exchange column as described elsewhere (30). All perpendicular and parallel mode EPR spectra were recorded using a Bruker ESP 300 Ez spectrophotometer (Bruker, Billerica, MA), interfaced with an Oxford Instruments ESR-9002 liquid helium continuous flow cryostat (Oxford Instruments, Oxon, UK). All spectra were recorded at 10 K using a microwave power of 50 milliwatt, a gain of 5 x 104, a modulation frequency of 100 kHz, and a modulation amplitude of 5 Gauss. The microwave frequencies of 9.62 and 9.39 GHz were used for the perpendicular (10 scans) and parallel (20 scans) mode EPR spectra, respectively. Spin quantitation of EPR signals was carried out as described in detail earlier (28).

Activity Assays and Metal Analysis—All nitrogenase activity assays were carried out as described previously (31). The products H2 and C2H4 were analyzed as published elsewhere (32). Ammonium was determined by a high performance liquid chromatography fluorescence method (33). FeMoco maturation assays were carried out as published earlier (14). Molybdenum (34) and iron (35) were determined as published elsewhere.

Protein Stability Experiments—Two approaches were used to determine the stability of the purified Av1 proteins: (i) heat treatment and (ii) prolonged storage at room temperature. A total amount of 100 mg of purified Av1 (in 25 mM Tris-HCl (pH 8.0), 10% glycerol, 250 mM NaCl, and 2 mM Na2S2O4) was incubated in a crimped anaerobic vial (volume, 8.7 ml; gas atmosphere, 100% Ar) at 56 °C for 30 s (i) or stored at room temperature for 8 h (ii). Precipitated protein was subsequently removed in both cases by centrifugation at 10,000 rpm for 10 min (Biofuge fresco, Heraeus, Germany) in a Vacuum Atmospheres dry box. Concentrations of Av1 proteins were subsequently determined using Bio-Rad protein assay, and enzymatic activities of these proteins were determined as described above.


Figure 2
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  		FIGURE 2.
Cell growth of AvYM13Awild-type (trace 1), AvYM21AW444Y-nifD (trace 2), AvYM19AW444F-nifD (trace 3), AvYM18AW444A-nifD (trace 4), and AvYM20AW444G-nifD (trace 5) under N2-fixing conditions. Strains were grown in 10-liter batches in a 12-liters New Brunswick fermentor on Burke's minimal medium. In all cases inoculums were 200 ml, with an approximate absorbance at 436 nm of 1.6. Growth of all strains was monitored by measuring the absorbance at 436 nm. The doubling time for each strain was determined by cell growth in the exponential phase as follows: (trace 1) ~5 h, (trace 2) ~5 h, (trace 3) ~5 h, and (trace 4) ~12 h. A negligible amount of cell growth was observed in the case of (trace 5).

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Trp-{alpha}444 is a highly conserved (~80%) residue among the currently known MoFe proteins from various organisms; the rest of them contain Tyr (~20%) in place of Trp.4 The 100% natural occurrence of an aromatic residue at this particular site, and the observation of a swapping in positions between this residue (Trp-{alpha}444) and one of the FeMoco ligands (His-{alpha}442) in Av1{Delta}nifB, have led to the hypothesis that a switch between the two residues is required for the large aromatic {alpha}444 residue to firmly pack the FeMoco into its final location (23). Mutations of this aromatic residue to small uncharged ones, therefore, may result in reduced capacities of the resulting Av1 variants in locking FeMoco in its binding site. A series of A. vinelandii strains expressing His-tagged Av1 protein variants with site-directed mutations at {alpha}444 was constructed, with Trp (W)-{alpha}444 replaced with Gly (G), Ala (A), Phe (F), or Tyr (Y). These strains (Table 1) are designated AvYM18AW444A-nifD (expressing Av1W444A-nifD and Av2W444A-nifD), AvYM19AW444F-nifD (expressing Av1W444F-nifD and Av2W444F-nifD), AvYM20AW444G-nifD (expressing Av1W444G-nifD and Av2W444G-nifD), and AvYM21AW444Y-nifD (expressing Av1W444Y-nifD and Av2W444Y-nifD).


Figure 3
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  		FIGURE 3.
Coomassie-stained SDS-PAGE of purified Av2 (A) and Av1 (B) proteins. A, 10–20% gradient SDS-PAGE of Av2 proteins: lane 1, protein standard, 15 µg; lane 2, Av2 wild-type, 4 µg; lane 3, Av2W444Y-nifD, 4 µg; lane 4, Av2 W444F-nifD, 4 µg; lane 5, Av2W444A-nifD, 4 µg; lane 6, Av2 W444G-nifD, 4 µg. The molecular masses of all purified Av2 proteins in this study appeared to be identical based on their elution profiles on gel filtration Sephacryl S-200 HR columns. B, 10–20% gradient SDS-PAGE of Av1 proteins: lane 1, protein standard, 15 µg; lane 2, Av1 wild-type, 5 µg; lane 3, Av1W444Y-nifD, 5 µg; lane 4, Av1W444F-nifD, 5 µg; lane 5, Av1W444A-nifD, 5 µg; lane 6, Av1W444G-nifD, 5 µg. The molecular masses of all purified Av1 proteins in this study appeared to be identical based on their elution profiles on gel filtration Sephacryl S-200 HR columns.

 
Under N2-fixing conditions,5 the doubling time of AvYM19AW444F-nifD or AvYM21AW444Y-nifD, like that of AvYM13Awild-type, is ~5 h (Fig. 2). However, the doubling time of AvYM18AW444A-nifD is ~12 h, which is significantly longer compared with that of AvYM13Awild-type (Fig. 2). In the case of AvYM20AW444G-nifD, only a negligible amount of cell growth is detected (Fig. 2). Given that cell growth under N2-fixing conditions is proportionately correlated with the amount of active nitrogenase, these observations indicate that a mutation of the aromatic Trp residue at {alpha}444 to a small uncharged residue (in the case of AvYM18AW444A-nifD or AvYM20AW444G-nifD) results in a significantly diminished enzymatic activity of nitrogenase; whereas nitrogenase reactivity is largely unaffected when the Trp-{alpha}444 is replaced by another aromatic residue (in the case of AvYM19AW444F-nifD or AvYM21AW444Y-nifD). Appar ently, the replacement of Trp-{alpha}444 with Gly has a more dramatic effect than that with Ala, which is not surprising, considering that Gly is a smaller residue than Ala. Meanwhile, although the doubling time of AvYM19AW444F-nifD or AvYM21AW444Y-nifD is roughly the same as that of AvYM13Awild-type, compared with AvYM13Awild-type, AvYM21AW444Y-nifD reaches nearly the same cell density while AvYM19AW444F-nifD achieves a slightly reduced cell mass, suggesting that the change of Trp-{alpha}444 to Phe (no natural occurrence) has at rifle more significant impact on the enzyme activity than that to Tyr (20% natural occurrence). This could be explained by the fact that the side chain of Phe is slightly smaller than that of the Tyr or Trp.

The decreased enzymatic activities do not originate from the Av2 proteins of these {alpha}444 variant strains.6 An approximate amount of 400 mg of non-tagged Av2 was purified from 200 g of cells of AvYM21AW444Y-nifD, AvYM20AW444F-nifD, AvYM18AW444A-nifD, or AvYM19AW444G-nifD, as was from AvYM13Awild-type (data not shown),7 suggesting that Av2 expression is unaffected in these variant strains. The monomer of Av2W444Y-nifD, Av2W444F-nifD, Av2W444A-nifD or Av2W444G-nifD, like that of Av2wild-type, is ~30 kDa (Fig. 3A). The molecular masses of all these Av2 proteins are ~60 kDa based on their elution profiles on gel filtration Sephacryl S-200 high resolution columns (data not shown), indicating that they are all homodimers. Compared with their wild-type counterpart, all Av2 proteins of the {alpha}444 variant strains have approximately the same metal content of 4 mol Fe/mol protein (Table 2) and exhibit, in the dithionite-reduced state, the same characteristic S = 1/2 EPR signal of rhombic line shape in the g = 2 region (Fig. 4). These data suggest that all Av2 proteins have a normal complement of [4Fe-4S] cluster and that the [4Fe-4S] cluster can be reduced by dithionite to an oxidation state of +1. All Av2 proteins show the same substrate reducing activities as Av2wild-type, with regard to C2H4 formation under C2H2/Ar, H2 formation under Ar, NH3 formation under N2, or H2 formation under N2 (Table 3), suggesting that they are all fully proficient in their catalytic capacities. Additionally, these Av2 proteins all function as normally as the wild-type protein in the FeMoco maturation assay (Table 3), indicating that they are perfectly competent in FeMoco assembly, a second function that has been definitively assigned to this multitask component of nitrogenase recently (14, 16, 17).


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  		TABLE 2
Metal contents of purified Av1 and Av2 proteins

 


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  		TABLE 3
Activities of purified Av2 proteins

 


Figure 4
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  		FIGURE 4.
Perpendicular mode EPR spectra of dithionite-reduced Av2wild-type (trace 1), Av2W444Y-nifD (trace 2), Av2W444F-nifD (trace 3), Av2W444A-nifD (trace 4), and Av2W444G-nifD (trace 5). EPR samples (20 mg/ml) were prepared and measured as described under "Experimental Procedures." All Av2 proteins show the characteristic S = 1/2 signal of the [4Fe-4S]1+ cluster at nearly the same intensity.

 
Like the Av2 proteins, the Av1 proteins of the Trp-{alpha}444 variant strains are expressed at practically the same level as Av1wild-type, as evidenced by Western blot analysis of Av1-specific antibody against the crude extracts of these variant strains under non-saturated conditions (supplemental Fig. S1). In addition, ~600 mg of His-tagged Av1 was purified from 200 g of cells of AvYM21AW444Y-nifD, AvYM20AW444F-nifD, AvYM18AW444A-nifD or AvYM19AW444G-nifD, as was from AvYM13Awild-type (data not shown),7 further supporting the notion that Av1 expression is unperturbed with point mutations at {alpha}444 of Av1. Moreover, like Av1wild-type, all these {alpha}444 Av1 variants are composed of {alpha} (~56 kDa)- and beta (~59 kDa)-subunits (Fig. 3B) and have the same, {alpha}2beta2-tetrameric molecular mass of ~230 kDa based on their elution profiles on gel filtration Sephacryl S-200 columns (data not shown). Given the presence of nearly the same amount of Av1 in the variant strains, the diminished nitrogenase activity, particularly in the case of AvYM18AW444A-nifD or AvYM20AW444G-nifD, must arise from a decrease in the activity of the variant Av1 protein. Indeed, compared with Av1wild-type, Av1W444A-nifD (of AvYM18AW444A-nifD) has ~20% of activity in terms of C2H4 formation under C2H2/Ar, H2 formation under Ar, NH3 formation under N2, or H2 formation under N2; whereas Av1W444G-nifD (of AvYM20AW444G-nifD) shows practically no activity at all (Table 4). On the other hand, Av1W444Y-nifD (of AvYM21AW444Y-nifD) has nearly 100% of the activity of Av1wild-type; while Av1W444F-nifD (of AvYM19AW444F-nifD) shows a moderately reduced activity that ranges between 81 and 92% of that of Av1wild-type (Table 4). The activities of Av1 proteins are consistent with the growth rates of the respective variant strains expressing them (Fig. 2); more importantly, they correlate well with their respective metal contents (Table 2). The amount of iron or molybdenum that is present in Av1 decreases as follows: Av1W444Y-nifD > Av1W444F-nifD > Av1W444A-nifD > Av1W444G-nifD, which agrees with an increase in the extent of Trp-{alpha}444 mutation regarding the size of the residue. In particular, the molybdenum content (Table 2), in the descending order of Av1W444Y-nifD (91%), Av1W444F-nifD (87%), Av1W444A-nifD (14%), and Av1W444G-nifD (3%), is remarkably consistent with the activity of the respective protein (Table 4), in the decreasing rank of Av1W444Y-nifD (91–105%), Av1W444F-nifD (81–92%), Av1W444A-nifD (18–21%), and Av1W444G-nifD (<1%). Because the molybdenum content correlates directly with the FeMoco content, these results provide compelling evidence that the reduced activities of Av1 proteins from {alpha}444 variant strains originate from decreased levels of FeMoco in the proteins. Furthermore, the substrate reducing activities of all substrates, namely, C2H4 formation under C2H2/Ar, H2 formation under Ar, NH3 formation under N2, and H2 formation under N2, are proportionally decreased (Table 4). This observation suggests that the FeMoco is correctly inserted in the Av1 variant protein and indicates, yet again, that the decreased activity of Av1 variant protein is caused by less FeMoco rather than the incorrectly positioned FeMoco that could change the activity profile of Av1.


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  		TABLE 4
Activities and EPR spin integration of purified Av1 proteins

 


Figure 5
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  		FIGURE 5.
Perpendicular mode EPR spectra of dithionite-reduced Av1wild-type (trace 1), Av1W444Y-nifD (trace 2), Av1W444F-nifD (trace 3), Av1W444A-nifD (trace 4), and Av1W444G-nifD (trace 5). EPR samples (20 mg/ml) were prepared and measured as described under "Experimental Procedures." The FeMoco-specific, S = 3/2 EPR signals of Av1W444Y-nifD, Av1W444F-nifD, Av1W444A-nifD, and Av1W444G-nifD integrate to 98, 80, 21, and 1% of that of Av1wild-type, respectively. The S = 1/2 EPR signals in the g {approx} 2 region of the spectra of Av1W444A-nifD and Av1W444G-nifD integrate to 0.08 and 0.1 spin per Av1wild-type, respectively.

 
EPR analyses of the Av1 variant proteins provide further support to our argument that a mutation of Trp-{alpha}444 to a small uncharged residue results in a decreased amount of FeMoco accumulation in Av1. In the dithionite-reduced state, Av1wild-type (Fig. 5, trace 1) exhibits a distinct, FeMoco-specific, S = 3/2 signal (1). This S = 3/2 signal is also observed in the case of Av1W444Y-nifD (Fig. 5, trace 2), Av1W444F-nifD (Fig. 5, trace 3), or Av1W444A-nifD (Fig. 5, trace 4), although spin integration data indicate that the intensities of the signals are 98, 80, and 21% of that of Av1wild-type for Av1W444Y-nifD, Av1W444F-nifD, and Av1W444A-nifD, respec tively (Table 4). In the case of Av1W444G-nifD, this character istic S = 3/2 signal is virtually non-existent, integrating to 1% of that of Av1wild-type (Fig. 5, trace 5). The spin integration data from these EPR experiments align well with those from the growth curves (Fig. 2), metal analyses (Table 2) and activity assays (Table 4), once again confirming the presence of less FeMoco in Av1 variant proteins with mutations from aromatic Trp-{alpha}444 to small uncharged residues like Ala and Gly. Thus, all results (summarized in supplemental Fig. S2) come together and corroborate with our theory that Trp-{alpha}444 is specifically involved in FeMoco insertion, that the large size and steric rigidity of Trp-{alpha}444 is crucial for locking FeMoco into its binding site, and that removal of this restraint compromises the ability of the Av1 to insert and/or retain FeMoco.

Interestingly, in the dithionite-reduced state, Av1W444A-nifD (Fig. 5, trace 4) and Av1W444G-nifD (Fig. 5, trace 5) show additional S = 1/2 signals that integrate to 0.08 and 0.1 spin per protein, respectively. This particular S = 1/2 signal has been previously assigned to a P-cluster analog comprising two [4Fe-4S]-like fragments, which may represent a physiologically relevant intermediate during P-cluster assembly (12, 13, 37). Consistent with this observation, in the IDS-oxidized state, Av1 variant proteins exhibit P-cluster specific (P2+ state), g = 11.8, parallel mode EPR signals (38, 39), which integrate to 106, 99, 92, and 93% of that of Av1wild-type (Fig. 6, trace 1) for Av1W444Y-nifD (Fig. 6, trace 2), Av1W444F-nifD (Fig. 6, trace 3), Av1W444A-nifD (Fig. 6, trace 4) and Av1W444G-nifD (Fig. 6, trace 5), respectively. The S = 1/2 signal-associated analog of P-cluster, therefore, could account for the missing portion of P-cluster in Av1W444A-nifD or Av1W444G-nifD. Apparently, when Trp-{alpha}444, one of the protein residues in a stretch of polypeptide that reorients with FeMoco insertion, is mutated to small uncharged residues, the [8Fe-7S] P-cluster is converted, partially, to a more open conformation consisting of a pair of [4Fe-4S]-like clusters. It is likely that the mutation of Trp-{alpha}444 to Ala or Gly leads to a conformational rearrangement that in turn affects the P-cluster site and forces the substructural units of P-cluster apart. This observation, therefore, suggests a potential association between the insertion of FeMoco and the assembly of P-cluster and, perhaps more significantly, places these seemingly independent events in an overall, interactive scheme of nitrogenase biosynthesis.


Figure 6
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  		FIGURE 6.
Parallel mode EPR spectra of IDS-oxidized Av1wild-type (trace 1), Av1W444Y-nifD (trace 2), Av1W444F-nifD (trace 3), Av1W444A-nifD (trace 4), and Av1W444G-nifD (trace 5). EPR samples (20 mg/ml) were prepared and measured as described under "Experimental Procedures." The P-cluster specific (P2+ state), g = 11.8, parallel mode EPR signals of Av1W444Y-nifD, Av1W444F-nifD, Av1W444A-nifD, and Av1W444G-nifD integrate to 106, 99, 92, and 93% of that of Av1wild-type, respectively.

 
It is important to note that, the mutations at Trp-{alpha}444 do not affect the overall stability of the Av1 variant proteins in this study. Like Av1wild-type, variant proteins Av1W444Y-nifD, Av1W444F-nifD, Av1W444A-nifD, and Av1W444G-nifD all exhibit considerable stability upon heat treatment or prolonged storage at room temperature (Table 5). In addition, in the presence of MgATP, each of these {alpha}444 Av1 variants can form a complex with Av2wild-type that is similarly stable compared with that formed between Av1wild-type and Av2wild-type (supplemental Fig. S3). Furthermore, structural predictions based on program MUMBO (40)8 show that the Trp-{alpha}444 mutations in this study present no steric problems that could lead to the instability of the Av1 protein (data not shown).


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  		TABLE 5
Comparison of the stability of purified Av1 proteins

 

In summary, using a combined mutational/bio-chemical/spectroscopic approach, we show that the Trp-{alpha}444 of Av1 is specifically involved in the FeMoco insertion/retention. Mutations of this aromatic residue to small uncharged ones result in dramatically decreased levels of FeMoco insertion/retention and drastically reduced activities of the Av1 proteins, suggesting that Trp-{alpha}444 may lock the FeMoco tightly into its binding site through the sterically restricting effect of its bulky, aromatic side chain. Additionally, these mutations cause partial conversion of the P-cluster to a more open conformation, indicating a potential connection between FeMoco insertion and P-cluster assembly. Our results provide some of the initial insights into the process of FeMoco insertion on the molecular basis. Future studies will focus on addressing the effects of other key residues involved in FeMoco insertion and investigating the link between the assembly processes of FeMoco and P-cluster, in hope of elucidating a more detailed mechanism of nitrogenase biosynthesis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM-67626 (to M. W. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3. Back

1 To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, University of California-Irvine, Irvine, CA 92697-3900. Tel.: 949-824-9509; Fax: 949-824-8551; E-mail: mribbe{at}uci.edu.

2 The abbreviations used are: Av2, Fe protein; FeMoco, iron-molybdenum cofactor; IDS, indigo disulfonate; Av1, MoFe protein. Back

3 The identity of X is unknown but it is considered to be C, O, or N (10). Back

4 These numbers are based on a sequence comparison of known nitrogenase MoFe proteins (SWISS-PROT) using the program BLAST (36). Back

5 Only organisms capable of expressing active nitrogenase enzymes are able to grow under the N2-fixing conditions, where no fixed or organic nitrogen (such as ammonia and urea) is present in the culture media. Back

6 Although point mutations of Av1 have no apparent effect on Av2, it has been well established that the latter is involved in FeMoco biosynthesis and insertion (11). Because this work deals with FeMoco insertion into Av1 variants, the activities of which are tested by assays that involve Av2, it is crucial to show that the Av2 of the variant strains have normal cluster compositions and function normally in both catalytic and biosynthetic capacities. Back

7 Note that for protein purification, all strains were grown in the presence of a limited amount of ammonia, so that a certain amount of cell mass could be accumulated regardless of the ability of the strain to fix nitrogen. After the consumption of ammonia, cells were derepressed for 3 h, during which process the nitrogenase protein (active or inactive) was expressed. Under these conditions, a consistent yield of ~200 g of cells per 180 liters of cell growth batch could be obtained for the wild-type and variant strains of A. vinelandii. Back

8 The protein-design program MUMBO (40) was used to predict the stability of Av1 variant proteins, where Trp-{alpha}444 was replaced by Tyr, Phe, Ala, or Gly, respectively. Initially, MUMBO was tested for its ability to find the correct side chain conformations of those residues located less than 10 Å away from Trp-{alpha}444 (with Trp-{alpha}444 included). The predicted side chain conformations were a close match to those found in the crystal structure (Protein Data Bank entry 1M1N) thus confirming the feasibility of the program for this particular protein (data not shown). Subsequently, the side chain conformations in the same 10 Å region of the Av1 variant proteins were calculated with MUMBO for each case, which again led to a close match to those of Av1wild-type. The mutated side chains were oriented similarly to the Trp side chain and energetically even more favorable (data not shown). Based on this prediction, no steric problems that could lead to the instability of the protein were observed. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Yves Muller (Friedrich-Alexander-Universität, Erlangen-Nürnberg, Germany) for his kind help on protein stability prediction using program MUMBO.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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