Studies on the Circular Dichroism (CD) and Electron Paramaguetic Resonance(EPR) Spe-ctroscopy of the Partially Oxidized FeMo Protein from Azotobacter vinelandii Nitrogenase

After nitrogenase FeMo protein from A. vinelandii was incubated with a large excess (5–6 equivalents) of indigo carmine for 30–60 min., all of P-clusters in the partially oxidized FeMo protein were oxidized, but all of FeMoCo in the protein were still in the reductive state. The oxidized P-clusters in the protein were able to be completely reduced by Na2S2O4(DT) and the reduction was accelerated by methyl viologen (MV). And all of FeMoCo in the protein were firstly oxidized by some oxidants such as methylene blue. The numbers of the redox equivalent of P-cluster and FeMoCo have been obtained by the CD reductive titration of and EPR/ ABS time course on the oxidation of the partially oxidizeA FeMo protein, respectivelly.     

The FeMoco-deficient MoFe protein produced by a nifH deletion strain of Azotobacter vinelandii shows unusual P-cluster features

The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (Delta(nifH) MoFe protein) was purified in large quantity. The alpha(2)beta(2) tetrameric Delta(nifH) MoFe protein is FeMoco-deficient based on metal analysis and the absence of the S = 3/2 EPR signal, which arises from the FeMo cofactor center in wild-type MoFe protein. The Delta(nifH) MoFe protein contains 18.6 mol Fe/mol and, upon reduction with dithionite, exhibits an unusually strong S = 1/2 EPR signal in the g approximately 2 region. The indigo disulfonate-oxidized Delta(nifH) MoFe protein does not show features of the P(2+) state of the P-cluster of the Delta(nifB) MoFe protein. The oxidized Delta(nifH) MoFe protein is able to form a specific complex with the Fe protein containing the [4Fe-4S](1+) cluster and facilitates the hydrolysis of MgATP within this complex. However, it is not able to accept electrons from the [4Fe-4S](1+) cluster of the Fe protein. Furthermore, the dithionite-reduced Delta(nifH) MoFe can be further reduced by Ti(III) citrate, which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.     

钼铁蛋白铁钼辅因子的有机组分对其功能的影响

棕色固氮菌(Azotobacter vinelandii)固氮酶的钼铁蛋白经邻菲啰啉在厌氧或有氧环境中处理后,变为 P-cluster 单一缺失或 P-cluster 和 FeMoco 同时缺失的失活钼铁蛋白。含柠檬酸盐或高柠檬酸盐的重组液都使这两种失活蛋白能恢复固氮酶重组的 H~ 和 C_2H_2还原活性,活性恢复程度随反映钼铁蛋白中金属原子簇含量变化的圆二色和磁圆二色谱及金属含量的恢复程度的提高而提高,但它们固 N_2能力的恢复程度则不相同:P-cluster 单一缺失的蛋白用两种重组液重组后均可恢复其固 N_2能力,而 P-cluster 和 FeMoco 同时缺失的蛋白,只有用含高柠檬酸盐的重组液重组才恢复其固 N_2能力,表明含不同有机组分的重组液所组装的 P-cluster 均与天然状态相同,只有含高柠檬酸盐的重组液所组装的 FeMoco 才与天然状态相同,从而证明高柠檬酸盐是 FeMoco 的必需的有机组分。     

Spectroscopic evidence for changes in the redox state of the nitrogenase P-cluster during turnover

Biological nitrogen fixation catalyzed by nitrogenase requires the participation of two component proteins called the Fe protein and the MoFe protein. Each alphabeta catalytic unit of the MoFe protein contains an [8Fe-7S] cluster and a [7Fe-9S-Mo-homocitrate] cluster, respectively designated the P-cluster and FeMo-cofactor. FeMo-cofactor is known to provide the site of substrate reduction whereas the P-cluster has been suggested to function in nitrogenase catalysis by providing an intermediate electron-transfer site. In the present work, evidence is presented for redox changes of the P-cluster during the nitrogenase catalytic cycle from examination of an altered MoFe protein that has the beta-subunit serine-188 residue substituted by cysteine. This residue was targeted for substitution because it provides a reversible redox-dependent ligand to one of the P-cluster Fe atoms. The altered beta-188(Cys) MoFe protein was found to reduce protons, acetylene, and nitrogen at rates approximately 30% of that supported by the wild-type MoFe protein. In the dithionite-reduced state, the beta-188(Cys) MoFe protein exhibited unusual electron paramagnetic resonance (EPR) signals arising from a mixed spin state system (S = 5/2, 1/2) that integrated to 0.6 spin/alphabeta-unit. These EPR signals were assigned to the P-cluster because they were also present in an apo-form of the beta-188(Cys) MoFe protein that does not contain FeMo-cofactor. Mediated voltammetry was used to show that the intensity of the EPR signals was maximal near -475 mV at pH 8.0 and that the P-cluster could be reversibly oxidized or reduced with concomitant loss in intensity of the EPR signals. A midpoint potential (Em) of -390 mV was approximated for the oxidized/resting state couple at pH 8.0, which was observed to be pH dependent. Finally, the EPR signals exhibited by the beta-188(Cys) MoFe protein greatly diminished in intensity under nitrogenase turnover conditions and reappeared to the original intensity when the MoFe protein returned to the resting state.     

Stoichiometry and spectral properties of the MoFe cofactor and noncofactor redox centers in the MoFe protein of nitrogenase from Azotobacter vinelandii

Reductive EPR and optical titrations of oxidized MoFe protein using reduced methyl viologen as reductant were used to quantitate the stoichiometry of the various spectroscopically and electrochemically distinct redox centers in the oxidized MoFe protein. Three centers were found to correlate with the EPR signal development (MoFe cofactor centers), and three centers were found to be independent of the EPR signal (P clusters) but to demonstrate distinct optical and kinetic properties. Oxidative EPR and optical titrations of reduced MoFe protein are reported which support the presence of three P-cluster centers. The optical titrations show a distinct change in kinetic behavior between the MoFe cofactor and P-cluster centers. Controlled potential coulometry demonstrates that incremental oxidation of reduced protein by methylene blue, thionine, or indigodisulfonate occurs specifically at three P-cluster sites. Subsequent oxidation by methylene blue and thionine (but not indigodisulfonate) causes the EPR signal to disappear. Three P-cluster sites, two EPR sites, and one presently uncharacterized site are suggested by the results of this study.     

Studies on Assembly of Vanadium-iron Protein in Vitro

By treating the reduced MoFe protein of nitrogenase from Azotobacter vinelandii with ophenanthroline under anaerobic or aerobic condition,inactive MoFe protein which was partialy deficient in both P-cluster and FeMoco could be obtained. After incubating the inactive MoFe protein with a reconstituent solution containing NaVO3,ferric homocitrate, Na2S and dithiothreitol, a reconstituted protein could be obtained. The proton reduction activity and absorption spectrum of the reconstituted protein could be well restored, but its C2H2-reduction activity could not be recovered. Its CD spectrum could be recovered except for the 550nm to 650nm region which differed from that of the reduced MoFe protein. The results showed that the reconstituted protein was different from MoFe protein,but was similar to vanadium-iron protein.     

Molecular insights into nitrogenase FeMoco insertion: TRP-444 of MoFe protein alpha-subunit locks FeMoco in its binding site

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-alpha444) 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-alpha444 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.     

Reactivation of Partially Metallocluster deficient MoFe Protein by Rhenium-containing Reconstituent Solution

When the reduced MoFe protein from Azotobacter vinelandii Lipmann was treated with ophenanthroline and air, an inactive protein partially deficient in both FeMoco and P-cluster could be obtained. After incubating the treated protein with a reconstituent solution containing Re2OT, ferric homocitrate, Na2S and dithiothreitol, which had no circular dichroism (CD) signal, the ultraviolet and visible CD spectra, the C2H2 and H+ -reduction activity of the incubated protein were significantly recovered. However, the spectra were somewhat different from those of the reduced MoFe protein. The results showed that: 1) in the incubated protein solution there was possibly a new recombined ReFe protein besides the intact MoFe protein which was not destroyed by the treatment with o-phenanthroline and air; 2) it might be possible that both ReFe protein and MoFe protein exhibited similar ability of nitrogen fixation, although they were somewhat different in structure.     

Studies on the Reactivation of Aerated Nitrogenase MoFe Protein from Azotobacter vinelandii by the Compounds of iron Molybdenum and Sulfur

After the exposure to air, the crystalline nitrogenase MoFe protein from Azotobacter vinelandii was resulted in the remarkable increase in its absorption (ABS) and the significant decrease in its activity and circular dichroism (CD). However, when the aerated MoFe protein was incubated with the reconstituting solution which consisted of Na2MoO4, ferric citrate, Na2S and dithiothreitol, the ABS and CD of the aerated. MoFe protein both were completely restored, simultaneously with the significant restoration of acetylene reduction. It is shown that the P-cluster and other parts related to the protein activity which was damaged by O2 are able to be repaired to a certain extent by the reconstituting solution.     

钼,铁,硫化合物激活曝氧的棕色固氮菌固氮酶钼铁蛋白的研究

曝氧后,棕色固氮菌(Azotobacter vinelandii)固氮酶钼铁蛋白的催化活性和圆二色信号都显著降低,而吸收光谱则显著增加。与钼、铁、硫化合物和二硫苏糖醇组成的重组溶液保温后,曝氢蛋白的圆二色信号和吸收光谱几乎完全恢复至天然状态的同时,乙炔还原活性也得到了显著的恢复,表明重组溶液可使曝氧蛋白中的 P-cluster和其它活性部位都得到了不同程度的修复。     

The Fe-only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus: a comparative study on the redox properties of the metal clusters present in the dinitrogenase components.

The dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe-only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox-titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1Mo) three significant S = 1/2 EPR signals deriving from oxidized states of the P-cluster were detected: (1) a rhombic signal (g = 2.07, 1.96 and 1.83), which showed a bell-shaped redox curve with midpoint potentials (Em) of -195 mV (appearance) and -30 mV (disappearance), (2) an axial signal (g(parallel) = 2.00, g perpendicular = 1.90) with almost identical redox properties and (3) a second rhombic signal (g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the 'low-potential' rhombic signal and the axial signal have been both attributed to the one-electron-oxidized P-cluster (P1+) present in two conformationally different proteins, the 'high-potential' rhombic signal has been suggested rather to derive from the P3+ state. Upon oxidation, the FeFe protein (Rc1Fe) exhibited three significant S = 1/2 EPR signals as well. However, the Rc1Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P-cluster states. (a) The most prominent feature is an unusually broad signal at g = 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: -80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption-shaped peak at g = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials (Em = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite-reduced state of Rc1Fe with the dinitrogenase reductase component (Rc2Fe) of the same enzyme system (turnover conditions in the presence of N2 and ATP). When the Rc1Mo protein was treated analogously, neither this 'turnover signal' nor any other S = 1/2 signal were detectable. All Rc1Fe-specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1Mo preparations.     

The Effect of Manganese on the Reconstitution of Partial Metallocluster-deficient Nitrogenase Molybdenum-Iron Protein

By treating the reduced MoFe protein of nitrogenase from Azotobacter vinelandii with O-phenanthroline (O-phen) and O2, inactive MoFe protein which was partialy deficient in both P-cluster and FeMoco could be obtained. After incubating the inactive protein with a reconstituent solution containing KMnO4, ferric homocitrate, Na2S and dithiothreitol, a reconstituted protein could be obtained. The absorption spectrum and C2H2, H+ and N2 reduction activity of the reconstituted protein could be well restored to the state of the reduced MoFe protein. However, the α-helix and CD spectrum at 380—550 nm and at 620—670 nm of the reconstituted protein were somewhat different from those of the reduced MoFe protein. The results showed that: (1) the reconstituted protein was composed of the assembled protein which might be a MnFe protein due to the reconstitution of the metalloclusterdeficient MoFe protein with Mn-containing solution and MoFe protein in which metalloclusters were still intact after the treatment with O-phen and O2; (2) It might be possible that the MnFe protein and MoFe protein were similar in the ability of nitrogen fixation, but were somewhat different in the structure from each other.     

Purification and Properties of the Constituents of the Nitrogenase Complex from Clostridium pasteurianum

A new procedure for a rapid and extensive purification of the FeMo protein and the Fe protein of the nitrogenase complex from Clostridium pasteurianum is described. Specific activities of 345 and 460 nmoles of N(2) reduced per mg of protein per min for the FeMo protein and for the Fe protein, respectively, have been obtained. Preparations of the FeMo protein contained 0.96 atom of molybdenum and 15 atoms of iron per molecule, whereas those of the Fe protein contained 2.86 atoms of iron per molecule. Experiments suggest that a definite association of two Fe proteins and one FeMo protein is functional in the active enzyme complex. No individual role could be ascribed to either of the two proteins, but the fact that hydrogenase inhibits N(2) fixation but not the reductant-dependent adenosine triphosphate hydrolysis supports the idea that there are two distinct sites on nitrogenase, one concerned with N(2) activation and the other with activated electron transport.     

Conformational differences between Azotobacter vinelandii nitrogenase MoFe proteins as studied by small-angle X-ray scattering

The nitrogenase MoFe protein is a heterotetramer containing two unique high-nuclearity metalloclusters, FeMoco and the P-cluster. FeMoco is assembled outside the MoFe protein, whereas the P-cluster is assembled directly on the MoFe protein polypeptides. MoFe proteins isolated from different genetic backgrounds have been analyzed using biochemical and spectroscopic techniques in attempting to elucidate the pathway of P-cluster biosynthesis. The DeltanifH MoFe protein is less stable than other MoFe proteins and has been shown by extended X-ray absorption fine structure studies to contain a variant P-cluster that most likely exists as two separate [Fe4S4]-like clusters instead of the subunit-bridging [Fe8S7] cluster found in the wild-type and DeltanifB forms of the MoFe protein [Corbett, M. C., et al. (2004) J. Biol. Chem. 279, 28276-28282]. Here, a combination of small-angle X-ray scattering and Fe chelation studies is used to show that there is a correlation between the state of the P-cluster and the conformation of the MoFe protein. The DeltanifH MoFe protein is found to be larger than the wild-type or DeltanifB MoFe proteins, an increase in size that can be modeled well by an opening of the subunit interface consistent with P-cluster fragmentation and solvent exposure. Importantly, this opening would allow for the insertion of P-cluster precursors into a region of the MoFe protein that is buried in the wild-type conformation. Thus, DeltanifH MoFe protein could represent an early intermediate in MoFe protein biosynthesis where the P-cluster precursors have been inserted, but P-cluster condensation and tetramer stabilization have yet to occur.     

Evidence that conserved residues Cys-62 and Cys-154 within the Azotobacter vinelandii nitrogenase MoFe protein α-subunit are essential for nitrogenase activity but conserved residues His-83 and Cys-88 are not

Metallocluster extrusion requirements, interspecies MoFe-protein primary sequence comparisons and comparison of the primary sequences of the MoFe-protein subunits with each other have been used to assign potential P-cluster (Fe-S cluster) domains within the MoFe protein. In each alpha-beta unit of the MoFe protein, alpha-subunit domains, which include potential Fe-S cluster ligands Cys-62, His-83, Cys-88 and Cys-154, and beta-subunit domains, which include potential Fe-S cluster ligands Cys-70, His-90, Cys-95 and Cys-153, are proposed to comprise nearly equivalent P-cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the alpha-subunit Cys-62, His-83, Cys-88 and Cys-154 positions by site-directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates, MoFe-protein acetylene-reduction activities, and whole-cell S = 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe-protein alpha-subunit residues, Cys-62 and Cys-154, are probably essential for MoFe-protein activity but that His-83 and Cys-88 residues are not. These results indicate either that His-83 and Cys-88 do not provide essential P-cluster ligands or that a new cluster-ligand arrangement is formed in their absence.     

Iron-molybdenum cofactor insertion into the Apo-MoFe protein of nitrogenase involves the iron protein-MgATP complex

Nitrogenase is composed of two component proteins, the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein). The Fe protein is a Mr 60,000 dimer of identical subunits with one bridging [4Fe-4S] center. It serves as a one-electron donor to the MoFe protein in a reaction that is coupled to MgATP hydrolysis. The MoFe protein is an alpha 2 beta 2 tetramer of Mr 220,000 which contains four [4Fe-4S] clusters and two iron-molybdenum cofactor (FeMo cofactor) centers. The exact structure of FeMo cofactor is not known, but it is believed to form the active site of the enzyme. Using specifically constructed deletion mutants of Azotobacter vinelandii, we have previously shown that the Fe protein, but not the MoFe protein, is required for FeMo cofactor biosynthesis (Robinson, A. C., Dean, D. R., and Burgess, B. K. (1987) J. Biol. Chem. 262, 14327-14332). During the partial purification of a FeMo cofactor-deficient form of the MoFe protein from one of these mutants (DJ54, delta nifH), we have discovered that, in addition to biosynthesis, the Fe protein-MgATP complex is involved in FeMo cofactor insertion into the MoFe protein. This insertion process is also sensitive to a number of other parameters (e.g. salt, pH, temperature, protein concentration). Based on our experimental data, we present a model for how this insertion reaction might take place, in which the Fe protein-MgATP complex binds the FeMo cofactor-deficient form of the MoFe protein and stabilizes a specific conformation of the MoFe protein that has the FeMo cofactor binding site exposed and available for coordination by preformed FeMo cofactor.     

Iron-molybdenum cofactor biosynthesis in Azotobacter vinelandii requires the iron protein of nitrogenase

Nitrogenase is composed of two separately purified proteins called the Fe protein and the MoFe protein. In Azotobacter vinelandii the genes encoding these structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). The MoFe protein contains an ironmolybdenum cofactor (FeMo cofactor) whose biosynthesis involves the participation of at least five gene products, nifQ, nifB, nifN, nifE, and nifV. In this study an A. vinelandii mutant strain, which contains a defined deletion within the nifH (Fe protein) gene, was isolated and studied. This mutant is still able to accumulate significant amounts of MoFe protein subunits. However, extracts of this nifH deletion strain have only very low levels of MoFe protein acetylene reduction activity. Fully active MoFe protein can be reconstituted by simply adding isolated FeMo cofactor to the extracts. Fe protein is not necessary to stabilize or insert this preformed FeMo cofactor into the FeMo cofactor-deficient MoFe protein synthesized by the nifH deletion strain. Extracts of the nifH deletion strain can carry out molybdate and ATP-dependent in vitro FeMo cofactor biosynthesis provided Fe protein is added, demonstrating that they contain the products encoded by the FeMo cofactor biosynthetic genes. These data demonstrate that the Fe protein is physically required for the biosynthesis of FeMo cofactor in A. vinelandii.     

The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein

We have examined three strains of Azotobacter vinelandii, which contain defined deletions within the nifH, nifB, or nifE genes. All three strains accumulate inactive FeMo cofactor-deficient forms of the MoFe protein of nitrogenase. These forms can be activated in vitro by addition of isolated FeMo cofactor in N-methylformamide. Although the phenotypes of these strains are superficially the same, our characterizations demonstrate that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is quite different from that synthesized by either the delta nifB or delta nifE strains. These differences include the following: 1) the activation of the delta nifH protein requires MgATP, whereas the activation of the delta nifB and delta nifE proteins does not; 2) the delta nifH extracts can be activated with FeMo cofactor to wild-type levels of activity, whereas delta nifB and delta nifE extracts cannot; 3) the delta nifH protein is markedly less heat stable than the delta nifB and delta nifE proteins; and 4) the migration of the delta nifH protein on native gels is very different when compared with delta nifB and delta nifE, which look like each other. These data can be explained if the nifB and nifE gene products are only involved in FeMo cofactor biosynthesis, whereas the nifH gene product is involved in both the initial synthesis of FeMo cofactor and in the insertion of preformed FeMo cofactor into the MoFe protein. A model is presented that suggests that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is the one that normally participates in MoFe protein assembly in wild-type cells.     

Comparative Studies on Mn,Cr and Mo Contained Reconstituent Solutions and Proteins Reconstituted with Partially Metallocluster-deficient MoFe Protein

By treating the reduced MoFe protein from Azotobacter vinelandii with o-phenanthroline and O2, an inactive protein partially deficient in both FeMoco and P-cluster could be obtained. The inactive protein could be reactivated by the reconsfituent solutions of different colours which were prepared from mixture in different proportions of ferric homocitrate, Na2S and dithiothreitol (DTT) with K2CrO4, KMnO4 and Na2MoO4, respectively, the inactive protein could be somewhat reactivated by DTF but not by the other compounds or their mixtures which were deficient in one or two of the above compounds. 99Mo was found in the reconstituted protein of the inactive MoFe protein with a reconstituent solution containing 99Mo. Its measurement of differential perturbed angular correlation indicated that 41% of 99Mo in the reconstituted protein were in a state similar to that of 99Mo in 99MoFe protein from K. pneumoniae. The results showed that the reactivation of the inactive MoFe rotein was mainly based on the restoration of its metallocluster content. It seemed to be reasonable to postulate that Mn- or Cr-containing nitrogenase could be obtained likewise by the reconstitution of the inactive protein with the Mn- or Cr-containing reconstituent solution.     

The Relationship Between the Metal Clusters and the Conformation of Molybdenum-Iron Protein from Azotobacter vinelandii

The ultraviolet CD spectrum of nitrogenase MoFe protein from Azotobacter vinelandii had a negative trough with double peaks at 208 nm and 222 nm, respectively, and the shape of the trough was similar to those of other proteins with a-helix structure. After treatment with o-phenanthroline under an aerobic or anaerobic condition, the height of the peak at 222 nm (h222 nm) decreased with the decrease of the C2H2-reduction activity, Fe content and CD spectra at both 450 nm and 660 nm, or at 450 nm of the treated proteins. However, after reconstituting with a reconstituent solution containing Na2MoO4, Na2S, dithiothreitol and either ferric homocitrate or ferric citrate, the h222 nm Of the reconstituted proteins could be restored as well as the activity, Fe content and CD spectra at both of 450 nm and 660 nm. The results show that there is a significant relationship between the metal clusters (FeMoco and P-cluster) and the conformation of MoFe protein.