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PubMed1999

Analysis of the nifHDK operon and structure of the NifH protein from the unicellular, diazotrophic cyanobacterium, Cyanothece strain sp. ATCC 51142(1).

Cyanothece sp. ATCC 51142 is a unicellular, diazotrophic cyanobacterium that demonstrates diurnal rhythms for photosynthesis and N(2) fixation, with peaks of O(2) evolution and nitrogenase activity approximately 12 h out of phase. We cloned and sequenced the nifHDK operon, and determined that the amino acid sequences of all three proteins were highly conserved relative to those of other cyanobacteria and bacteria. However, the Fe-protein, encoded by the nifH gene, demonstrated two differences from the related protein in Azotobacter vinelandii, for which a 3-D structure has been determined. First, the Cyanothece Fe-protein contained a 37 amino acid extension at the N-terminus. This approximately 4 kDa addition to the protein appeared to fold as a separate domain, but remained a part of the active protein, as was verified by migration on acrylamide gels. In addition, the Cyanothece Fe-protein had amino acid differences at positions involved in formation of the Fe-protein dimer-dimer contacts in A. vinelandii nitrogenase. There were also changes in residues involved with interaction between the Fe-protein and the MoFe-protein when compared with A. vinelandii. Since the Cyanothece Fe-protein is quickly degraded after activity, it is suggested that the extension and the amino acid alterations were somehow involved in this degradative process.

Amino Acid Sequence↗
PubMed1995

Role of the MoFe protein alpha-subunit histidine-195 residue in FeMo-cofactor binding and nitrogenase catalysis.

Site-directed mutagenesis and gene-replacement procedures were used to isolate mutant strains of Azotobacter vinelandii that produce altered MoFe proteins in which the alpha-subunit residue-195 position, normally occupied by a histidine residue, was individually substituted by a variety of other amino acids. Structural studies have revealed that this histidine residue is associated with the FeMo-cofactor binding domain and probably provides an NH-->S hydrogen bond to a central bridging sulfide located within FeMo-cofactor. Substitution by a glutamine residue results in an altered MoFe protein that binds but does not reduce N2, the physiological substrate. Although N2 is not a substrate for the altered MoFe protein, it is a potent inhibitor of both acetylene and proton reduction, both of which are otherwise effectively reduced by the altered MoFe protein. This result provides evidence that N2 inhibits proton and acetylene reduction by simple occupancy of a common active site. N2 also uncouples MgATP from proton reduction catalyzed by the altered MoFe protein but does so without lowering the overall rate of MgATP hydrolysis. Thus, the quasi-unidirectional flow of electrons from the Fe protein to the MoFe protein that occurs during nitrogenase turnover is controlled, in part, by the substrate serving as an effective electron sink. Substitution of the alpha-histidine-195 residue by glutamine also imparts to the altered MoFe protein hypersensitivity of both its acetylene reduction and N2 binding to inhibition by CO, indicating that the imidazole group of the alpha-histidine-195 residue might protect an Fe contained within the FeMo-cofactor from attack by CO. Finally, comparisons of the catalytic and spectroscopic properties of altered MoFe proteins produced by various mutant strains suggest that the alpha-histidine-195 residue has a structural role, which serves to keep FeMo-cofactor attached to the MoFe protein and to correctly position FeMo-cofactor within the polypeptide matrix, such that N2 binding is accommodated.

Azotobacter vinelandii↗
PubMed1995

Electron spin echo envelope modulation spectroscopic analysis of altered nitrogenase MoFe proteins from Azotobacter vinelandii.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to study changes in the polypeptide environment of the FeMo-cofactor that were elicited by amino-acid substitutions within the nitrogenase MoFe protein alpha-subunit. A previous ESEEM study [Thomann et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6620] detected modulation arising from nitrogen coupled to the S = 3/2 spin system of the FeMo-cofactor (Fe7S9Mo:homocitrate). Such modulation was found to be sensitive to the substitution of alpha-195His by alpha-195Asn as indicated by whole-cell ESEEM analysis of mutant strains from Azotobacter vinelandii. Subsequent structural studies revealed that the alpha-195His residue does not provide direct N-coordination to the cluster but is within hydrogen-bonding distance of one of a set of three sulfides that bridge the FeMo-cofactor subcluster fragments. In the present work, the ESEEM analysis is extended to both partially purified alpha-195Asn MoFe protein and purified MoFe protein from an additional mutant strain in which alpha-195His is replaced by alpha-195Gln. The dramatic decrease in the intensity of the ESEEM signal resulting from the alpha-195Asn substitution in whole cells was confirmed for the case of the isolated alpha-195Asn MoFe protein. In contrast, substitution of alpha-195His by alpha-195Gln caused no detectable change in the modulation. Simulations of the alpha-195His and alpha-195Gln ESEEM data give quadrupole parameters of e2qQ = 2.2 MHz and eta = 0.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Azotobacter vinelandii↗
PubMed2000

Azotobacter vinelandii nitrogenases with substitutions in the FeMo-cofactor environment of the MoFe protein: effects of acetylene or ethylene on interactions with H+, HCN, and CN-.

Wild-type and three altered Azotobacter vinelandii nitrogenase MoFe proteins, with substitutions either at alpha-195(His) (replaced by alpha-195(Asn) or alpha-195(Gln)) or at alpha-191(Gln) (replaced by alpha-191(Lys)), were used to probe the interactions of HCN and CN(-), both of which are present in NaCN solutions at pH 7.4, with nitrogenase. The first goal was to determine how added C(2)H(2) enhances the rate of CH(4) production from HCN reduction by wild-type nitrogenase. In the absence of C(2)H(2), wild-type Mo-nitrogenase showed a declining total electron flux, which is an overall measure of all products formed, as the NaCN concentration was increased from 1 to 5 mM, whereas the rates of both CH(4) and NH(3) production increased with increasing NaCN concentration. The NH(3) production rate exceeded the CH(4) production rate up to 5 mM NaCN, at which point they became equal. The "excess NH(3)" likely arises from the two-electron reduction of HCN to CH(2)=NH, some of which is released and hydrolyzed to HCHO plus NH(3). With added C(2)H(2), the rate of CH(4) production increased but only until it equaled that of NH(3) production, which remained unchanged. In addition, total electron flux was decreased even more at each NaCN concentration by C(2)H(2). The increased CH(4) production did not arise from the added C(2)H(2). The lowered total electron flux with C(2)H(2) present would decrease the affinity of the enzyme for HCN, making it a poorer competitor for the binding site. Thus, less CH(2)=NH would be displaced, more CH(2)=NH would undergo the full six-electron reduction, and the rate of CH(4) production would be enhanced. A second goal was to gain mechanistic insight into the roles of the amino acid residues in the alpha-subunit of the MoFe protein at positions alpha-191 and alpha-195 in substrate reduction. At 5 mM NaCN and in the presence of excess wild-type Fe protein, the specific activity for CH(4) production by the alpha-195(Asn), alpha-195(Gln), and alpha-191(Lys) MoFe proteins was 59%, 159%, and 6%, respectively, of that of wild type. For the alpha-195(Asn) MoFe protein, total electron flux decreased with increasing NaCN concentration like wild type. However, the rates of both CH(4) and NH(3) production were maximal at 1 mM NaCN, and they remained unequal even at 5 mM NaCN. With the alpha-195(Gln) MoFe protein, the rates of production of both CH(4) and NH(3) were equal at all NaCN concentrations, and total electron flux was hardly affected by changing the NaCN concentration. With the alpha-191(Lys) MoFe protein, the rates of both CH(4) and NH(3) production were very low, but the rate of NH(3) production was higher, and both rates slowly increased with increasing NaCN concentration. A hypothesis, which is based on the varying apparent affinities of the altered MoFe proteins for HCN and CN(-), is advanced to explain the higher rate of NH(3) production versus the rate of CH(4) production and the effect of increasing NaCN concentration on electron flux to products. A new method for CH(3)NH(2) quantification showed that all four MoFe proteins produced CH(3)NH(2). Added CO significantly inhibited both CH(4) and NH(3) production from HCN with all MoFe proteins except for the alpha-191(Lys) MoFe protein, which still manifested its very low rate of NH(3) production but without CH(4) production. All of the MoFe proteins responded differently to the addition of C(2)H(2) to reactions containing NaCN. With the alpha-195(Asn) MoFe protein, added C(2)H(2) decreased the rates of both CH(4) and NH(3) production, but the rate of NH(3) production decreased much less. C(2)H(2) also exacerbated the inhibition of electron flux. With the alpha-195(Gln) MoFe protein, added C(2)H(2) decreased the rates of both CH(4) and NH(3) production substantially and about equally. C(2)H(2) also eliminated the slight decrease in total electron flux that was caused by NaCN. Added C(2)H(2) hardly affected the alpha-191(Lys) MoFe protein. (ABSTRACT TRUNCA

Acetylene↗
PubMed2000

Construction and characterization of a heterodimeric iron protein: defining roles for adenosine triphosphate in nitrogenase catalysis.

One molecule of MgATP binds to each subunit of the homodimeric Fe protein component of nitrogenase. Both MgATP molecules are hydrolyzed to MgADP and P(i) in reactions coupled to the transfer of one electron into the MoFe protein component. As an approach to assess the contributions of individual ATP binding sites, a heterodimeric Fe protein was produced that has an Asn substituted for residue 39 in the ATP binding domain in one subunit, while the normal Asp(39) residue within the other subunit remains unchanged. Separation of the heterodimeric Fe protein from a mixed population with homodimeric Fe proteins contained in crude extracts was accomplished by construction of a seven His tag on one subunit and a differential immobilized-metal-affinity chromatography technique. Three forms of the Fe protein (wild-type homodimeric Fe protein [Asp(39)/Asp(39)], altered homodimeric Fe protein [Asn(39)/Asn(39)], and heterodimeric Fe protein [Asp(39)/Asn(39)]) were compared on the basis of the biochemical and biophysical changes elicited by nucleotide binding. Among those features examined were the MgATP- and MgADP-induced protein conformational changes that are manifested by the susceptibility of the [4Fe-4S] cluster to chelation and by alterations in the electron paramagnetic resonance, circular dichroism, and midpoint potential of the [4Fe-4S] cluster. The results indicate that changes in the [4Fe-4S] cluster caused by nucleotide binding are the result of additive conformational changes contributed by the individual subunits. The [Asp(39)/Asn(39)] Fe protein did not support substrate reduction activity but did hydrolyze MgATP and showed MgATP-dependent primary electron transfer to the MoFe protein. These results support a model where each MgATP site contributes to the rate acceleration of primary electron transfer, but both MgATP sites must be functioning properly for substrate reduction. Like the altered homodimeric [Asn(39)/Asn(39)] Fe protein, the heterodimeric [Asp(39)/Asn(39)] Fe protein was found to form a high affinity complex with the MoFe protein, revealing that alteration on one subunit is sufficient to create a tight complex.

Adenosine Triphosphate↗
PubMed2000

Formation of a tight 1:1 complex of Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein: evidence for long-range interactions between the Fe protein binding sites during catalytic hydrogen evolution.

It has been well documented that the combination of the MoFe protein of Azotobacter vinelandii nitrogenase (Av1) with the Fe protein (Cp2) from Clostridium pasteurianum nitrogenase produces an inactive, stable complex. However, we report that this heterologous nitrogenase has a low level of activity for H(2) evolution, with a specific activity of 12 nmol min(-)(1) mg(-)(1) of Av1. This activity does not arise from contaminating hydrogenase since it required the presence of both Cp2 and Av1 and showed saturation kinetics when increasing amounts of Cp2 were added to the assay. Incubation of the two proteins at a 4:1 Cp2:Av1 ratio in the absence of MgATP followed by analytical gel filtration showed, surprisingly, that the stoichiometry of the isolated complex was Av1.Cp2 instead of Av1.(Cp2)(2) as determined previously. The presence of MgATP in the elution buffer did not change the elution profile of the complex. The hydrodynamic radius of the isolated complex determined by dynamic light scattering was 5.93 +/- 0.14 nm, intermediate between Av1 and a stable 2:1 nitrogenase complex, consistent with a 1:1 assignment for the Av1.Cp2 complex. When assayed with Av2, the isolated Av1.Cp2 complex showed full half-site reactivity with a specific activity of 750 nmol of C(2)H(2) reduced min(-)(1) mg(-)(1) of Av1. The EPR spectrum of the isolated complex showed the Cp2 to be oxidized and the Av1 to retain the S = (3)/(2) signal characteristic of FeMoco. In the presence of MgATP, under turnover conditions at a 2:1 ratio of Cp2:Av1, the [4Fe-4S] center of Cp2 was protected from the chelator 2,2'-bipyridyl. This is consistent with the formation of a tight 2:1 complex of Av1.(Cp2)(2) which is more stable than the homologous Cp nitrogenase. Assuming that the Lowe-Thorneley model for nitrogenase applies and that a rate-limiting dissociation of the complex is required for H(2) evolution, then with a rate of 0.032 s(-)(1) the 1:1 complex is too stable to be involved in catalysis. The differences in the stability of the 2:1 and 1:1 complexes indicate cooperativity between the Fe protein binding sites of Av1, which structural data show to be separated by 105 A. On the basis of these observations, we propose a model for nitrogenase catalysis in which the stable 1:1 complex formed between oxidized Fe protein and the one-electron-reduced MoFe protein plays an essential role. In this scheme, the two Fe protein binding sites of the MoFe protein alternately bind and release Fe protein in a shuttle mechanism associated with long-range conformational changes in the MoFe protein.

Adenosine Triphosphate↗
PubMed1993

Using dysprosium complexes to probe the nitrogenase paramagnetic centers.

EPR progressive power saturation techniques were used to monitor relaxation enhancement of the nitrogenase paramagnetic centers produced by interaction with Dy3+ complexes. Three models are presented for the relationship between the degree of enhancement and the distance of closest approach of Dy3+ to the intrinsic metal cluster. In the first model, the perturbing dysprosium ions are represented as a single average site. The second and third models are variations of the treatment of Innes and Brudvig [(1989) Biochemistry 28, 1116-1125] and assume the unknown protein to be spherical with Dy3+ dispersed either randomly over the surface of the protein or randomly in solution. Using these models, the distance of closest approach of the Dy3+ complex to the [4Fe-4S] cluster in the Fe-protein from Azotobacter vinelandii was determined to be 5.0-6.5 A. Similarly, the distance of closest approach to FeMoco in the MoFe-protein was determined to be 0.0-1.2 A, which, when corrected to the fact that FeMoco exists as an S = 3/2 spin state, indicates that the distance is > or = 6 A. These distances did not change when (1) either protein was in the presence of the other, (2) both proteins were cross-linked to each other, or (3) the Fe-protein from A. vinelandii was mixed with the MoFe-protein from Clostridium pasteurianum. On the other hand, formation of the inactive complex of the Fe-protein from C. pasteurianum with the MoFe-protein from A. vinelandii blocked dysprosium-induced relaxation enhancement, implying that each component protein overlaps the metal cluster in the complementing protein.

Azotobacter vinelandii↗
PubMed1993

X-ray crystal structure of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum at 3.0-A resolution.

The crystal structure of the nitrogenase molybdenum-iron (MoFe) protein from Clostridium pasteurianum (Cp1) has been determined at 3.0-A resolution by a combination of isomorphous replacement, molecular replacement, and noncrystallographic symmetry averaging. The structure of Cp1, including the two types of metal centers associated with the protein (the FeMo-cofactor and the P-cluster pair), is similar to that previously described for the MoFe-protein from Azotobacter vinelandii (Av1). Unique features of the Cp1 structure arise from the presence of an approximately 50-residue insertion in the alpha subunit and an approximately 50-residue deletion in the beta subunit. As a consequence, the FeMo-cofactor is more buried in Cp1 than in Av1, since the insertion is located on the surface above the FeMo-cofactor. The location of this insertion near the putative nitrogenase iron protein binding site provides a structural basis for the observation that the nitrogenase proteins from C. pasteurianum have low activity with complementary nitrogenase proteins isolated from other organisms. Mechanistic implications of the Cp1 structure for substrate entry/product release, substrate binding to the FeMo-cofactor, and electron- and proton-transfer reactions of nitrogenase are discussed.

Binding Sites↗
PubMed2001

Electron paramagnetic resonance analysis of different Azotobacter vinelandii nitrogenase MoFe-protein conformations generated during enzyme turnover: evidence for S = 3/2 spin states from reduced MoFe-protein intermediates.

Rapid-freezing experiments elicited two transient EPR signals, designated 1b and 1c, during Azotobacter vinelandii nitrogenase turnover at 23 degrees C and pH 7.4. The first of the signals to form, signal 1b, exhibited g values of 4.21 and 3.76. Its formation was at the expense of the starting EPR signal (signal 1a with g values of 4.32, 3.66, and 2.01). The second signal to arise, signal 1c, with a characteristic g value of 4.69, formed very slowly and was always of low intensity. Both signals occurred independently of the substrate being reduced. Increased electron flux through the MoFe protein caused these signals to form more rapidly. Moreover, after a MoFe-protein solution had been pretreated (using conditions of extremely low electron flux) to set up an equimolar mixture of its resting state and one-electron reduced state, these signals appeared even more rapidly when this mixture was exposed to an excess of the Fe protein. We have simulated the kinetics of formation of these EPR features using the published kinetic model for nitrogenase catalysis [Lowe, D. J., and Thorneley, R. N. F. (1984) Biochem. J. 224, 887-909] and propose that they arise from reduced states of the MoFe protein and reflect different conformations of the FeMo cofactor with different protonation states.

Azotobacter vinelandii↗
PubMed2001

Mechanistic features and structure of the nitrogenase alpha-Gln195 MoFe protein.

EPR signals observed under CO and C(2)H(2) during nitrogenase turnover were investigated for the alpha-Gln(195) MoFe protein, an altered form for which the alpha-His(195) residue has been substituted by glutamine. Under CO, samples show S = 1/2 hi- and lo-CO EPR signals identical to those recognized for the wild-type protein, whereas the S = 3/2 signals generated under high CO/high flux conditions differ. Previous work has revealed that the EPR spectrum generated under C(2)H(2) exhibits a signal (S(EPR1)) originating from the FeMo-cofactor having two or more bound C(2)H(2) adducts and a second signal (S(EPR2)) arising from a radical species [Sørlie, M., Christiansen, J., Dean, D. R., and Hales, B. J. (1999) J. Am. Chem. Soc. 121, 9457-9458]. Pressure-dependent studies show that the intensity of these signals has a sigmoidal dependency at low pressures and maximized at 0.1 atm C(2)H(2) with a subsequent decrease in steady-state intensity at higher pressures. Analogous signals are not recognized for the wild-type MoFe protein. Analysis of the principal g-factors of S(EPR2) suggests that it either represents an unusual metal cluster or is a carboxylate centered radical possibly originating from homocitrate. Both S(EPR1) and S(EPR2) exhibit similar relaxation properties that are atypical for S = 1/2 signals originating from Fe-S clusters or radicals and indicate a coupled relaxation pathway. The alpha-Gln(195) MoFe protein also exhibits these signals when incubated under turnover conditions in the presence of C(2)H(4). Under these conditions, additional inflections in the g 4-6 region assigned to ground-state transitions of an S = 3/2 spin system are also recognized and assigned to turnover states of the MoFe protein without C(2)H(4) bound. The structure of alpha-Gln(195) was crystallographically determined and found to be virtually identical to that of the wild-type MoFe protein except for replacement of an NuH-S hydrogen bond interaction between FeMo-cofactor and the imidazole side chain of alpha-His(195) by an analogous interaction involving Gln.

Amino Acid Substitution↗
PubMed2001

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.

Adenosine Triphosphate↗
PubMed2000

Insights into nucleotide signal transduction in nitrogenase: structure of an iron protein with MgADP bound.

Coupling the energy of nucleoside triphosphate binding and hydrolysis to conformational changes is a common mechanism for a number of proteins with disparate cellular functions, including those involved in DNA replication, protein synthesis, and cell differentiation. Unique to this class of proteins is the dimeric Fe protein component of nitrogenase in which the binding and hydrolysis of MgATP controls intermolecular electron transfer and reduction of nitrogen to ammonia. In the work presented here, the MgADP-bound (or "off") conformational state of the nitrogenase Fe protein has been captured and a 2.15 A resolution X-ray crystal structure is presented. The structure described herein reveals likely mechanisms for long-range communication from the nucleotide-binding sites for controlling the affinity of association with the MoFe protein component. Two pathways, termed switches I and II, appear to be integral to this nucleotide signal transduction mechanism. In addition, the structure provides the basis for the changes in the biophysical properties of the [4Fe-4S] cluster observed when Fe protein binds nucleotides. The structure of the MgADP-bound Fe protein provides important insights into the respective contributions of nucleotide interaction and complex formation in defining the conformational states that are the keys to nitrogenase catalysis.

Adenosine Diphosphate↗
PubMed2000

Differential effects on N(2) binding and reduction, HD formation, and azide reduction with alpha-195(His)- and alpha-191(Gln)-substituted MoFe proteins of Azotobacter vinelandii nitrogenase.

In contrast to the wild-type MoFe protein, neither the alpha-195(Asn) nor the alpha-191(Lys) MoFe protein catalyzed N(2) reduction to NH(3), when complemented with wild-type Fe protein. However, N(2) was bound by the alpha-195(Asn) MoFe protein and inhibited the reduction of both protons and C(2)H(2). The alpha-191(Lys) MoFe protein did not interact with N(2). With the alpha-195(Asn) MoFe protein, the N(2)-induced inhibition of substrate reduction was reversed by removing the N(2). Surprisingly, even though added H(2) also relieved N(2) inhibition of substrate reduction, the alpha-195(Asn) MoFe protein did not catalyze HD formation under a N(2)/D(2) atmosphere. This observation is the first indication that these two reactions have different chemical origins, prompting a revision of the current hypothesis that these two reactions are consequences of the same nitrogenase chemistry. A rationale that accounts for the dichotomy of the two reactions is presented. The two altered MoFe proteins also responded quite differently to azide. It was a poor substrate for both but, in addition, azide was an electron-flux inhibitor with the 195(Asn) MoFe protein. The observed reactivity changes are correlated with likely structural changes caused by the amino acid substitutions and provide important details about the interaction(s) of N(2,) H(2), D(2), and azide with Mo-nitrogenase.

Amino Acid Substitution↗
PubMed1994

Purification and characterization of a FeMo cofactor-deficient MoFe protein.

Previous studies have shown that the nifH gene product is required for FeMo cofactor biosynthesis and insertion and that a delta nifH strain of Azotobacter vinelandii designated DJ54 accumulates a FeMo cofactor-deficient MoFe protein that is distinct from the FeMo cofactor-deficient protein synthesis by Nif B-, N-, or E- strains [Tal, S., Chun, T., Gavini, N., & Burgess, B. K. (1991) J. Biol. Chem. 266, 10654-10657]. Here we report the purification and activation of the MoFe protein from DJ54. The purified protein is an alpha 2 beta 2 tetramer that is indistinguishable from the wild-type MoFe protein by the criteria of SDS-polyacrylamide gel electrophoresis, native gel electrophoresis, and two-dimensional gel electrophoresis. It binds normally to its redox partner, the Fe protein, by the criterion of chemical cross-linking. It does not contain FeMo cofactor and does not catalyze significant C2H2 reduction or reduction-independent MgATP hydrolysis. It can, however, be activated with FeMo cofactor following the addition of the Fe protein and MgATP when an additional required component(s) is supplied by cell-free extracts from a delta nifD strain of A. vinelandii. The purified DJ54 MoFe protein does contain P-clusters by the criteria of metal analysis, CD spectroscopy, cluster extrusion, and electrochemical reduction of the POX state. In the presence of dithionite it exhibits an axial S = 1/2 EPR signal that integrates to 0.1-0.3 spin per alpha 2 beta 2 tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1982

Nitrogenase reactivity: cyanide as substrate and inhibitor.

We have examined the reduction of cyanide by using the purified component proteins of nitrogenase (Av1 and Av2). The previously reported self-inhibition phenomenon was found to be an artifact. One of the two species present in cyanide solutions, CN-, was shown to be a potent reversible inhibitor (Ki = 27 microM) of total electron flow, apparently uncoupling MgATP hydrolysis and electron transfer. There appears to be no differential effect of CN- on the specific activities of Av1 and Av2 nor is there any apparent irreversible physical damage to Av2. CN- inhibition is completely reversed by low levels of CO, implying a common binding site. Azide partially relieves the inhibitory effect, but other substrates and inhibitors (N2, C2H2, N2O, H2) have no effect. The other species present in cyanide solutions, HCN, was shown to be the substrate (Km = 4.5 mM at Av2/Av1 = 8), and extrapolation of the data indicates that at high enough HCN concentration H2 evolution can be eliminated. The products are methane plus ammonia (six electrons), and methylamine (four electrons). There is an excess (relative to methane) of ammonia formed, which, according to electron balance studies, may arise from a two-electron intermediate. Both nitrous oxide and acetylene (but not N2) influence the distribution of cyanide reduction products, implying simultaneous binding. HCN appears to bind to and be reduced at an enzyme state more oxidized than the one responsible for either H2 evolution or N2 reduction.

Adenosine Triphosphate↗
PubMed1987

Characterization of the metal clusters in the nitrogenase molybdenum-iron and vanadium-iron proteins of Azotobacter vinelandii using magnetic circular dichroism spectroscopy.

Low-temperature magnetic circular dichroism (MCD) spectroscopy has been used to investigate the metal clusters in the conventional nitrogenase MoFe protein and alternative VFe protein from Azotobacter vinelandii. In the dithionite-reduced state, the MCD spectrum of the MoFe protein is extremely similar to that previously observed for the S = 3/2 spin state of the M clusters in the MoFe protein of Klebsiella pneumoniae. A paramagnetic cluster with an S = 3/2 ground state is also responsible for the temperature-dependent MCD transitions of dithionite-reduced VFe protein. However, the electronic and magnetic properties of this cluster are quite distinct from those of M centers in conventional nitrogenase. When these proteins are oxidized with thionine, the MoFe protein exhibits MCD spectra and magnetization characteristics identical with those observed for the P clusters in K. pneumoniae, while those of the VFe protein are only similar. However, the paramagnetism in the thionine-oxidized VFe protein, like the conventional enzyme, probably arises from an S = 5/2 spin system with near-axial symmetry and a negative zero-field splitting. Novel clusters with electronic, magnetic, and redox properties similar to those of conventional P clusters are, therefore, present in the VFe protein.

Azotobacter↗
PubMed1988

Distinct structural features of the alpha and beta subunits of nitrogenase molybdenum-iron protein of Clostridium pasteurianum: an analysis of amino acid sequences.

Nitrogenase is composed of two separately purified proteins, a molybdenum-iron (MoFe) protein and an iron (Fe) protein. Structural genes (nifD and nifK) encoding alpha and beta subunits of the MoFe protein of Clostridium pasteurianum (Cp) have been cloned and sequenced. The deduced amino acid sequences were analyzed for structures that could be related to the unique properties of the Cp protein, particularly its low capacity to form an active enzyme with a heterologous Fe protein. Cp nifK is located immediately downstream from Cp nifD, with the start codon of nifK overlapping by one base with the stop codon of nifD. An open reading frame following nifK was identified as nifE. The amino acid sequence deduced from nifK encompasses the partial amino acid sequences previously reported from the isolated beta subunit. Cp nifK encodes a polypeptide of 458 amino acid residues (Mr 50 115) whose amino-terminal region is about 50 residues shorter than the otherwise conserved corresponding polypeptides from four other organisms. In contrast, Cp alpha subunit (nifD product) contains an additional stretch of 50 amino acid residues in the 380-430 region, which is unique to the Cp protein. It therefore appears that the combined size of the alpha and beta subunits could be important to nitrogenase function. An analysis of the predicted secondary structure from the amino acid sequence of each subunit from three species (C. pasteurianum, Azotobacter vinelandii, and Rhizobium japonicum) further revealed structural features, including regions adjacent to some of the conserved cysteine residues, differentiating the Cp MoFe protein from others. These different regions may be further tested for correlation with distinct properties of Cp nitrogenase.

Amino Acid Sequence↗
PubMed1988

Dinitrogenase with altered substrate specificity results from the use of homocitrate analogues for in vitro synthesis of the iron-molybdenum cofactor.

The in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires homocitrate (2-hydroxy-1,2,4-butanetricarboxylic acid). Homocitrate is apparently synthesized by the nifV gene product. In the absence of homocitrate, no FeMo-co is formed in vitro, as determined from coupled C2H2 reduction assays and the lack of 99Mo label incorporation into apodinitrogenase. Several organic acids were tested for their ability to replace homocitrate in the FeMo-co synthesis system. With appropriate homocitrate analogues, aberrant forms of FeMo-co are synthesized that exhibit altered substrate specificity and inhibitor susceptibility. Homoisocitrate (1-hydroxy-1,2,4-butanetricarboxylic acid) and 2-oxoglutarate facilitated the incorporation of 99Mo into apodinitrogenase in the FeMo-co synthesis system, yielding a dinitrogenase that effectively catalyzed the reduction of protons but not C2H2 or N2. Citrate also promoted the incorporation of 99Mo into apodinitrogenase, and the resulting holodinitrogenase reduced protons and C2H2 effectively but not N2. In addition, proton reduction from this enzyme was inhibited by CO. The properties of the homodinitrogenase formed in the presence of citrate were reminiscent of those of the Klebsiella pneumoniae NifV- dinitrogenase. We also observed low rates of HD formation from NifV- dinitrogenase compared to those from the wild-type enzyme. No HD formation was observed with the dinitrogenase activated in vitro in the presence of citrate. We propose that in vivo NifV- mutants utilize citrate for FeMo-co synthesis.

Enzyme Activation↗