Biomedical subjects

D R Dean

Publications and source records attributed to D R Dean.

At least 37 records · Page 2Linked to original sources

PubMed1998

14N electron spin-echo envelope modulation of the S = 3/2 spin system of the Azotobacter vinelandii nitrogenase iron-molybdenum cofactor.

Wild-type nitrogenase MoFe protein shows a deep 14N electron spin-echo envelope modulation (ESEEM) arising from a nitrogen nucleus (N1) coupled to the S = 3/2 spin system of the FeMo-cofactor of the MoFe protein. A previous ESEEM study on altered MoFe proteins generated by substitutions at the alpha-195-histidine position suggested that alpha-195-histidine provides a hydrogen bond to the FeMo-cofactor but is not the source of the 14N1 modulation [DeRose et al. (1995) Biochemistry 34, 2809-2814]. This study also raised the possibility of a correlation between ESEEM spectroscopic properties and the nitrogenase phenotype. We now report ESEEM studies on altered MoFe proteins with substitutions at residues alpha-96-arginine, alpha-359-arginine, and alpha-381-phenylalanine to (i) assign the first-shell hydrogen bonding as revealed by the 14N modulation; (ii) explore the mechanistic relevance of the ESEEM signatures to nitrogenase activity; and (iii) study microscopic changes within the polypeptide environment of the FeMo-cofactor. Present ESEEM data reveals that two kinds of 14N modulations are present in wild-type MoFe protein. A new 2-dimensional procedure for high-precision analysis of the ESEEM data of the MoFe proteins shows that the deep wild-type ESEEM modulation (denoted N1) has a hyperfine-coupling constant of Aiso = 1.05 MHz and nuclear quadrupole coupling parameters of e2qQ = 2.17 MHz, eta = 0.59; the other (denoted N2) has a smaller hyperfine coupling of Aiso = approximately 0.5 MHz and e2qQ = approximately 3.5 MHz, eta = approximately 0.4. The N2 ESEEM pattern is more obvious when unmasked by substitutions that result in the loss of the deep N1 modulation. Correlations of the ESEEM properties and catalytic activities of the altered MoFe proteins suggest that (i) the side chain of the alpha-359-arginine is the source of the deep ESEEM N1 modulation; (ii) one or both of the amide nitrogens of alpha-356-glycine/alpha-357-glycine are responsible for the weak N2 modulation; (iii) substitution of the nonpolar alpha-381-phenylalanine residue, as well as substitution of either the alpha-195-histidine or alpha-359-arginine residues, can eliminate the N1 interaction with FeMo-cofactor; and (iv) ESEEM can be used to detect slight reorientations of FeMo-cofactor within its polypeptide pocket, although the mechanistic relevance of the loss or perturbation of the hydrogen-bonding interactions between FeMo-cofactor and polypeptide environment has not yet been established.

Azotobacter vinelandii↗

PubMed1998

Catalytic and biophysical properties of a nitrogenase Apo-MoFe protein produced by a nifB-deletion mutant of Azotobacter vinelandii.

A Zn-immobilized metal-affinity chromatography technique was used to purify a poly-histidine-tagged, FeMo-cofactorless MoFe protein (apo-MoFe protein) from a nifB-deletion mutant of Azotobacter vinelandii. Apo-MoFe protein prepared in this way was obtained in sufficient concentrations for detailed catalytic, kinetic, and spectroscopic analyses. Metal analysis and electron paramagnetic resonance spectroscopy (EPR) were used to show that the apo-MoFe protein does not contain FeMo-cofactor. The EPR of the as-isolated apo-MoFe protein is featureless except for a minor S = 1/2 signal probably arising from the presence of either a damaged P cluster or a P cluster precursor. The apo-MoFe protein has an alpha2beta2 subunit composition and can be activated to 80% of the theoretical MoFe protein value by the addition of isolated FeMo-cofactor. Oxidation of the as-isolated apo-MoFe protein by indigodisulfonate was used to elicit the parallel mode EPR signal indicative of the two-electron oxidized form of the P cluster (P2+). The midpoint potential of the PN/P2+ redox couple for the apo-MoFe protein was shown to be shifted by -63 mV when compared to the same redox couple for the intact MoFe protein. Although the apo-MoFe protein is not able to catalyze the reduction of substrates under turnover conditions, it does support the hydrolysis of MgATP at 60% of the rate supported by the MoFe protein when incubated in the presence of Fe protein. The ability of the apo-MoFe protein to specifically interact with the Fe protein was also shown by stopped-flow techniques and by formation of an apo-MoFe protein-Fe protein complex. Finally, the two-electron oxidized form of the apo-MoFe protein could be reduced to the one-electron oxidized form (P1+) in a reaction that required Fe protein and MgATP. These results are interpreted to indicate that the apo-MoFe protein produced in a nifB-deficient genetic background [corrected] contains intact P clusters and P cluster polypeptide environments. Small changes in the electronic properties of P clusters contained within the apo-MoFe protein are most likely caused by slight perturbations in their polypeptide environments.

Alkylation↗

PubMed1998

Evidence for coupled electron and proton transfer in the [8Fe-7S] cluster of nitrogenase.

Substrate reduction by nitrogenase requires electron transfer from a [4Fe-4S] cluster in the iron (Fe) protein component to an FeMo cofactor in the molybdenum-iron (MoFe) protein component in a reaction that is coupled to MgATP hydrolysis and component protein association and dissociation. An [8Fe-7S] (or P-) cluster in the MoFe protein has been proposed as an intermediate electron-transfer site, although how this cluster functions in electron-transfer remains unclear. In the present work, it is demonstrated that one redox couple of the P-cluster (P2+/1+) undergoes coupled electron and proton transfer, whereas a more reduced couple (P1+/N) does not involve a coupled proton transfer. Redox titrations of the MoFe protein P-cluster were performed, and the midpoint potential of the P2+/1+ couple (Em2) was found to be pH dependent, ranging from -224 mV at pH 6.0 to -348 mV at pH 8.5. A plot of Em2 versus the pH for this redox couple was linear and revealed a change of -53 mV/pH unit, indicating a single protonation event associated with reduction. From this plot, it was concluded that p is <6.0 and p is >8.5 in a proton-modified Nernst equation. In contrast, the midpoint potential for the P1+/N couple (Em1) was found to be -290 mV and was invariant over the pH range 6.0-8.5. These results indicate that the protonated species does not influence either the P1+ or the PN oxidation states. In addition, at physiological pH values, electron transfer is coupled to proton transfer for the P2+/1+ couple. The P-clusters are unique among [Fe-S] clusters in that they appear to be ligated to the protein through a serinate-gammaO ligand (betaSer188) and a peptide bond amide-N ligand (alphaCys88), in addition to cysteinate-S ligands. Elimination of the serinate ligand by replacement with a glycine was found to shift the Em values for both P-cluster couples by greater than +60 mV, however the pH dependence of Em2 was unchanged. These results rule out Ser188 as the protonated ligand responsible for the pH dependence of Em2. The implications of these results in understanding the nitrogenase electron-transfer mechanism are discussed.

Amino Acid Substitution↗

PubMed1998

The Azotobacter vinelandii NifEN complex contains two identical [4Fe-4S] clusters.

The nifE and nifN gene products from Azotobacter vinelandii form an alpha2beta2 tetramer (NifEN complex) that is required for the biosynthesis of the nitrogenase FeMo cofactor. In the current model for NifEN complex organization and function, the complex is structurally analogous to the nitrogenase MoFe protein and provides an assembly site for a portion of FeMo cofactor biosynthesis. In this work, gene fusion and immobilized metal-affinity chromatography strategies were used to elevate the in vivo production of the NifEN complex and to facilitate its rapid and efficient purification. The NifEN complex produced and purified in this way exhibits an FeMo cofactor biosynthetic activity similar to that previously described for the NifEN complex purified by traditional chromatography methods. UV-visible, EPR, variable-temperature magnetic circular dichroism, and resonance Raman spectroscopies were used to show that the NifEN complex contains two identical [4Fe-4S]2+ clusters. These clusters have a predominantly S = 1/2 ground state in the reduced form, exhibit a reduction potential of -350 mV, and are likely to be coordinated entirely by cysteinyl residues on the basis of spectroscopic properties and sequence comparisons. A model is proposed where each NifEN complex [4Fe-4S] cluster is bridged between a NifE-NifN subunit interface at a position analogous to that occupied by the P clusters in the nitrogenase MoFe protein. In contrast to the MoFe protein P clusters, the NifEN complex [4Fe-4S] clusters are proposed to be asymmetrically coordinated to the NifEN complex where NifE cysteines-37, -62, and -124 and NifN cysteine-44 are the coordinating ligands. On the basis of a homology model of the three-dimensional structure of the NifEN complex, the [4Fe-4S] cluster sites are likely to be remote from the proposed FeMo cofactor assembly site and are unlikely to become incorporated into the FeMo cofactor during its assembly.

Artificial Gene Fusion↗

PubMed1998

Assembly of iron-sulfur clusters. Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii.

An enzyme having the same L-cysteine desulfurization activity previously described for the NifS protein was purified from a strain of Azotobacter vinelandii deleted for the nifS gene. This protein was designated IscS to indicate its proposed role in iron-sulfur cluster assembly. Like NifS, IscS is a pyridoxal-phosphate containing homodimer. Information gained from microsequencing of oligopeptides obtained by tryptic digestion of purified IscS was used to design a strategy for isolation and DNA sequence analysis of a 7,886-base pair A. vinelandii genomic segment that includes the iscS gene. The iscS gene is contained within a gene cluster that includes homologs to nifU and another gene contained within the major nif cluster of A. vinelandii previously designated orf6. These genes have been designated iscU and iscA, respectively. Information available from complete genome sequences of Escherichia coli and Hemophilus influenzae reveals that they also encode iscSUA gene clusters. A wide conservation of iscSUA genes in nature and evidence that NifU and NifS participate in the mobilization of iron and sulfur for nitrogenase-specific iron-sulfur cluster formation suggest that the products of the iscSUA genes could play a general role in the formation or repair of iron-sulfur clusters. The proposal that IscS is involved in mobilization of sulfur for iron-sulfur cluster formation in A. vinelandii is supported by the presence of a cysE-like homolog in another gene cluster located immediately upstream from the one containing the iscSUA genes. O-Acetylserine synthase is the product of the cysE gene, and it catalyzes the rate-limiting step in cysteine biosynthesis. A similar cysE-like gene is also located within the nif gene cluster of A. vinelandii. The likely role of such cysE-like gene products is to increase the cysteine pool needed for iron-sulfur cluster formation. Another feature of the iscSUA gene cluster region from A. vinelandii is that E. coli genes previously designated as hscB, hscA, and fdx are located immediately downstream from, and are probably co-transcribed with, the iscSUA genes. The hscB, hscA, and fdx genes are also located adjacent to the iscSUA genes in both E. coli and H. influenzae. The E. coli hscA and hscB gene products have previously been shown to bear primary sequence identity when respectively compared with the dnaK and dnaJ gene products and have been proposed to be members of a heat-shock-cognate molecular chaperone system of unknown function. The close proximity and apparent co-expression of iscSUA and hscBA in A. vinelandii indicate that the proposed chaperone function of the hscBA gene products could be related to the maturation of iron-sulfur cluster-containing proteins. Attempts to place non-polar insertion mutations within either A. vinelandii iscS or hscA revealed that such mutations could not be stably maintained in the absence of the corresponding wild-type allele. These results reveal a very strong selective pressure against the maintenance of A. vinelandii iscS or hscA knock-out mutations and suggest that such mutations are either lethal or highly deleterious. In contrast to iscS or hscA, a strain having a polar insertion mutation within the cysE-like gene was readily isolated and could be stably maintained. These results show that the cysE-like gene located upstream from iscS is not essential for cell growth and that the cysE-like gene and the iscSUA-hscBA-fdx genes are contained within separate transcription units.

Amino Acid Sequence↗

PubMed1997

Evidence for multiple substrate-reduction sites and distinct inhibitor-binding sites from an altered Azotobacter vinelandii nitrogenase MoFe protein.

The arginine-277 residue of the alpha-subunit of the nitrogenase MoFe protein was targeted for substitution because it is (i) a close neighbor of alpha-cysteine-275, which is one of only two residues anchoring the FeMo cofactor to the polypeptide, and (ii) a component of a potential channel for entry/exit of substrates/products and for accepting FeMo cofactor during MoFe-protein maturation. Several of the eight mutant strains constructed were capable of good diazotrophic growth and also contained FeMo cofactor as indicated by its biologically unique S = 3/2 EPR spectrum. These observations indicate that the positively charged alpha-arginine-277 residue is not required for acceptance of the negatively charged FeMo cofactor by the separately synthesized, cofactor-deficient, apo-MoFe protein. The wide range of nitrogen-fixation phenotypes shown by these mutant strains generally correlated well with their C2H2- and proton-reduction activities, which range from 5 to 65% of wild-type activity. One notable exception is the histidine-substituted strain, DJ788 (alpha-277His). This strain, although unable to fix N2 and grow diazotrophically, elaborates an altered alpha-277His MoFe protein that catalyzes the reduction of the alternative substrates, C2H2, HCN, HN3, and protons. These observations are best explained if multiple redox levels are available to the MoFe protein but the alpha-277His MoFe protein is incapable of reaching the more-reduced redox levels required for nitrogen fixation. Under nonsaturating CO concentrations, the alpha-277His MoFe-protein-catalyzed reduction of C2H2 showed sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity among two C2H4-evolving sites and indicates the presence of three sites, which can be simultaneously occupied, on the MoFe protein. Similar kinetics were not observed for alpha-277His MoFe-protein-catalyzed reduction of either HCN or HN3 with nonsaturating CO levels, indicating that these substrates are unlikely to share common binding sites with C2H2. Further, CN- did not induce cooperativity in C2H2 reduction and, therefore, CO and CN- are unlikely to share a common binding site. These changed substrate specificities, reinforced by changes in the FeMo-cofactor-derived S = 3/2 EPR spectrum, clearly indicate the importance of the alpha-277 residue in catalysis and the delicate control exerted on the properties of bound FeMo cofactor by its polypeptide environment.

Arginine↗

PubMed1997

Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase.

The nifV gene product (NifV) from Azotobacter vinelandii was recombinantly expressed at high levels in Escherichia coli and purified. NifV is a homodimer that catalyzes the condensation of acetyl coenzyme A (acetyl-CoA) and alpha-ketoglutarate. Although alpha-ketoglutarate supports the highest level of activity, NifV will also catalyze the condensation of acetyl-CoA and certain other keto acids. E. coli cells in which a high level of nifV expression is induced excrete homocitrate into the growth medium.

Azotobacter vinelandii↗

PubMed1997

Role of NifS in maturation of glutamine phosphoribosylpyrophosphate amidotransferase.

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is synthesized as an inactive precursor that requires two maturation steps: incorporation of a [4Fe-4S] center and cleavage of an 11-residue NH2-terminal propeptide. Overproduction from a multicopy plasmid in Escherichia coli leads to the formation of soluble proenzyme and mature enzyme forms as well as a small fraction of insoluble proenzyme. Heterologous expression of Azotobacter vinelandii nifS from a compatible plasmid increased the maturation of the soluble proenzyme three- to fourfold without influencing the content of the insoluble fraction. These results support a role for NifS in heterologous Fe-S cluster assembly and enzyme maturation.

Amidophosphoribosyltransferase↗

PubMed1995

Involvement of the P cluster in intramolecular electron transfer within the nitrogenase MoFe protein.

Nitrogenase is the catalytic component of biological nitrogen fixation, and it is comprised of two component proteins called the Fe protein and MoFe protein. The Fe protein contains a single Fe4S4 cluster, and the MoFe protein contains two metallocluster types called the P cluster (Fe8S8) and FeMo-cofactor (Fe7S9Mo-homocitrate). During turnover, electrons are delivered one at a time from the Fe protein to the MoFe protein in a reaction coupled to component-protein association-dissociation and MgATP hydrolysis. Under conditions of optimum activity, the rate of component-protein dissociation is rate-limiting. The Fe protein's Fe4S4 cluster is the redox entity responsible for intermolecular electron delivery to the MoFe protein, and FeMo-cofactor provides the substrate reduction site. In contrast, the role of the P cluster in catalysis is not well understood although it is believed to be involved in accumulating electrons delivered from the Fe protein and brokering their intramolecular delivery to the substrate reduction site. A nitrogenase component-protein docking model, which is based on the crystallographic structures of the component proteins and which pairs the 2-fold symmetric surface of the Fe protein with the exposed surface of the MoFe protein's pseudosymmetric alpha beta interface, is now available. During component-protein interaction, this model places the P cluster between the Fe protein's Fe4S4 cluster and FeMo-cofactor, which implies that the P cluster is involved in mediating intramolecular electron transfer between the clusters. In the present study, evidence supporting this idea was obtained by demonstrating that it is possible to alter the rate of substrate reduction by perturbing the polypeptide environment between the P cluster and FeMo-cofactor without necessarily disrupting the metallocluster polypeptide environments or altering component-protein interaction.

Amino Acid Sequence↗

PubMed1995

Characterization of the gamma protein and its involvement in the metallocluster assembly and maturation of dinitrogenase from Azotobacter vinelandii.

Dinitrogenase, the enzyme capable of catalyzing the reduction of N2, is a heterotetramer (alpha 2 beta 2) and contains the iron-molybdenum cofactor (FeMo-co) at the active site of the enzyme. Mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase, which is enzymatically inactive but can be activated in vitro by the addition of purified FeMo-co. Apodinitrogenase from certain mutant strains of Azotobacter vinelandii has a subunit composition of alpha 2 beta 2 gamma 2. The gamma subunit has been implicated as necessary for the efficient activation of apodinitrogenase in vitro. Characterization of gamma protein in crude extracts and partially pure fractions has suggested that it is a chaperone-insertase required by apodinitrogenase for the insertion of FeMo-co. These are three major forms of gamma protein detectable by Western analysis of native gels. An apodinitrogenase-associated form is found in extracts of nifB or nifNE strains and dissociates from the apocomplex upon addition of purified FeMo-co. A second form of gamma protein is unassociated with other proteins and exists as a homodimer. Both of these forms of gamma protein can be converted to a third form by the addition of purified FeMo-co. This conversion requires the addition of active FeMo-co and correlates with the incorporation of iron into gamma protein. Crude extracts that contain this form of gamma protein are capable of donating FeMo-co to apodinitrogenase, thereby activating the apodinitrogenase. These data support a model in which gamma protein is able to interact with both FeMo-co and apodinitrogenase, facilitate FeMo-co insertion into apodinitrogenase, and then dissociate from the activated dinitrogenase complex.

Azotobacter vinelandii↗

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↗

PubMed1995

Characteristics of NIFNE in Azotobacter vinelandii strains. Implications for the synthesis of the iron-molybdenum cofactor of dinitrogenase.

The products of the nifN and nifE genes of Azotobacter vinelandii function as a 200-kDa alpha 2 beta 2 tetramer (NIFNE) in the synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase, the enzyme system required for biological nitrogen fixation. NIFNE was purified using a modification of the published protocol. Immunoblot analysis of anoxic native gels indicated that distinct forms of NIFNE accumulate in strains deficient in either NIFB (delta nifB::kan delta nifDK) or NIFH (delta nifHDK). During the purification of NIFNE from the delta nifHDK mutant, its mobility in these gels changed, becoming similar to that of NIFNE from the delta nifB::kan delta nifDK mutant. While NIFB activity initially co-purified with the NIFNE activity from the delta nifHDK mutant, further purification of NIFNE activity resulted in the loss of the co-purifying NIFB activity; this loss correlated with the change in NIFNE mobility on native gels. These results suggest that the form of NIFNE accumulated in the delta nifHDK mutant is associated with NIFB activity in crude extract but loses this association during NIFNE purification. Addition of the purified metabolic product of NIFB, termed NifB-co, to either NIFNE purified from the delta nifHDK strain or to the NIFNE in crude extract of the delta nifB::kan delta nifDK strain caused a change in the mobility of NIFNE on anoxic native gels to that of the form accumulated in a delta nifHDK mutant. These results support a model where both NifB-co and dinitrogenase reductase participate in FeMo-co synthesis through NIFNE, which serves as a scaffold for this process.

Azotobacter vinelandii↗

PubMed1995

Nitrogenase structure and function: a biochemical-genetic perspective.

Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme composed of two component proteins called the Fe protein and the MoFe protein. During catalysis, electrons are delivered one at a time from the Fe protein to the MoFe protein in a process involving component-protein association and dissociation and hydrolysis of at least two MgATP for each electron transfer. The Fe protein contains the sites for MgATP binding and hydrolysis, whereas the site for substrate binding and reduction is located on the MoFe protein. Among the important aspects of nitrogenase enzymology discussed here are (a) the structures of the metal centers that participate in electron transfer, (b) the organization of the metalloclusters within the polypeptides and their contributions to substrate binding and electron transfer, (c) the nature of the dynamic interactions between the two component proteins that lead to nucleotide hydrolysis and intermolecular electron transfer, (d) the mechanism by which the multiple electrons necessary for substrate reduction are distributed within the MoFe protein, (e) the nature of the intramolecular electron path within the MoFe protein, and (f) where and how substrate and various inhibitors become bound to the substrate-reduction site. This chapter summarizes biochemical-genetic strategies used to address these questions and discussed them in the context of the recently proposed three-dimensional models for both the Fe protein and MoFe protein from Azotobacter vinelandii.

Metalloproteins↗

PubMed1994

nifU gene product from Azotobacter vinelandii is a homodimer that contains two identical [2Fe-2S] clusters.

The nifU gene product is required for the full activation of the metalloenzyme nitrogenase, the catalytic component of biological nitrogen fixation. In the present work, a hybrid plasmid that contains the Azotobacter vinelandii nifU gene was constructed and used to hyperexpress the NIFU protein in Escherichia coli. Recombinant NIFU was purified to homogeneity and was found to be a homodimer of 33-kDa subunits with approximately two Fe atoms per subunit. The combination of UV/visible absorption, variable-temperature magnetic circular dichroism, EPR, and resonance Raman spectroscopies shows the presence of a [2Fe-2S]2+,+ center (Em = -254 mV) with complete cysteinyl coordination in each subunit. The electronic, magnetic, and vibrational properties of the [2Fe-2S]2+,+ center do not conform to those established for any of the spectroscopically distinct types of 2Fe ferredoxins. These distinctive properties appear to be a consequence of a novel arrangement of coordinating cysteinyl residues in NIFU, and the residues likely to be involved in cluster coordination are discussed in light of primary sequence comparisons to other putative [2Fe-2S] proteins. The observed physicochemical properties of NIFU and its constituent [2Fe-2S] cluster also provide insight into the role of this protein in nitrogenase metallocluster biosynthesis.

Azotobacter vinelandii↗

PubMed1994

Identification of a nitrogenase protein-protein interaction site defined by residues 59 through 67 within the Azotobacter vinelandii Fe protein.

During nitrogenase catalysis the Fe protein and the MoFe protein associate and dissociate in a MgATP-dependent process involving electron transfer from the Fe protein to the MoFe protein. A docking model, based primarily on the crystal structures of the separate components from Azotobacter vinelandii, was previously proposed in which the 2-fold symmetric surface of the homodimeric Fe protein interacts with the exposed surface of a MoFe protein pseudosymmetric alpha beta-unit interface. In this model, a loop, which is included within residues 59 through 67 of the Fe protein primary sequence, is likely to interact with the MoFe protein during component protein docking. In the present study, evidence supporting the component protein docking model was obtained by construction of an A. vinelandii strain that produces a hybrid Fe protein for which residues 59 through 67 have been replaced by the corresponding residues from the Fe protein of Clostridium pasteurianum. Biochemical analyses of the hybrid Fe protein revealed the following features when compared with the unaltered Fe protein. First, the hybrid Fe protein exhibited half the maximum specific activity of the normal Fe protein and was insensitive to inhibition by low levels of NaCl. Second, the hybrid Fe protein activity was hypersensitive to a molar excess of MoFe protein, which also resulted in the uncoupling of MgATP hydrolysis from substrate reduction. Third, stopped-flow spectrophotometry experiments showed that during catalysis the hybrid Fe protein dissociates from the MoFe protein at only half the normal rate of Fe protein-MoFe protein dissociation. Thus, the salient feature of the hybrid Fe protein is that it appears to form a relatively tighter complex with the MoFe protein. This property is in line with previous biochemical reconstitution experiments where it was shown that a heterologous mixture of Fe protein from C. pasteurianum and MoFe protein from A. vinelandii form a tight, inactive complex and supports the proposal that a region defined by residues 59 through 67 within the Fe protein is involved in component protein interaction.

Adenosine Triphosphate↗

PubMed1994

Catalytic formation of a nitrogenase iron-sulfur cluster.

Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme comprised of two component proteins called the Fe protein and the MoFe protein. Both nitrogenase component proteins contain metalloclusters. The Azotobacter vinelandii nifS gene product (NifS), which is required for full activation of the nitrogenase component proteins, is a pyridoxal phosphate enzyme and is able to catalyze the desulfurization of L-cysteine to yield sulfur and L-alanine (Zheng, L., White, R. H., Cash, V.L., Jack, R.F., and Dean, D.R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2754-2758). An enzyme-bound persulfide that was identified as an intermediate in the cysteine desulfurization reaction catalyzed by NifS has been suggested as a possible S-donor in formation of the iron-sulfide cores of the nitrogenase metalloclusters. In the present work it is shown that NifS is able to effectively catalyze activation of an apo-form of the Fe protein that was prepared by removal of its Fe4S4 cluster using the chelator, alpha,alpha'-dipyridyl. The reconstitution reaction includes apo-Fe protein, NifS, L-cysteine, ferrous ion, dithiothreitol, and MgATP. Reconstitution of the inactive apo-Fe protein catalyzed by NifS results in 80-95% recovery of the original activity and yields an Fe protein having the normal electron paramagnetic resonance spectra properties associated with the Fe protein's Fe4S4 cluster. An altered NifS protein, NifS-Ala325, which lacks the desulfurase activity and is unable to from the NifS-bound persulfide, is not able to catalyze reactivation of the apo-Fe protein. These in vitro results support the proposal that NifS activity provides the inorganic sulfide necessary for in vivo formation of the nitrogenase metalloclusters. Moreover, because NifS has recently been shown to be a member of a highly homologous gene family, it appears that pyridoxal phosphate chemistry might play a general role in iron-sulfur cluster assembly.

Apoproteins↗

PubMed1994

Mechanism for the desulfurization of L-cysteine catalyzed by the nifS gene product.

The nifS gene product (NIFS) is a pyridoxal phosphate binding enzyme that catalyzes the desulfurization of L-cysteine to yield L-alanine and sulfur. In Azotobacter vinelandii this activity is required for the full activation of the nitrogenase component proteins. Because the nitrogenase component proteins, Fe protein and MoFe protein, both contain metalloclusters which are required for their respective activities, it is suggested that NIFS participates in the biosynthesis of the nitrogenase metalloclusters by providing the inorganic sulfur required for Fe-S core formation [Zheng, L., White, R. H., Cash, V. L. Jack, R. F., & Dean, D. R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2754-2758]. In the present study the mechanism for the desulfurization of L-cysteine catalyzed by NIFS was determined in the following ways. First, the substrate analogs, L-allylglycine and vinylglycine, were shown to irreversibly inactivate NIFS by formation of a gamma-methylcystathionyl or cystathionyl residue, respectively, through nucleophilic attack by an active site cysteinyl residue on the corresponding analog-pyridoxal phosphate adduct. Second, this reactive cysteinyl residue, which is required for L-cysteine desulfurization activity, was identified as Cys325 by the specific alkylation of that residue and by site-directed mutagenesis experiments. Third, the formation of an enzyme-bound cysteinyl persulfide was identified as an intermediate in the NIFS-catalyzed reaction. Fourth, evidence was obtained for an enamine intermediate in the formation of L-alanine.(ABSTRACT TRUNCATED AT 250 WORDS)

Allylglycine↗