Probing the MgATP-bound conformation of the nitrogenase Fe protein by solution small-angle X-ray scattering

The MgATP-bound conformation of the Fe protein of nitrogenase from Azotobacter vinelandii has been examined in solution by small-angle X-ray scattering (SAXS) and compared to existing crystallographically characterized Fe protein conformations. The results of the analysis of the crystal structure of an Fe protein variant with a Switch II single-amino acid deletion recently suggested that the MgATP-bound state of the Fe protein may exist in a conformation that involves a large-scale reorientation of the dimer subunits, resulting in an overall elongated structure relative to the more compact structure of the MgADP-bound state. It was hypothesized that the Fe protein variant may be a conformational mimic of the MgATP-bound state of the native Fe protein largely on the basis of the observation that the spectroscopic properties of the [4Fe-4S] cluster of the variant mimicked in part the spectroscopic signatures of the native nitrogenase Fe protein in the MgATP-bound state. In this work, SAXS studies reveal that the large-scale conformational differences between the native Fe protein and the variant observed by X-ray crystallography are also observed in solution. In addition, comparison of the SAXS curves of the Fe protein nucleotide-bound states to the nucleotide-free states indicates that the conformation of the MgATP-bound state in solution does not resemble the structure of the variant as initially proposed, but rather, at the resolution of this experiment, it resembles the structure of the nucleotide-free state. These results provide insights into the Fe protein conformations that define the role of MgATP in nitrogenase catalysis.     

Homology modelling of the Frankia nitrogenase iron protein

The NifH protein contains an iron-sulfur cluster performing different functions during nitrogen fixation. Frankia is an actinomycete, entering into symbiotic association with a number of dicotyledonous plants and fixing nitrogen. The structure of the Frankia NifH protein was determined using homology modelling technique. Metal binding sites and functionally important regions of the protein were analyzed. Thiol ligands and active sites help in protein functioning and conformations. Structurally important nests were recognized. Clefts and cavities contain biologically important residues. Site-directed mutagenesis results reveal that mutations in functional residues hamper nitrogen fixation. The structure is rigid with an accessible surface for solvents. The structure is reliable offering insights into the 3D structural framework as well as structure-function relation of NifH protein.     

Studies on the activating enzyme for iron protein of nitrogenase from Rhodospirillum rubrum

Removal of ADP-ribose from the iron protein of nitrogenase by activating enzyme resulted in the activation of the inactive iron protein. A radioassay that directly measured the initial velocity of the activation was developed using iron protein radiolabeled with either [8-3H]- or [G-32P]ADP-ribose. The release of radiolabeled ADP-ribose by activating enzyme was linearly correlated with the increase in the specific activity of the iron protein as measured by acetylene reduction. Both ATP and MnCl2 were required for the activation of inactive iron protein. The optimal ratio of [MnCl2]/[ATP] in the radioassay was 2:1, and the optimal concentrations were 4 mM and 2 mM for [MnCl2] and [ATP], respectively. The Km for inactive iron protein was 74 microM and the Vmax was 628 pmol of [32P] ADP-ribose released min-1 microgram of activating enzyme-1. Adenosine, cytidine, guanosine, or uridine mono-, di-, or triphosphates did not substitute for ATP in the activation of native iron protein. Activating enzyme removed ADP-ribose from oxygen-denatured iron protein in the absence of ATP. ADP, ADP-ribose, pyrophosphate, and high concentrations of NaCl inhibited activating enzyme activity.     

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.     

Nucleotide sequence of the gene encoding the nitrogenase iron protein of Thiobacillus ferrooxidans.

The DNA sequence was determined for the cloned Thiobacillus ferrooxidans nifH and part of the nifD genes. A putative T. ferrooxidans nifH promoter was identified whose sequences showed perfect consensus with those of the Klebsiella pneumoniae nif promoter. Two putative consensus upstream activator sequences were also identified. The amino acid sequence was deduced from the DNA sequence. In a comparison of nifH DNA sequences from T. ferrooxidans and eight other nitrogen-fixing microbes, a Rhizobium sp. isolated from Parasponia andersonii showed the greatest homology (74%) and Clostridium pasteurianum (nifH 1) showed the least homology (54%). In a comparison of the amino acid sequences of the Fe proteins, the Rhizobium sp. and Rhizobium japonicum showed the greatest homology (both 86%) and C. pasteurianum (nifH 1 gene product) demonstrated the least homology (56%) to the T. ferrooxidans Fe protein.     

Nucleotide sequence of the gene coding for the nitrogenase iron protein from Klebsiella pneumoniae

We report the complete DNA sequence of the Klebsiella pneumoniae nifH gene, the gene which codes for component 2 (Fe protein or nitrogenase reductase) of the nitrogenase enzyme complex. The amino acid sequence of the K. pneumoniae nitrogenase Fe protein is deduced from the DNA sequence. The K. pneumoniae Fe protein contains 292 amino acids, has a Mr = 31,753, and contains 9 cysteine residues. We compare the amino acid sequence of the K. pneumoniae protein with available amino acid sequence data on nitrogenase Fe proteins from two other species, Clostridium pasteurianum and Azotobacter vinelandii. The C. pasteurianum Fe protein, for which the complete sequence is known, shows 67% homology with the K. pneumoniae Fe protein. Extensive regions of strong conservation (90-95%) are found, while other regions show relatively poor conservation (30-35%). It is suggested that these strongly conserved regions are of special importance to the function of this enzyme, and the findings are discussed in the light of evolutionary theories on the origin of nif genes.     

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.     

Change in subunit composition of the iron protein of nitrogenase from Rhodospirillum rubrum during activation and inactivation of iron protein.

The subunit composition of the Fe protein of nitrogenase from Rhodospirillum rubrum during activation and inactivation was investigated. It was found that the upper subunit (on gel electrophoresis) of the two-subunit Fe protein was converted into the lower subunit during activation in vitro. When the Fe protein was inactivated in vivo by the addition of NH4Cl and alpha-oxoglutarate to the cells, a phosphate-labelled upper band appeared. During activation in vitro by the activating enzyme, some of the phosphate of the upper band remained with the protein and appeared in the lower band. Activations in vitro were performed on inactive Fe protein obtained from cells grown with glutamate as the nitrogen source. Both native and oxygen-denatured Fe protein exhibited the loss of upper band during treatment with activating enzyme.     

A substrate channel in the nitrogenase MoFe protein

Nitrogenase catalyzes the six electron/six proton reduction of N₂ to two ammonia molecules at a complex organometallocluster called “FeMo cofactor.” This cofactor is buried within the α-subunit of the MoFe protein, with no obvious access for substrates. Examination of high-resolution X-ray crystal structures of MoFe proteins from several organisms has revealed the existence of a water-filled channel that extends from the solvent-exposed surface to a specific face of FeMo cofactor. This channel could provide a pathway for substrate and product access to the active site. In the present work, we examine this possibility by substituting four different amino acids that line the channel with other residues and analyze the impact of these substitutions on substrate reduction kinetic parameters. Each of the MoFe protein variants was purified and kinetic parameters were established for the reduction of the substrates N₂, acetylene, azide, and propyne. For each MoFe protein, V _max values for the different substrates were found to be nearly unchanged when compared with the values for the wild-type MoFe protein, indicating that electron delivery to the active site is not compromised by the various substitutions. In contrast, the K _m values for these substrates were found to increase significantly (up to 22-fold) in some of the MoFe protein variants compared with the wild-type MoFe protein values. Given that each of the amino acids that were substituted is remote from the active site, these results are consistent with the water-filled channel functioning as a substrate channel in the MoFe protein.     

Analysis of steady state Fe and MoFe protein interactions during nitrogenase catalysis

Steady state kinetic measurements are reported for nitrogenase from Azotobacter vinelandii (Av) and Clostridium pasteurianum (Cp) under a variety of conditions, using dithionite as reductant. The specific activities of Av1 and Cp1 are determined as functions of Av2:Av1 and Cp2:Cp1, respectively, at component protein ratios from 0.4 to 50, and conform to a simple hyperbolic rate law for the interaction of Av2 with Av1 and Cp2 with Cp1. The specific activities of Av2 and Cp2 are also measured as a function of increasing Av1 and Cp1 concentrations, producing 'MoFe inhibition' at large MoFe:Fe ratios. When the rate of product formation under MoFe inhibited conditions is re-plotted as increasing Av2:Av1 or Cp2:Cp1 ratios, sigmoidal kinetic behavior is observed, suggesting that the rate constants in the Thorneley and Lowe (T&L) model are more dependent upon the oxidation level of MoFe protein than previously suspected [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 224 (1984) 887-894], at least when applied to Av and Cp. Calculation of Hill coefficients gave values of 1.7-1.9, suggesting a highly cooperative Fe-MoFe protein interaction in both Av and Cp nitrogenase catalysis. The T&L model lacks analytical solutions [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 215 (1983) 393-404], so the ease of its application to experimental data is limited. To facilitate the study of steady state kinetic data for H(2) evolution, analytical equations are derived from a different mechanism for nitrogenase activity, similar to that of Bergersen and Turner [Biochem. J. 131 (1973) 61-75]. This alternative cooperative model assumes that two Fe proteins interact with one MoFe protein active site. The derived rate laws for this mechanism were fitted to the observed sigmoidal behavior for low Fe:MoFe ratios (<0.4), as well as to the commonly observed hyperbolic behavior for high values of Fe:MoFe for both Av and Cp.     

Insights into the role of nucleotide-dependent conformational change in nitrogenase catalysis: Structural characterization of the nitrogenase Fe protein Leu127 deletion variant with bound MgATP

In the present work, determination of the structure of the nitrogenase Leu 127 deletion variant Fe protein with MgATP bound is presented, along with density functional theory calculations, to provide insights into the roles of MgATP in the nitrogenase reaction mechanism. Comparison of the MgATP-bound structure of this Fe protein to the nucleotide-free form indicates that the binding of MgATP does not alter the overall structure of the variant significantly with only small differences in the conformation of amino acids in direct contact with the two bound MgATP molecules being seen. The earlier observation of splitting of the [4Fe-4S] cluster into two [2Fe-2S] clusters was observed to be unaltered upon binding MgATP. Density functional theory was used to probe the assignment of ligands to the two [2Fe-2S] rhombs. The Mg(2+) environment in the MgATP-bound structure of the Leu127 deletion Fe protein is similar to that observed for the Fe protein in the nitrogenase Fe protein: MoFe protein complex stabilized by MgADP and tetrafluoroaluminate suggesting that large scale conformational change implicated for the Fe protein may not be mediated by changes in the Mg(2+) coordination. The results presented here indicated that MgATP may enhance the stability of an open conformation and prohibit intersubunit interactions, which have been implicated in promoting nucleotide hydrolysis. This could be critical to the tight control of MgATP hydrolysis observed within the nitrogenase complex and may be important for maintaining unidirectional electron flow toward substrate reduction.     

Reversible regulation of the nitrogenase iron protein from Rhodospirillum rubrum by ADP-ribosylation in vitro.

Nitrogenase activity in the photosynthetic bacterium Rhodospirillum rubrum is reversibly regulated by interconversion of the Fe protein between a modified and an unmodified form. Since the discovery of the activation process in 1976, investigators have been unable to demonstrate the inactivation (modification) reaction in vitro. In this study, NAD-dependent modification and concomitant inactivation of the Fe protein were demonstrated in crude extracts of R. rubrum. Activation of the in vitro-modified Fe protein by activating enzyme and structural similarity between the in vivo and in vitro modifications are presented as evidence that the in vitro modification is the physiologically relevant ADP-ribosylation reaction. Using a partially purified preparation, we showed that the inactivating enzyme activity is stimulated by divalent metal ions and ADP, that O2-denatured Fe protein will not serve as a substrate, and that dithionite inhibits the modification reaction.     

Effects of topology and excluded volume on protein denatured state conformational properties

The conformational constraints on protein denatured states are of prime importance in modulating early events in protein folding. Although structural studies have demonstrated residual structure in protein denatured states, much remains poorly understood with regard to the conformational properties of this state. Here, we investigate topological effects on loop formation probabilities in denatured iso-1-cytochrome c by comparing histidine-heme binding affinities for histidines on the N- versus the C-terminal side of the heme. For histidines N-terminal to the heme (preceding cysteine 14), the polypeptide emerges from the edge of the heme and must simply fold over to bind to the heme. For histidines C-terminal to the heme (following histidine 18), the polypeptide emerges from the back side of the heme and must wrap around the heme for the histidine to bind to the heme. Thus, the steric constraints on this wrap-around topology are expected to be much more demanding than for the heme-edge topology of the N-terminal histidines. Evaluation of loop formation probabilities in 3 M guanidine hydrochloride, conditions that fully denature the variants studied, demonstrates that N-terminal histidine-heme loop formation is 10-25-fold more favorable than C-terminal histidine-heme loop formation, for similar loop sizes. A two-dimensional square lattice model indicates that excluded volume is important in this topological preference. These data provide direct evidence that denatured state topology affects contact probability, and thus probable folding pathways, in a disordered protein.     

Transition state complexes of the Klebsiella pneumoniae nitrogenase proteins. Spectroscopic properties of aluminium fluoride-stabilized and beryllium fluoride-stabilized MgADP complexes reveal conformational differences of the Fe protein.

Stable inactive 2 : 1 complexes of the Klebsiella pneumoniae nitrogenase components (Kp2/Kp1) were prepared with ADP or the fluorescent ADP analogue, 2'(3')-O-[N-methylanthraniloyl] ADP and AlF(4)(-) or BeF(3)(-) ions. By analogy with published crystallographic data [Schindelin et al. (1997) Nature 387, 370-376)], we suggest that the metal fluoride ions replaced phosphate at the two ATP-binding sites of the iron protein, Kp2. The beryllium (BeF(x)) and aluminium (AlF(4)(-)) containing complexes are proposed to correspond to the ATP-bound state and the hydrolytic transition states, respectively, by analogy with the equivalent complexes of myosin [Fisher et al. (1995) Biochemistry 34, 8960-8972]. (31)P NMR spectroscopy showed that during the initial stages of complex formation, MgADP bound to the complexed Kp2 in a manner similar to that reported for isolated Kp2. This process was followed by a second step that caused broadening of the (31)P NMR signals and, in the case of the AlF4- complex, slow hydrolysis of some of the excess ADP to AMP and inorganic phosphate. The purified BeFx complex contained 3.8 +/- 0.1 MgADP per mol Kp1. With the AlF(4)(-) complex, MgAMP and adenosine (from MgAMP hydrolysis) replaced part of the bound MgADP although four AlF(4)(-) ions were retained, demonstrating that full occupancy by MgADP is not required for the stability of the complex. The fluorescence emission maximum of 2'(3')-O-[N-methylanthraniloyl] ADP was blue-shifted by 6-8 nm in both metal fluoride complexes and polarization was 6-9 times that of the free analogue. The fluorescence yield of bound 2'(3')-O-[N-methylanthraniloyl] ADP was enhanced by 40% in the AlF(4)(-) complex relative to the solvent but no increase in fluorescence was observed in the BeFx complex. Resonance energy transfer from conserved tyrosine residues located in proximity to the Kp2 nucleotide-binding pocket was marked in the AlF(4)(-) complex but minimal in the BeFx fluoride complex, illustrating a clear conformational difference in the Fe protein of the two complexes. Our data indicate that complex formation during the nitrogenase catalytic cycle is a multistep process involving at least four conformational states of Kp2: similar to the free Fe protein; as initially complexed with detectable (31)P NMR; as detected in mature complexes with no detectable (31)P NMR; in the AlF(4)(-) complex in which an altered tyrosine interaction permits resonance energy transfer with 2'(3')-O-[N-methylanthraniloyl] ADP.     

Incorporation of adenine into the modifying group of inactive iron protein of nitrogenase from Rhodospirillum rubrum

Adenine was fed to cells of Rhodospirillum rubrum grown on glutamate. The adenine was found to be incorporated into the modifying group of the inactive form of iron protein. Adenine labelled in the 8-position ([8-3H]adenine) and the 2-position ([2-3H]adenine) was specifically incorporated into the electrophoretic 'upper-band' subunit of iron protein. Incorporation of label from the 2-position into many proteins was observed if histidine was not present in the medium. Label was removed by the activating enzyme for iron protein.     

Evidence for conformational protection of nitrogenase against oxygen in Gluconacetobacter diazotrophicus by a putative FeSII protein

The mechanisms protecting nitrogenase in Gluconacetobacter diazotrophicus from damage by oxygen were studied. Evidence is provided suggesting that in G. diazotrophicus these mechanisms include respiratory protection as well as conformational protection in which a putative FeSII Shethna protein is involved.     

A hybrid Azotobacter vinelandii-Clostridium pasteurianum nitrogenase iron protein that has in vivo and in vitro catalytic activity

Site-directed mutagenesis and gene replacement procedures were used to construct a mutant strain of Azotobacter vinelandii which expresses a hybrid nitrogenase Fe protein. This hybrid Fe protein has its carboxyl-terminal 18 residues replaced with the 5 analogous residues from the Clostridium pasteurianum Fe protein sequence. The hybrid Fe protein is 13 amino acids smaller than the wild-type A. vinelandii Fe protein and has a net loss of 4 negatively charged residues, resulting in a change in size and charge. The strain which produces the hybrid Fe protein remained capable of diazotrophic growth, albeit at a reduced rate. Also, the purified hybrid Fe protein exhibited a maximum activity about one-half that of native Fe protein. These results demonstrate that the tight, inactive complex which is formed when A. vinelandii MoFe protein and C. pasteurianum Fe protein are mixed in heterologous reconstitution experiments cannot be accounted for only by differences in the A. vinelandii and C. pasteurianum Fe protein primary sequences located at their respective carboxyl termini.