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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

Crystallographic and enzymatic investigations on the role of Ser558, His610, and Asn614 in the catalytic mechanism of Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2p).

Dihydrolipoamide acetyltransferase (E2p) is the structural and catalytic core of the pyruvate dehydrogenase multienzyme complex. In Azotobacter vinelandii E2p, residues Ser558, His610', and Asn614' are potentially involved in transition state stabilization, proton transfer, and activation of proton transfer, respectively. Three active site mutants, S558A, H610C, and N614D, of the catalytic domain of A. vinelandii E2p were prepared by site-directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants have been determined at 2.7, 2.5, and 2.6 A resolution, respectively. The S558A and H610C mutants exhibit a strongly (200-fold and 500-fold, respectively) reduced enzymatic activity whereas the substitution of Asn614' by aspartate results in a moderate (9-fold) reduced activity. The decrease in enzymatic activity of the S558A and H610C mutants is solely due to the absence of the hydroxyl and imidazole side chains, respectively, and not due to major conformational rearrangements of the protein. Furthermore the sulfhydryl group of Cys610' is reoriented, resulting in a completely buried side chain which is quite different from the solvent-exposed imidazole group of His610' in the wild-type enzyme. The presence of Asn614' in A. vinelandii E2p is exceptional since all other 18 known dihydrolipoamide acyltransferase sequences contain an aspartate in this position. We observe no difference in conformation of Asp614' in the N614D mutant structure compared with the conformation of Asn614' in the wild-type enzyme. Detailed analysis of all available structures and sequences suggests two classes of acetyltransferases: one class with a catalytically essential His-Asn pair and one with a His-Asp-Arg triad as present in chloramphenicol acetyltransferase [Leslie, A. G. W. (1990) J. Mol. Biol. 213, 167-186] and in the proposed active site models of Escherichia coli and yeast E2p.

Acetyltransferases↗

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

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↗

PubMed1995

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.

2,2'-Dipyridyl↗

PubMed1995

The "nitrogenase-protective" FeSII protein of Azotobacter vinelandii: overexpression, characterization, and crystallization.

The Azotobacter vinelandii FeSII protein confers conformational protection to nitrogenase by binding to the MoFe and Fe proteins under periods of oxidative stress to create an inactive but O2-stabilized tripartite complex. In this work the FeSII protein has been overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity, crystallized, and characterized in terms of its functional, spectroscopic, and redox properties. The recombinant protein is a homodimer and is expressed as a holoprotein with one [2Fe-2S]2+,+ cluster in each subunit. It is shown to be functional in reconstituting an O2-stable nitrogenase complex in vitro. Spectroscopic studies using the combination of UV-visible absorption, CD, and variable temperature MCD, EPR, and resonance Raman indicate that the [2Fe-2S]2+,+ cluster is coordinated exclusively by cysteine residues. The arrangement of coordinating cysteines in the primary sequence and the EPR properties of the [2Fe-2S]+ cluster (g = 2.04, 1.95, 1.88) are very similar to those of chloroplast ferredoxins. However, the variable-temperature MCD, resonance Raman, and redox properties (Em = -262 +/- 10 mV based on dye-mediated EPR redox titrations) are more characteristic of hydroxylase-type ferredoxins such as adrenodoxin. In contrast to chloroplast-type ferredoxins, the vibrational properties of the [2Fe-2S]2+,+ cluster in the FeSII protein indicate that none of the cysteinyl Fe-S-C-C dihedral angles are close to 180 degrees and that the cluster is not exposed to solvent. Preliminary X-ray diffraction analysis indicates that the protein crystallizes in an orthorhombic space group with unit cell dimensions a = 135 A, b = 135 A, and c = 38 A and that there are at least two dimers per asymmetric unit.

Azotobacter vinelandii↗

PubMed1995

Proton NMR investigation of the [4Fe--4S]1+ cluster environment of nitrogenase iron protein from Azotobacter vinelandii: defining nucleotide-induced conformational changes.

This work presents the complete assignment of the isotropically shifted 1H NMR resonances of Azotobacter vinelandii nitrogenase iron protein (Fe protein) to beta-CH2 and alpha-CH protons of the [4Fe--4S]1+ cluster cysteinyl ligands. Four resonances were observed for the reduced Fe protein with chemical shifts of 49, 23, 17, and 13 ppm. T1 measurements and analysis of relative peak areas coupled with one-dimensional nuclear Overhauser effect (NOE) difference spectra were used to assign the two most downfield-shifted resonances (49 and 23 ppm) to cysteinyl ligand beta-CH2 protons and the 17 and 14 ppm resonances to cysteinyl ligand alpha-CH protons. Temperature dependent studies of the isotropically shifted protons revealed both Curie and anti-Curie behavior. These results, along with previous Mossbauer studies of the Fe protein, allowed the assignment of signal A (49 ppm) to four beta-CH2 protons and signal C (17 ppm) to 2 alpha-CH protons of two cysteinyl ligands bound to a mixed-valence iron pair (Fe3(+)--Fe2+) of the [4Fe--4S]1+ cluster. Signal B (23 ppm) was assigned to four beta-CH2 protons, and signal C (17 ppm) and D (13 ppm) were assigned to two alpha-CH protons of two cysteinyl ligands bound to a ferrous pair of irons (2Fe2+). The effects of MgATP, MgADP, and Mg-adenosine-beta, gamma-methylene-5'-triphosphate binding to the Fe protein on the assigned resonances were established and are discussed in the context of nucleotide-induced changes in the protein environment of the [4Fe--4S] cluster.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

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↗

PubMed1992

Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase.

The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 20.3) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Av1) were more complex than for Av2. A time-dependent inactivation of Av1 activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1992

Acetylene inhibition of Azotobacter vinelandii hydrogenase: acetylene binds tightly to the large subunit.

Acetylene is a slow-binding inhibitor of the Ni- and Fe-containing dimeric hydrogenase isolated from Azotobacter vinelandii. Acetylene was released from hydrogenase during the recovery from inhibition. This indicates that no transformation of acetylene to another compound occurred as a result of the interaction with hydrogenase. However, the release of C2H2 proceeds more rapidly than the recovery of activity, which indicates that release of C2H2 is not sufficient for recovery of activity. Acetylene binds tightly to native hydrogenase; hydrogenase and radioactivity coelute from a gel permeation column following inhibition with 14C2H2. Acetylene, or a derivative, remains bound to the large 65,000 MW subunit (and not to the small 35,000 MW subunit) of hydrogenase following denaturation as evidenced by SDS-PAGE and fluorography of 14C2H2-inhibited hydrogenase. This result suggests that C2H2, and by analogy H2, binds to and is activated by the large subunit of this dimeric hydrogenase. Radioactivity is lost from 14C2H2-inhibited protein during recovery. The inhibition is remarkably specific for C2H2: propyne, butyne, and ethylene are not inhibitors.

Acetylene↗

PubMed1992

Fe2+ and phosphate interactions in bacterial ferritin from Azotobacter vinelandii.

Fe2+ binding to both apo- and holo- bacterial ferritin from Azotobacter vinelandii (AVBF) was measured as a function of pH under carefully controlled anaerobic conditions. Fe2+ binding to apo-AVBF is strongly pH dependent with 25 Fe2+ ions/apo-AVBF binding tightly at pH 5.5 and over 150 Fe2+/apo-AVBF at pH 9.0. Holo-AVBF gave a similar pH-dependent binding profile with over 400 Fe2+/AVBF binding at pH of 9.0. Proton release per Fe2+ bound to either AVBF protein increases with increasing pH until a total of about two protons are released at pH 9.0. These binding results are both qualitatively and quantitatively different from corresponding measurements (Jacobs et al., 1989) on apo- and holo- mammalian ferritin (MF) where less Fe2+ binds in both cases. The high level of Fe2+ binding to holo-AVBF relative to that of mammalian ferritin is a consequence of the higher phosphate content in the core of AVBF. Reduction of AVBF by either dithionite or methyl viologen in the absence of chelating agents demonstrated that phosphate, but not Fe2+, is released from the AVBF core in amounts commensurate with the degree of iron reduction, although even at 100% reduction considerable phosphate remains associated with the reduced mineral core. Fe2+ binding to holo-AVBF made deficient in phosphate was lower than that of native AVBF, while the addition of phosphate to native holo-AVBF increased the Fe2+ binding capacity. These results clearly support the role of phosphate as the site of interaction of Fe2+ with the AVBF mineral core.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaerobiosis↗

PubMed1992

Molecular relaxation spectroscopy of flavin adenine dinucleotide in wild type and mutant lipoamide dehydrogenase from Azotobacter vinelandii.

The temperature dependence of the fluorescence emission spectra of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii shows that the protein matrix in the vicinity of the prosthetic group is rigid on a nanosecond time scale in a medium of high viscosity (80% glycerol). The active site of a deletion mutant of this enzyme, which lacks 14 C-terminal amino acids, is converted from a solid-state environment (on the nanosecond time scale of fluorescence) into a state where efficient dipolar relaxation takes place at temperatures between 203 and 303 K. In aqueous solution, fast dipolar fluctuations are observed in both proteins. It is shown from fluorescence quenching of the flavin by iodide ions that the prosthetic groups of the mutant protein are partially iodide accessible in contrast to the wild type enzyme. A detailed analysis of the temperature dependence of spectral energies according to continuous relaxation models reveals two distinct relaxation processes in the deletion mutant, which were assigned to solvent and protein dipoles, respectively. From the long-wavelength shifts of the emission spectra upon red-edge excitation, it is demonstrated that the active site of the wild type enzyme has high structural homogeneity in comparison to the deletion mutant. In combination with results obtained by X-ray diffraction studies on crystals of the wild type enzyme, it can be concluded that the C-terminal polypeptide of the A. vinelandii enzyme interacts with the dehydrolipoamide binding site, thereby shielding the flavins from the solvent.

Azotobacter vinelandii↗

PubMed2001

Crystal structure of the all-ferrous [4Fe-4S]0 form of the nitrogenase iron protein from Azotobacter vinelandii.

The structure of the nitrogenase iron protein from Azotobacter vinelandii in the all-ferrous [4Fe-4S](0) form has been determined to 2.25 A resolution by using the multiwavelength anomalous diffraction (MAD) phasing technique. The structure demonstrates that major conformational changes are not necessary either in the iron protein or in the cluster to accommodate cluster reduction to the [4Fe-4S](0) oxidation state. A survey of [4Fe-4S] clusters coordinated by four cysteine ligands in proteins of known structure reveals that the [4Fe-4S] cluster of the iron protein has the largest accessible surface area, suggesting that solvent exposure may be relevant to the ability of the iron protein to exist in three oxidation states.

Amides↗

PubMed1994

Calculation of the redox potentials of iron-sulfur proteins: the 2-/3-couple of [Fe4S*4Cys4] clusters in Peptococcus aerogenes ferredoxin, Azotobacter vinelandii ferredoxin I, and Chromatium vinosum high-potential iron protein.

Calculations of the redox potentials of the 2-/3-couples of [Fe4S*4Cys4] clusters in the iron-sulfur proteins Peptococcus aerogenes ferredoxin (PaFd), Azotobacter vinelandii ferredoxin I (AvFdI) and Chromatium vinosum high potential iron protein (CvHiPIP) based on the Protein Dipoles Langevin Dipoles (PDLD) method are reported. The structures of these proteins have been determined by X-ray crystallography; in the case of PaFd the structure has recently been revised due to a change in the sequence close to Cluster II. The large differences between the potentials of the [Fe4S*4Cys4] clusters of PaFd and AvFdI and the potential of the [Fe4S*4Cys4] cluster of CvHiPIP are successfully modeled and originate principally in differences in the configuration of main-chain amide groups near the clusters. The small difference between the potentials of PaFd and AvFdI is also satisfactorily modeled in the case of Cluster I of PaFd. Solvent dipoles close to the cluster in PaFd are an important contributor to its higher potential. The two X-ray structures of PaFd yield similar results for Cluster I of PaFd. In contrast, the results for Cluster II differ substantially; for reasons not yet clear, the recently revised structure leads to results in worse agreement with experiment.

Amino Acid Sequence↗

PubMed1991

Circular dichroism and magnetic circular dichroism of Azotobacter vinelandii ferredoxin I.

Room temperature circular dichroism (CD) and low temperature magnetic circular dichroism (MCD) spectra of air-oxidized and dithionite-reduced Azotobacter vinelandii ferredoxin I (FdI), a [( 4Fe-4S]2+/1+, [3Fe-4S]1+/0) protein, are reported. Unlike the CD of oxidized FdI, the CD of dithionite-reduced FdI exhibits significant pH dependence, consistent with protonation-deprotonation at or near the cluster reduced: the [3Fe-4S] cluster. The MCD of reduced FdI, which originates in the paramagnetic reduced [3Fe-4S]0 cluster, is also pH-dependent. Detailed studies of the field dependence and temperature dependence of the MCD of oxidized and reduced FdI, in the latter case at pH 6.0 and 8.3, are reported. The low-field temperature dependence of the MCD of oxidized FdI, which originates in the paramagnetic oxidized [3Fe-4S]1+ cluster, establishes the absence of a significant population of excited electronic states of this cluster up to 60 K. The low-field temperature dependence of the MCD of reduced FdI establishes that the ground-state manifold of the reduced [3Fe-4S]0 cluster possesses S greater than or equal to 2 at both pH 6.0 and 8.3. Analysis, assuming S = 2 and an axial zero-field splitting Hamiltonian, leads to D = -2.0 and -3.5 cm-1 at pH 6.0 and 8.3, respectively. The site of the (de)protonation affecting the spectroscopic properties of the [3Fe-4S] cluster remains unknown.

Azotobacter↗

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

Mössbauer and EPR studies of Azotobacter vinelandii ferredoxin I.

Azotobacter vinelandii ferredoxin I (FdI) is a small protein that contains one Fe4S4 cluster and one Fe3S4 cluster. Previous studies of FdI have shown that the redox potential of the Fe3S4 cluster and the MCD and CD spectra of the reduced Fe3S4 cluster are pH-dependent. Using Mössbauer and EPR spectroscopy, we have studied FdI in different oxidation states and at different pH values. Here, we report the spin Hamiltonian parameters of the oxidized (S = 1/2) Fe3S4 cluster at pH 7.4 and the reduced (S = 2) Fe3S4 cluster at pH 6.0 and 8.5. The pH dependence observed by MCD is also evident in the Mössbauer spectra which show a change of the magnetic hyperfine tensor for one Fe site of the valence-delocalized pair. The Fe4S4 cluster is ligated by cysteines 20, 39, 42, and 45, but not by the adjacent cysteine 24. Treatment of FdI with 3 equiv of ferricyanide alters the Fe4S4 cluster, yielding a new species, [Fe4S4]'. The S = 1/2 EPR signal of [Fe4S4]' has previously been attributed to the formation of a cysteine disulfide radical from Cys24 and cluster sulfide. Here we show that the EPR signal is broadened by 57Fe, indicating that the electronic spin is significantly coupled to the cluster iron. Consistent with this, substantial magnetic hyperfine interactions are observed by Mössbauer spectroscopy. In addition, the average isomer shift of the four Fe sites is smaller for [Fe4S4]' than for [Fe4S4]2+, indicating that the oxidation is iron-based to at least some extent. Incubation of FdI with excess ferricyanide destroys the Fe4S4 cluster but leaves the Fe3S4 cluster intact. Our studies of (3Fe)FdI show that the S = 1/2 spin of the Fe3S4 cluster interacts with another paramagnet, presumably a radical generated at the site left vacant by the removal of the Fe4S4 cluster.

Azotobacter vinelandii↗

PubMed1984

Reactions with the oxidized iron protein of Azotobacter vinelandii nitrogenase: formation of a 2Fe center.

The Fe-S center of oxidized Fe protein from Azotobacter vinelandii nitrogenase is decomposed by alpha,alpha'-dipyridyl in a biphasic process. In the presence of MgATP, 2 Fe are immediately removed by chelation while the additional irons are removed only after several hours. A slower biphasic Fe release also was observed in the presence of chelator alone. MgADP prevented the Fe release by chelator. An intermediate in the reaction was isolated containing 2 Fe. The visible spectrum of the intermediate was similar to that of 2Fe-2S ferredoxins (epsilon max at 325, 416, and 460 nm of 16.1, 11.3, and 9.0 mM-1 cm-1). The 2Fe form was electron paramagnetic resonance (EPR) silent until partially reduced with sodium dithionite. The EPR spectral properties were similar to 2Fe-2S ferredoxins; namely, the Fe center had resonances at g = 2.00, 1.94, and 1.92 which were detectable, essentially unbroadened at 70 K. The results suggest that in the oxidized (2+) state Fe protein can undergo a 4Fe to 2Fe conversion.

2,2'-Dipyridyl↗