Molybdenum-iron Protein Research Articles

Climbing Nitrogenase: Toward a Mechanism of Enzymatic Nitrogen Fixation

"Nitrogen fixation", the reduction of dinitrogen (N2) to two ammonia (NH3) molecules, by the Mo-dependent nitrogenase is essential for all life. Despite four decades of research, a daunting number of unanswered questions about the mechanism of nitrogenase activity make it the "Everest of enzymes". This Account describes our efforts to climb one "face" of this mountain by meeting two interdependent challenges central to determining the mechanism of biological N2 reduction. The first challenge is to determine the reaction pathway: the composition and structure of each of the substrate-derived moieties bound to the catalytic FeMo cofactor (FeMo-co) of the molybdenum-iron (MoFe) protein of nitrogenase. To overcome this challenge, it is necessary to discriminate between the two classes of potential reaction pathways: (1) a "distal" (D) pathway, in which H atoms add sequentially at a single N or (2) an "alternating" (A) pathway, in which H atoms add alternately to the two N atoms of N2. Second, it is necessary to characterize the dynamics of conversion among intermediates within the accepted Lowe-Thorneley kinetic scheme for N2 reduction. That goal requires an experimental determination of the number of electrons and protons delivered to the MoFe protein as well as their "inventory", a partition into those residing on each of the reaction components and released as H2 or NH3. The principal obstacle to this "climb" has been the inability to generate N2 reduction intermediates for characterization. A combination of genetic, biochemical, and spectroscopic approaches recently overcame this obstacle. These experiments identified one of the four-iron Fe-S faces of the active-site FeMo-co as the specific site of reactivity, indicated that the side chain of residue alpha70V controls access to this face, and supported the involvement of the side chain of residue alpha195H in proton delivery. We can now freeze-quench trap N2 reduction pathway intermediates and use electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies to characterize them. However, even successful trapping of a N2 reduction intermediate occurs without synchronous electron delivery to the MoFe protein. As a result, the number of electrons and protons, n, delivered to MoFe during its formation is unknown. To determine n and the electron inventory, we initially employed ENDOR spectroscopy to analyze the substrate moiety bound to the FeMo-co and 57Fe within the cofactor. Difficulties in using that approach led us to devise a robust kinetic protocol for determining n of a trapped intermediate. This Account describes strategies that we have formulated to bring this "face" of the nitrogenase mechanism into view and afford approaches to its climb. Although the summit remains distant, we look forward to continued progress in the ascent.

Testing if the Interstitial Atom,X, of the Nitrogenase Molybdenum−Iron Cofactor Is N or C: ENDOR, ESEEM, and DFT Studies of theS=3/2Resting State in Multiple Environments

A high-resolution (1.16 A) X-ray structure of the nitrogenase molybdenum-iron (MoFe) protein revealed electron density from a single N, O, or C atom (denoted X) inside the central iron prismane ([6Fe]) of the [MoFe7S9:homocitrate] FeMo-cofactor (FeMo-co). We here extend earlier efforts to determine the identity of X through detailed tests of whether X = N or C by interlocking and mutually supportive 9 GHz electron spin echo envelope modulation (ESEEM) and 35 GHz electron-nuclear double resonance (ENDOR) measurements on 14/15N and 12/13C isotopomers of FeMo-co in three environments: (i) incorporated into the native MoFe protein environment; (ii) extracted into N-methyl formamide solution; and (iii) incorporated into the NifX protein, which acts as a chaperone during FeMo-co biosynthesis. These measurements provide powerful evidence that X not equal N/C, unless X in effect is magnetically decoupled from the S = 3/2 electron spin system of resting FeMo-co. They reveal no signals from FeMo-co in any of the three environments that can be assigned to X from either 14/15N or 13C: If X were either element, its maximum observed hyperfine coupling at all fields of measurement is estimated to be A(14/15NX) < 0.07/0.1 MHz, A(13CX) < 0.1 MHz, corresponding to intrinsic couplings of about half these values. In parallel, we have explicitly calculated the hyperfine tensors for X = 14/15N/13C/17O, nuclear quadrupole coupling constant e2qQ for X = 14N, and hyperfine constants for the Fe sites of S = 3/2 FeMo-co using density functional theory (DFT) in conjunction with the broken-symmetry (BS) approach for spin coupling. If X = C/N, then the decoupling required by experiment strongly supports the "BS7" spin coupling of the FeMo-co iron sites, in which a small X hyperfine coupling is the result of a precise balance of spin density contributions from three spin-up and three spin-down (3 upward arrow:3 downward arrow) iron atoms of the [6Fe] prismane "waist" of FeMo-co; this would rule out the "BS6" assignment (4 upward arrow:2 downward arrow for [6Fe]) suggested in earlier calculations. However, even with the BS7 scheme, the hyperfine couplings that would be observed for X near g2 are sufficiently large that they should have been detected: we suggest that the experimental results are compatible with X = N only if aiso(14/15NX) < 0.03-0.07/0.05-0.1 MHz and aiso(13CX) < 0.05-0.1 MHz, compared with calculated values of aiso(14/15NX) = 0.3/0.4 MHz and aiso(13CX) = 1 MHz. However, the DFT uncertainties are large enough that the very small hyperfine couplings required by experiment do not necessarily rule out X = N/C.

Molecular insights into nitrogenase FeMo cofactor insertion: the role of His 362 of the MoFe protein α subunit in FeMo cofactor incorporation

The assembly of the complex iron-molybdenum cofactor (FeMoco) of nitrogenase molybdenum-iron (MoFe) protein has served as one of the central topics in the field of bioinorganic chemistry for decades. Here we examine the role of a MoFe protein residue (His alpha362) in FeMoco insertion, the final step of FeMoco biosynthesis where FeMoco is incorporated into its binding site in the MoFe protein. Our data from combined metal, activity and electron paramagnetic resonance analyses show that mutations of His alpha362 to small uncharged Ala or negatively charged Asp result in significantly reduced FeMoco accumulation in MoFe protein, indicating that His alpha362 plays a key role in the process of FeMoco insertion. Given the strategic location of His alpha362 at the entry point of the FeMoco insertion funnel, this residue may serve as one of the initial docking points for FeMoco insertion through transient ligand coordination and/or electrostatic interaction.

How many metals does it take to fix N 2 ? A mechanistic overview of biological nitrogen fixation

During the process of biological nitrogen fixation, the enzyme nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia. Nitrogenase consists of two component metalloproteins, the iron (Fe) protein and the molybdenum-iron (MoFe) protein; the Fe protein mediates the coupling of ATP hydrolysis to interprotein electron transfer, whereas the active site of the MoFe protein contains the polynuclear FeMo cofactor, a species composed of seven iron atoms, one molybdenum atom, nine sulfur atoms, an interstitial light atom, and one homocitrate molecule. This Perspective provides an overview of biological nitrogen fixation and introduces three contributions to this special feature that address central aspects of the mechanism and assembly of nitrogenase.

Trapping H-Bound to the Nitrogenase FeMo-Cofactor Active Site during H2Evolution: Characterization by ENDOR Spectroscopy

We here show that the iron-molybdenum (FeMo)-cofactor of the nitrogenase alpha-70(Ile) molybdenum-iron (MoFe) protein variant accumulates a novel S = (1)/(2) state that can be trapped during the reduction of protons to H(2). (1,2)H-ENDOR measurements disclose the presence of two protons/hydrides (H(+/)(-)) whose hyperfine tensors have been determined from two-dimensional field-frequency (1)H ENDOR plots. The two H(+/)(-) have large isotropic hyperfine couplings, A(iso)( )() approximately 23 MHz, which shows they are bound to the cofactor. The favored analysis for these plots indicates that the two H(+/)(-) have the same principal values, which indicates that they are chemically equivalent. The tensors are further related to each other by a permutation of the tensor components, which indicates an underlying symmetry of binding relative to the cofactor. At present, no model for the structure of the iron-molybdenum (FeMo)-cofactor in the S = (1)/(2) state trapped during the reduction of H(+) can be shown unequivocally to satisfy all of the constraints generated by the ENDOR analysis. The data disfavors any model that involves protonation of sulfides, and thus suggests that the intermediate instead contains two chemically equivalent bound hydrides; it appears unlikely that these are terminal monohydrides.

The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase.

It is known that an E146D site-directed variant of the Azotobacter vinelandii iron protein (Fe protein) is specifically defective in its ability to participate in iron-molybdenum cofactor (FeMoco) insertion. Molybdenum-iron protein (MoFe protein) from the strain expressing the E146D Fe protein is partially ( approximately 45%) FeMoco deficient. The "free" FeMoco that is not inserted accumulates in the cell. We were able to insert this "free" FeMoco into the partially pure FeMoco-deficient MoFe protein. This insertion reaction required crude extract of the DeltanifHDK A. vinelandii strain CA12, Fe protein and MgATP. We used this as an assay to purify a required "insertion" protein. The purified protein was identified as GroEL, based on the molecular mass of its subunit (58.8 kDa), crossreaction with commercially available antibodies raised against E. coli GroEL, and its NH(2)-terminal polypeptide sequence. The NH(2)-terminal polypeptide sequence showed identity of up to 84% to GroEL from various organisms. Purified GroEL of A. vinelandii alone or in combination with MgATP and Fe protein did not support the FeMoco insertion into pure FeMoco-deficient MoFe protein, suggesting that there are still other proteins and/or factors missing. By using GroEL-containing extracts from a DeltanifHDK strain of A. vinelandii CA12 along with FeMoco, Fe protein, and MgATP, we were able to supply all required proteins and/or factors and obtained a fully active reconstituted E146D nifH MoFe protein. The involvement of the molecular chaperone GroEL in the insertion of a metal cluster into an apoprotein may have broad implications for the maturation of other metalloenzymes.

MgATP-Bound and nucleotide-free structures of a nitrogenase protein complex between the Leu 127 Delta-Fe-protein and the MoFe-protein.

A mutant form of the nitrogenase iron protein with a deletion of residue Leu 127, located in the switch II region of the nucleotide binding site, forms a tight, inactive complex with the nitrogenase molybdenum iron (MoFe) protein in the absence of nucleotide. The structure of this complex generated with proteins from Azotobacter vinelandii (designated the L127Delta-Av2-Av1 complex) has been crystallographically determined in the absence of nucleotide at 2.2 A resolution and with bound MgATP (introduced by soaking) at 3.0 A resolution. As observed in the structure of the complex between the wild-type A. vinelandii nitrogenase proteins stabilized with ADP.AlF(4-), the most significant conformational changes in the L127Delta complex occur in the Fe-protein component. While the interactions at the interface between the MoFe-protein and Fe-proteins are conserved in the two complexes, significant differences are evident at the subunit-subunit interface of the dimeric Fe-proteins, with the L127Delta-Av2 structure having a more open conformation than the wild-type Av2 in the complex stabilized by ADP.AlF(4-). Addition of MgATP to the L127Delta-Av2-Av1 complex results in a further increase in the separation between Fe-protein subunits so that the structure more closely resembles that of the wild-type, nucleotide-free, uncomplexed Fe-protein, rather than the Fe-protein conformation in the ADP.AlF(4-) complex. The L127Delta mutation precludes key interactions between the Fe-protein and nucleotide, especially, but not exclusively, in the region corresponding to the switch II region of G-proteins, where the deletion constrains Gly 128 and Asp 129 from forming hydrogen bonds to the gamma-phosphate and activating water for attack on this group, respectively. These alterations account for the inability of this mutant to support mechanistically productive ATP hydrolysis. The ability of the L127Delta-Av2-Av1 complex to bind MgATP demonstrates that dissociation of the nitrogenase complex is not required for nucleotide binding.

Hydrolysis of Nucleoside Triphosphates Other than ATP by Nitrogenase

The hydrolysis of ATP to ADP and P(i) is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron (Fe) protein and the molybdenum-iron (MoFe) protein. The oxidation state of nitrogenase was found to greatly influence the nucleotide hydrolysis rates. MgATP hydrolysis rates were 20 times higher under dithionite reducing conditions (approximately 4,000 nmol of MgADP formed per min/mg of Fe protein) as compared with indigo disulfonate oxidizing conditions (200 nmol of MgADP formed per min/mg of Fe protein). In contrast, MgGTP, MgITP, and MgUTP hydrolysis rates were significantly higher under oxidizing conditions (1,400-2,000 nmol of MgNDP formed per min/mg of Fe protein) as compared with reducing conditions (80-230 nmol of MgNDP formed per min/mg of Fe protein). The K(m) values for MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330-540 microM) for both the reduced and oxidized states of nitrogenase. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF(4)(-) resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF(4)(-) x MgNDP. The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed.

Evidence That MgATP Accelerates Primary Electron Transfer in aClostridium pasteurianum Fe Protein-Azotobacter vinelandii MoFe Protein Nitrogenase Tight Complex

The nitrogenase catalytic cycle involves binding of the iron (Fe) protein to the molybdenum-iron (MoFe) protein, transfer of a single electron from the Fe protein to the MoFe protein concomitant with the hydrolysis of at least two MgATP molecules, followed by dissociation of the two proteins. Earlier studies found that combining the Fe protein isolated from the bacterium Clostridium pasteurianum with the MoFe protein isolated from the bacterium Azotobacter vinelandii resulted in an inactive, nondissociating Fe protein-MoFe protein complex. In the present work, it is demonstrated that primary electron transfer occurs within this nitrogenase tight complex in the absence of MgATP (apparent first-order rate constant k = 0.007 s-1) and that MgATP accelerates this electron transfer reaction by more than 10,000-fold to rates comparable to those observed within homologous nitrogenase complexes (k = 100 s-1). Electron transfer reactions were confirmed by EPR spectroscopy. Finally, the midpoint potentials (Em) for the Fe protein [4Fe-4S]2+/+ cluster and the MoFe protein P2+/N cluster were determined for both the uncomplexed and complexed proteins and with or without MgADP. Calculations from electron transfer theory indicate that the measured changes in Em are not likely to be sufficient to account for the observed nucleotide-dependent rate accelerations for electron transfer.

Conformational variability in structures of the nitrogenase iron proteins from Azotobacter vinelandii and Clostridium pasteurianum

The nitrogenase iron (Fe) protein performs multiple functions during biological nitrogen fixation, including mediating the mechanistically essential coupling between ATP hydrolysis and electron transfer to the nitrogenase molybdenum iron (MoFe) protein during substrate reduction, and participating in the biosynthesis and insertion of the FeMo-cofactor into the MoFe-protein. To establish a structural framework for addressing the diverse functions of Fe-protein, crystal structures of the Fe-proteins from Azotobacter vinelandii and Clostridium pasteurianum have been determined at resolutions of 2.2 Å and 1.93 Å, respectively. These two Fe-proteins are among the more diverse in terms of amino acid sequence and biochemical properties. As described initially for the A. vinelandii Fe-protein in a different crystal form at 2.9 Å resolution, each subunit of the dimeric Fe-protein adopts a polypeptide fold related to other mononucleotide-binding proteins such as G-proteins, with the two subunits bridged by a 4Fe:4S cluster. The overall similarities in the subunit fold and dimer arrangement observed in the structures of the A. vinelandii and C. pasteurianum Fe-proteins indicate that they are representative of the conformation of free Fe-protein that is not in complex with nucleotide or the MoFe-protein. Residues in the cluster and nucleotide-binding sites are linked by a network of conserved hydrogen bonds, salt-bridges and water molecules that may conformationally couple these regions. Significant variability is observed in localized regions, especially near the 4Fe:4S cluster and the MoFe-protein binding surface, that change conformation upon formation of the ADP·AlF 4 −stabilized complex with the MoFe-protein. A core of 140 conserved residues is identified in an alignment of 59 Fe-protein sequences that may be useful for the identification of homologous proteins with functions comparable to that of Fe-protein in non-nitrogen fixing systems.

Changes in the midpoint potentials of the nitrogenase metal centers as a result of iron protein-molybdenum-iron protein complex formation.

All nitrogenase-catalyzed substrate reduction reactions require the transient association between the iron (Fe) protein component and the molybdenum-iron (MoFe) protein component with concomitant intercomponent electron transfer and MgATP hydrolysis. Understanding the effects of Fe protein-MoFe protein complex formation on the properties of the nitrogenase metal centers is thus essential to understanding the electron transfer reactions. This work presents evidence for significant shifts in midpoint potentials for two of the three nitrogenase metal centers as a result of Fe protein binding to the MoFe protein. The midpoint potentials for the three nitrogenase metal centers, namely the [4Fe-4S] cluster of the Fe protein, and the [8Fe-7S] (or P-) clusters and FeMo cofactors (or M-centers) of the MoFe protein, were determined within a nondissociating nitrogenase complex prepared with a site-specifically altered Fe protein (Leu at position 127 deleted, L127Delta). The midpoint potential for each metal center was determined by mediated redox titrations, with the redox state of each center being monitored by parallel and perpendicular mode EPR spectroscopy. The midpoint potential of the Fe protein [4Fe-4S]2+/1+ cluster couple was observed to change by -200 mV from -420 mV in the uncomplexed L127Delta Fe protein to -620 mV in the L127Delta Fe protein-MoFe protein complex. The midpoint potential of the two electron oxidized couple of the P-clusters (P2+/N) of the MoFe protein was observed to shift by -80 mV upon protein-protein complex formation. No significant change in the midpoint potential of an oxidized state of FeMoco (Mox/N) was observed upon complex formation. These results provide insights into the energetics of intercomponent electron transfer in nitrogenase, suggesting that the energy of protein-protein complex formation is coupled to an increase in the driving force for electron transfer. The results are interpreted in light of the expected changes in the protein environments of the metal centers within the nitrogenase complex.

Reconstitution activity of partial metallocluster-deficient molybdenum-iron protein of nitrogenase with a series of molybdenum-sulfur compounds

with a homocitrate ligand coordinated to Mo atom,bridged by three nonprotein lig-ands,two of which are inorganic sulfur atoms and the other is nitrogen- or oxygen-containing

Covalent modification of nitrogenase MoFe protein by ADP.

MgADP- reacted with the nitrogenase molybdenum-iron (MoFe) protein of Klebsiella pneumoniae (Kp1) over a period of 2 h to yield a stable, catalytically active conjugate. The isolated protein exhibited a new, broad 31P NMR resonance at -1 p.p.m. lacking phosphorus J coupling. The adenine ring of [8-14C]ADP remained associated with the conjugate. A covalently bound nucleotide was identified as AMP by NMR and TLC. Extended dialysis of Kp1 against MgADP- resulted in further AMP binding at the protein surface. ADP was initially bound tightly to Kp1 at a site distinct from the AMP sites. ATP did not replace ADP. The time course of the formation of the Kp1-AMP was altered by the nitrogenase iron protein (Kp2) and was dependent on redox potential. Kp1-AMP was stable to concentration and oxidation with ferricyanide ion at -350 mV. Slow hydrolysis of Kp1-AMP over a period of 6 h yielded AMP and unaltered Kp1. The adenine ring of ADP exchanged with adenine of MgATP2- during reductant-limited turnover of nitrogenase under N2, indicating reversibility of ATP hydrolysis at 15 degrees C. [32P]Pi exchanged with the terminal phosphate group of both ADP and ATP on incubation with Kp1. 32P exchange and the catalytic activity of Kp1 were inhibited by a 20-fold molar excess of the lysine-modifying reagent, o-phthalaldehyde (OPT). Preincubation with MgADP- protected against OPT inactivation. Two potentially reactive lysine residues on the alpha chain of the MoFe protein near a putative hydrophobic docking site for the nitrogenase Fe protein are proposed as sites of OPT and nucleotide binding. Azotobacter vinelandii MoFe protein (Av1) also formed an AMP adduct but Kp2 did not. Catalase did not interact with ADP. The reactions of the nitrogenase MoFe protein with adenine nucleotides have no counterpart in known protein-nucleotide interactions.

Evidence for Electron Transfer-dependent Formation of a Nitrogenase Iron Protein-Molybdenum-Iron Protein Tight Complex: THE ROLE OF ASPARTATE 39

Nitrogenase-catalyzed substrate reduction reactions require the association of the iron (Fe) protein and the molybdenum-iron (MoFe) protein, electron transfer from the Fe protein to the MoFe protein coupled to the hydrolysis of MgATP, followed by protein-protein complex dissociation. This work examines the role of MgATP hydrolysis and electron transfer in the dissociation of the Fe protein-MoFe protein complex. Alteration of aspartate 39 to asparagine (D39N) in the nucleotide binding site of Azotobacter vinelandii Fe protein by site-directed mutagenesis resulted in an Fe protein-MoFe protein complex that did not dissociate after electron transfer. While the D39N Fe protein-MoFe protein complex was inactive in all substrate reduction reactions, the complex catalyzed both reductant-dependent and reductant-independent MgATP hydrolysis. Once docked to the MoFe protein, the D39N Fe protein was found to transfer one electron to the MoFe protein requiring MgATP hydrolysis, with an apparent first order rate constant of 0.02 s-1 compared with 140 s-1 for the wild-type Fe protein. Only following electron transfer to the MoFe protein did the D39N Fe protein form a tight complex with the MoFe protein, with no detectable dissociation rate. This was in contrast with the dissociation rate constant of the wild-type Fe protein from the MoFe protein following electron transfer of 5 s-1. Chemically oxidized D39N Fe protein with MgADP-bound did not form a tight complex with the MoFe protein, showing a dissociation rate similar to chemically oxidized wild-type Fe protein (3 s-1 for D39N Fe protein and 6 s-1 for wild-type Fe protein). These results suggest that electron transfer from the Fe protein to the MoFe protein within the protein-protein complex normally induces conformational changes which increase the affinity of the Fe protein for the MoFe protein. A model is presented in which Asp-39 participates in a nucleotide signal transduction pathway involved in component protein-protein dissociation.

Electron transfer from the nitrogenase iron protein to the [8Fe-(7/8)S] clusters of the molybdenum-iron protein.

The reduction of substrates catalyzed by nitrogenase requires electron transfer between the iron (Fe) protein and the molybdenum-iron (MoFe) protein in a reaction that is coupled to the hydrolysis of MgATP. The [4Fe-4S] cluster of the Fe protein transfers one electron ultimately to the M-clusters (FeMoco) of the MoFe protein for substrate reduction, with the P-clusters ([8Fe-(7/8)S]) of the MoFe protein as proposed electron transfer intermediates. This work presents direct EPR evidence for primary electron transfer from the [4Fe-4S] cluster of the Fe protein to the P-clusters of the MoFe protein in a reaction that requires the MgATP-bound state of the Fe protein. An oxidized state of the MoFe protein was prepared in which the P-clusters were oxidized by 2 equiv of electrons to the P2+ state. In this oxidation state, the M-clusters (S = 3/2) and the P(2+-clusters (S > or = 3) are paramagnetic and can be observed by perpendicular and parallel mode EPR, providing the opportunity to follow electron transfer from the Fe protein to either cluster type in the MoFe protein. Electron transfer from the reduced [4Fe-4S]1+ cluster of two different Fe proteins to the P2+ clusters of the MoFe protein was observed by the disappearance of the [4Fe-4S]1+ cluster EPR signal and the conversion of the MoFe protein P-clusters from the P2+ to the P1+ oxidation state. In the first case, stoichiometric quantities of the wild-type Fe protein transferred one electron to the P-clusters only in the presence of MgATP. MgADP would not support this electron transfer reaction. In the second case, an altered Fe protein (L127 delta) that is in a conformation resembling the MgATP-bound state was found to transfer an electron to the P-clusters in the absence of MgATP. These results suggest that the first electron transferred from the Fe protein goes to the P-cluster and that the MgATP-bound protein conformation of the Fe protein, not MgATP hydrolysis, is required for this electron transfer reaction.

Evidence for electron transfer from the nitrogenase iron protein to the molybdenum-iron protein without MgATP hydrolysis: characterization of a tight protein-protein complex.

MgA TP hydrolysis has been proposed to be absolutely required for electron transfer from the nitrogenase iron (Fe) protein to the molybdenum-iron (MoFe) protein. This work presents evidence for primary electron transfer from the Azotobacter vinelandii nitrogenase Fe protein to the MoFe protein in the absence of MgATP hydrolysis. Deletion of an amino acid (Leu 127) in a signal transduction pathway in the Fe protein resulted in an Fe protein conformation resembling the MgATP-bound state. This altered Fe protein (L127delta) was found to bind to the MoFe protein in the absence of MgATP, forming a tight protein complex. Both steady state and stopped-flow transient kinetic measurements suggest that two L127delta Fe proteins bind to one MoFe protein with an extremely high affinity. From pre-steady state kinetic determinations of the rate of complex dissociation, the affinity was found to be at least 350 times tighter than that of the wild-type A. vinelandii nitrogenase complex and at least 20 times tighter than that of the heterologous Clostridium pasteurianum Fe protein-A. vinelandii MoFe protein complex. The L127delta Fe protein-MoFe protein complex was isolated by gel filtration liquid chromatography. Scanning densitometry of an SDS gel of the complex isolated from the gel filtration column revealed a stoichiometry of 1.7 L 127 delta Fe proteins bound per MoFe protein. The L 127 delta Fe protein was found to transfer a single electron from its [4Fe-4S] cluster to the MoFe protein at a rate of 0.2s-1. This compares with the MgATP dependent electron transfer rate of 140 s-1 observed for transfer of an electron from the wild-type Fe protein to the MoFe protein. No substrate reduction (H+ or C2H2) was detected when wild-type MoFe protein was complemented with L 127 delta Fe protein. The MgATP-independent electron transfer from the L 127 delta Fe protein to the MoFe protein required active MoFe protein and was not inhibited by MgADP. EPR spectroscopy of the complex was employed to confirm the electron transfer reaction. These results show that Fe protein in a conformation resembling the MgATP-bound state can transfer at least one electron to the MoFe protein without the need for MgATP hydrolysis.

The [4Fe-4S] cluster domain of the nitrogenase iron protein facilitates conformational changes required for the cooperative binding of two nucleotides.

MgATP binding and hydrolysis are central to all reduction reactions catalyzed by nitrogenase. The iron (Fe) protein component of nitrogenase is a homodimeric protein with a bridging [4Fe-4S] cluster and two nucleotide binding sites, one on each subunit. This work presents evidence that the [4Fe-4S] cluster domain of the nitrogenase Fe protein functions as a hinge region between the two nucleotide binding domains, participating in the cooperative binding of two nucleotides. Alanine residues at position 98 (located near the [4Fe-4S] cluster) of the Azotobacter vinelandii Fe protein were changed by means of site-directed mutagenesis to Val (V) and Gly (G), and the resulting altered proteins were purified and characterized. While the wild-type and A98G Fe proteins were found to bind two nucleotides (MgATP or MgADP) with strong cooperativity (Hill coefficient of 2), the A98V Fe protein was found to bind one nucleotide with no apparent cooperativity. The binding of two nucleotides to the wild-type Fe protein is known to induce protein conformational changes which are reflected as changes in the properties of the [4Fe-4S] cluster, including a change in the redox potential of the [4Fe-4S] cluster of -120 mV for MgATP binding (-300 to -420 mV) and of -160 mV for MgADP binding (-300 to -460 mV). The binding of one nucleotide to the A98V Fe protein was found to result in only half the lowering of the redox potential, with MgATP binding resulting in a -80 mV change (-280 to -360 mV) and MgADP binding resulting in a -50 mV change (-280 to -330 mV). Results from 1H NMR, EPR, and CD spectra, along with Fe chelation rates, were all consistent with the binding of a single nucleotide to the A98V Fe protein inducing a partial conformational change. Finally, the A98V Fe protein with one nucleotide bound, still bound to the molybdenum-iron protein but did not support MgATP hydrolysis, electron transfer, or substrate reduction. A model is discussed in which the [4Fe-4S] cluster domain can be viewed as a hinge region between the two nucleotide binding domains which facilitates conformational rearrangements required for the cooperative binding of a second nucleotide.

Reductant-Independent ATP Hydrolysis Catalyzed by Homologous Nitrogenase Proteins fromAzotobacter vinelandiiand Heterologous Crosses withClostridium pasteuranium

Reductant-independent ATPase activity was initiated and studied forAzotobacter vinelandiiandClostridium pasteuraniumnitrogenase proteins (Av1, Cp1 and Av2, Cp2, 1 designating the iron molybdenum protein and 2 the iron protein) and their heterologous crosses by two methods: (1) allowing dithionite to be depleted from a normal assay in the presence of substrate levels of MgATP and (2) using reduced but reductant-free nitrogenase proteins in the presence of substrate levels of MgATP. In both cases, at a 1:1 protein ratio, MgATP is converted initially to MgADP with a specific activity of 400–500 nmol MgATP hydrolyzed/min·mg Av1, but in slower steps the MgADP is converted to AMP and, after 12 h, AMP is ultimately converted to adenosine. This reactivity requires the presence of both proteins, increases with increasing Av2/Av1 ratio, and is not a result of unique redox states of either protein. For Av1–Av2, ATP hydrolysis in the absence of Mg2+occurred at nearly the same rate as reductant-dependent MgATP hydrolysis. Reductant-independent ATPase activity also occurred for the Av1–Cp2 and Cp1–Av2 heterologous crosses and was 2-fold and 18-fold slower than the Av1–Av2 or Cp1–Cp2 combinations. In both cases further hydrolysis of MgADP to AMP and AMP to adenosine occurred. A unique nucleotide hydrolysis system is apparently operating in the complex formed between the two nitrogenase proteins in the absence of reductant. The relationship between the reductant-independent and reductant-dependent activities of nitrogenase catalysis is explored.

Nucleotide hydrolysis and protein conformational changes in Azotobacter vinelandii nitrogenase iron protein: defining the function of aspartate 129.

The biological reduction of dinitrogen catalyzed by nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. The present work examines the role of a strictly conserved aspartic acid residue of nitrogenase iron protein (Fe protein) in coupling MgATP hydrolysis to electron transfer and substrate reduction. The aspartic acid residue at position 129 in the Azotobacter vinelandii Fe protein has been suggested to participate in nucleotide interactions from its location in the X-ray structure near several amino acids previously identified to participate in nucleotide binding and protein conformational changes. The function of this amino acid was probed by changing aspartic acid to glutamic acid (D129E) and asparagine (D129N) by site-directed mutagenesis. The D129N Fe protein proved to be unstable and could not be purified. Characterization of the purified D129E Fe protein revealed a central role for Asp 129 in the nucleotide-induced protein conformational changes in the Fe protein and possibly in the mechanism of MgATP hydrolysis. Data from EPR, circular dichroism spectroscopy, and Fe2+ chelation rates and the chemical shifts of isotropically shifted protons in the 1H NMR spectra implicate Asp 129 in the nucleotide-induced conformational changes in the Fe protein, which are reflected in changes in the environment of the [4Fe-4S] cluster. The D129E Fe protein was found to bind both MgATP and MgADP with high affinity. The Kd determined for MgADP binding (Kd = 131 microM) was comparable to that found for wild-type Fe protein (128 microM). The affinity for MgATP binding was 1.6 times tighter than that for wild-type Fe protein (370 compared to 580 microM). The midpoint reduction potential of the [4Fe-4S] cluster was similar to that determined for the wild-type Fe protein (-290 mV for wild-type Fe protein and -300 mV for D129E Fe protein). Upon the addition of MgATP or MgADP, the midpoint potentials for wild-type and D129E Fe proteins shifted to -430 and -440 mV, respectively. The D129E Fe protein was also found to bind to the molybdenum-iron protein (MoFe protein) with normal affinity, although it could not support electron transfer to the MoFe protein or MoFe protein-stimulated MgATP hydrolysis.

Friction and wear of boron carbide-titanium boride composites: K. Nishiyama, K. Taguchi (Science University of Tokyo, Tokyo, Japan)

Purified iron protein (Fe protein) and molybdenum-iron protein (Mo-Fe protein) of nitrogenase from Clostridium pasteurianum were used for studies of ATP hydrolysis and electron transfer in the nitrogenase reaction. Total electron transfer was determined by measuring dithionite oxidation spectrophotometrically. Comparison with manometric determination of H2 evolution showed that dithionite serves as a two electron donor. The studies of different combinations of the Fe protein and the Mo-Fe protein showed that excess Mo-Fe protein inhibited electron transfer but not ATP hydrolysis, whereas excess Fe protein did not inhibit either reaction. The ATP:2 electron ratios ranged from around 4 with excess Fe protein to more than 20 with excess Mo-Fe protein. With relatively low levels of Mo-Fe protein, reductant-independent ATP hydrolysis was around 6% of the reductant-dependent value, and with a large excess of Mo-Fe protein it increased to about 10%. We conclude that because the ATP:2 electron ratio varies with experimental conditions, no single value can be ascribed to this ratio.