The [2Fe-2S] protein I (Shetna protein I) from Azotobacter vinelandii is homologous to the [2Fe-2S] ferredoxin from Clostridium pasteurianum

 The [2Fe-2S] protein from Azotobacter vinelandii that was previously known as iron-sulfur protein I, or Shethna protein I, has been shown to be encoded by a gene belonging to the major nif gene cluster. Overexpression of this gene in Escherichia coli yielded a dimeric protein of which each subunit comprises 106 residues and contains one [2Fe-2S] cluster. The sequence of this protein is very similar to that of the [2Fe-2S] ferredoxin from Clostridium pasteurianum (2FeCpFd), and the four cysteine ligands of the [2Fe-2S] cluster occur in the same positions. The A. vinelandii protein differs from the C. pasteurianum one by the absence of the N-terminal methionine, the presence of a five-residue C-terminal extension, and a lesser number of acidic and polar residues. The UV-visible absorption and EPR spectra, as well as the redox potentials of the two proteins, are nearly identical. These data show that the A. vinelandii FeS protein I, which is therefore proposed to be designated 2FeAvFdI, is the counterpart of the [2Fe-2S] ferredoxin from C. pasteurianum. The occurrence of the 2FeAvFdI-encoding gene in the nif gene cluster, together with the previous demonstration of a specific interaction between the 2FeCpFd and the nitrogenase MoFe protein, suggest that both proteins might be involved in nitrogen fixation, with possibly similar roles. Received: 21 December 1998 / Accepted: 1 March 1999     

Resonance Raman spectroscopy of Azotobacter vinelandii ferredoxin I. Vibrational features of the [3Fe-3S] cluster

Low temperature resonance Raman spectra have been obtained for Clostridium pasteurianum and Bacillus stearothermophilus ferredoxins. Several heretofore undetected fundamental bands have been observed and these data have been used to discriminate the vibrational contribution of the [3Fe-3S] cluster to the spectrum of Azotobacter vinelandii ferredoxin I. The vibrational features of the [3Fe-3S] core distinguish it from other 3-iron clusters and imply structural differences among this class of iron-sulfur clusters.     

Crystal structures of ferricyanide-oxidized [Fe-S] clusters in Azotobacter vinelandii ferredoxin I

Crystals of Azotobacter vinelandii ferredoxin I (FdI) have been soaked in solutions containing K₃Fe(CN)₆ in order to study the oxidation of the [3Fe-4S] and [4Fe-4S] clusters in the protein. Ferricyanide treatment results in partial loss of Fe and S from each cluster accompanied by alteration of Fe-S bonds. The effects of oxidation can be quantitated by crystallographic refinement when each [Fe-S] cluster is modeled as having a single, average structure with non-standard geometry. The oxidized clusters refined at 2.1-Å resolution display statistically significant deviations from geometric ideality. If interpreted in terms of atomic shifts these deviations indicate that each cluster first loses an inorganic S atom. In each case an Fe atom bonded to this S separates from the remaining atoms of the cluster such that the [3Fe-4S] and [4Fe-4S] clusters partially decompose into a single Fe plus 2Fe and 3Fe fragments. The extent of structural changes observed are essentially the same in crystals soaked at 3?:?1, 9?:?1 and 30?:?1 mole ratio of K₃ Fe(CN)₆?:?FdI, suggesting that the crystal lattice permits limited oxidation reactions to occur at a low mole ratio but restricts conformational changes from occurring that may be required for more extensive oxidative reactions at higher mole ratio. The results are relevant to understanding the transformations which may take place when [Fe-S] proteins are deliberately oxidized with ferricyanide.     

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I. Changes in [4Fe-4S] cluster reduction potential and reactivity.

We have used site-directed mutagenesis to obtain two variants of Azotobacter vinelandii ferredoxin I (AvFdI), whose x-ray structures are now available. In the C20A protein, a ligand to the [4Fe-4S] cluster was removed whereas in the C24A mutant a free cysteine next to that cluster was removed. Like native FdI, both mutants contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. The structure of C24A is very similar to that of native FdI, while the structure of C20A is rearranged in the region of the [4Fe-4S] cluster to allow it to use the free Cys-24 as a replacement ligand. Here we compare the properties of the native, C20A, and C24A proteins. Although all three proteins are O2 stable in vitro, the C20A protein is much less stable toward proteolysis than the other two in vivo. Spectroscopic results show that all three proteins exhibit the same general redox behavior during O2-oxidation and dithionite reduction. Electrochemical data show that the [3Fe-4S] clusters in all three proteins have the same pH-dependent reduction potentials (-425 mV versus SHE, pH 7.8), whereas the [4Fe-4S] cluster potentials vary over a approximately 150 mV range from -600 mV (C24A) to -647 mV (native) to -746 mV (C20A). Despite this variation in potential both the C20A and C24A proteins appear to be functional in vivo. Native FdI reacts with three equivalents of Fe(CN)3-(6) to form a paramagnetic species previously proposed to be a cysteinyl-disulfide radical. Neither the C20A nor the C24A variant undergoes this reaction, strongly suggesting that it involves the free Cys-24.     

Alteration of the reduction potential of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I

The [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E(0')) relative to other structurally similar ferredoxins. Previous attempts to raise that E(0') by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter the E(0') by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E(0') measurements show that all had altered E(0') relative to native FdI. For P21G FdI and I40Q FdI, the E(0') increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control of E(0'). Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E(0') while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a +168 mV change in E(0') while a -33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 A, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E(0'). Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change in E(0').     

Nfu2: a scaffold protein required for [4Fe-4S] and ferredoxin iron-sulphur cluster assembly in Arabidopsis chloroplasts

Nfu proteins are candidates to act as scaffold protein in vivo for iron-sulphur cluster biogenesis. In this work, Nfu2 protein function in the chloroplast was investigated in vivo using T-DNA insertion lines disrupted in AtNfu2 gene. Both alleles characterized presented the same dwarf phenotype due to photosynthetic and metabolic limitations. Nfu2 cDNA expression in nfu2.1 mutant rescued this phenotype. Photosynthesis study of these mutants revealed an altered photosystem I (PSI) activity together with a decrease in PSI amount confirmed by immunodetection experiments, and leading to an over reduction of the plastoquinol pool. Decrease of plastid 4Fe-4S sulphite reductase activity correlates with PSI amount decrease and supports an alteration of 4Fe-4S cluster biogenesis in nfu2 chloroplasts. The decrease of electron flow from the PSI is combined with a decrease in ferredoxin amount in nfu2 mutants. Our results are therefore in favour of a requirement of Nfu2 protein for 4Fe-4S and 2Fe-2S ferredoxin cluster assembly, conferring to this protein an important function for plant growth and photosynthesis as demonstrated by nfu2 mutant phenotype. As glutamate synthase and Rieske Fe-S proteins are not affected in nfu2 mutants, these data indicate that different pathways are involved in Fe-S biogenesis in Arabidopsis chloroplasts.     

Azotobacter vinelandii ferredoxin I: a sequence and structure comparison approach to alteration of [4Fe-4S]2+/+ reduction potential.

The reduction potential (E(0)') of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is approximately 200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences in E(0)' do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S](2+/+) clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized Chromatium vinosum ferredoxin) was shown to have a [4Fe-4S](2+/+) cluster with a very negative E(0)' similar to that of AvFdI. The observation that the sequences and structures surrounding the very negative E(0)' clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S](2+/+) cluster of AvFdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higher E(0)' P. asaccharolyticus ferredoxin all led to increases in E(0)' for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH-S bond between the introduced Ser side chain and the Sgamma atom of Cys ligand 42 and an accompanying movement of water.     

Purification and biochemical characterization of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough).

A novel iron-sulfur protein has been isolated from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough). It is a stable monomeric protein, which has a molecular mass of 52 kDa, as determined by sedimentation-equilibrium centrifugation. Analysis of the metal and acid-labile sulfur content of the protein revealed the presence of 6.3 +/- 0.4 Fe/polypeptide and 6.2 +/- 0.7 S2-/polypeptide. Non-iron transition metals, heme, flavin and selenium were absent. Combining these data with the observation of a very anisotropic S = 1/2 [6Fe-6S]3+ prismane-like EPR signal in the dithionite-reduced protein, we believe that we have encountered the first example of a prismane-cluster-containing protein. The prismane protein has a slightly acidic amino acid composition and isoelectric point (pI = 4.9). The ultraviolet/visible spectrum is relatively featureless (epsilon 280 = 81 mM-1.cm-1, epsilon 400 = 25 mM-1.cm-1, epsilon 400,red = 14 mM-1.cm-1). The shape of the protein is approximately globular (S20.w = 4.18 S). The N-terminal amino acid sequence is MFS/CFQS/C QETAKNTG. Polyclonal antibodies against the protein were raised. Cytoplasmic localization was inferred from subcellular fractionation studies. Cross-reactivity of antibodies against this protein indicated the occurrence of a similar protein in D. vulgaris (Monticello) and Desulfovibrio desulfuricans (ATCC 27774). We have not yet identified a physiological function for the prismane protein despite trials for some relevant enzyme activities.     

Structure of a thioredoxin-like [2Fe-2S] ferredoxin from Aquifex aeolicus

The 2.3 A resolution crystal structure of a [2Fe-2S] cluster containing ferredoxin from Aquifex aeolicus reveals a thioredoxin-like fold that is novel among iron-sulfur proteins. The [2Fe-2S] cluster is located near the surface of the protein, at a site corresponding to that of the active-site disulfide bridge in thioredoxin. The four cysteine ligands are located near the ends of two surface loops. Two of these ligands can be substituted by non-native cysteine residues introduced throughout a stretch of the polypeptide chain that forms a protruding loop extending away from the cluster. The presence of homologs of this ferredoxin as components of more complex anaerobic and aerobic electron transfer systems indicates that this is a versatile fold for biological redox processes.     

Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli.

Escherichia coli contains a soluble, [2Fe-2S] ferredoxin of unknown function (Knoell, H.-E., and Knappe, J. (1974) Eur. J. Biochem. 50, 245-252). Using antiserum to the purified protein to screen E. coli genomic expression libraries, we have cloned a gene (designated fdx) encoding this protein. The DNA sequence of the gene predicts a polypeptide of 110 residues after removal of the initiator methionine (polypeptide M(r) = 12,186, holoprotein M(r) = 12,358). The deduced amino acid sequence is strikingly similar to those of the ferredoxins found in animal mitochondria which function with cytochrome P450 enzymes and to the ferredoxin from Pseudomonas putida which functions with P450cam. The overall sequence identity is approximately 36% when compared with human mitochondrial and P. putida ferredoxins, and the identities include 4 cysteine residues proposed to coordinate the iron cluster. The protein was overproduced approximately 500-fold using an expression plasmid, and the holoprotein was assembled and accumulated in amounts exceeding 30% of the total cell protein. The overexpressed ferredoxin exhibits absorption, circular dichroism, and electron paramagnetic resonance spectra closely resembling those of the animal ferredoxins and P. putida ferredoxin.     

Structure of C42D Azotobacter vinelandii FdI. A Cys-X-X-Asp-X-X-Cys motif ligates an air-stable [4Fe-4S]2+/+ cluster

All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys motifs contain [4Fe-4S](2+/+) clusters that can be easily and reversibly converted to [3Fe-4S](+/0) clusters. In contrast, ferredoxins with unmodified Cys-X-X-Cys-X-X-Cys motifs assemble [4Fe-4S](2+/+) clusters that cannot be easily interconverted with [3Fe-4S](+/0) clusters. In this study we changed the central cysteine of the Cys(39)-X-X-Cys(42)-X-X-Cys(45) of Azotobacter vinelandii FdI, which coordinates its [4Fe-4S](2+/+) cluster, into an aspartate. UV-visible, EPR, and CD spectroscopies, metal analysis, and x-ray crystallography show that, like native FdI, aerobically purified C42D FdI is a seven-iron protein retaining its [4Fe-4S](2+/+) cluster with monodentate aspartate ligation to one iron. Unlike known clusters of this type the reduced [4Fe-4S](+) cluster of C42D FdI exhibits only an S = 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.     

Structural stabilization of [2Fe-2S] ferredoxin from Halobacterium salinarum

The ferredoxin of the extreme haloarchaeon Halobacterium salinarum requires high (>2 M) concentration of salt for its stability. We have used a variety of spectroscopic probes for identifying the structural elements which necessitate the presence of high salt for its stability. Titration of either the fluorescence intensity of the tryptophan residues or the circular dichroism (CD) at 217 nm with salt has identified a structural form at low (<0.1 M) concentration of salt. This structural form (L) exhibits increased solvent exposure of W side chain(s) and decreased level of secondary structure compared to the native (N) protein at high concentrations of salt. The L-form, however, contains significantly higher levels of both secondary and tertiary structures compared to the form (U) found in highly denaturing conditions such as 8 M urea. The structural integrity of the L-form was highly pH dependent while that of N- or U-form was not. The pH dependence of either fluorescence intensity or CD of the L-form showed the presence of two apparent pK values: approximately 5 and approximately 10. The structural integrity of the L-form at low (<5) pH was very similar to that of the N-form. However, titration with denaturants showed that the low pH L-form is significantly less stable than the N-form. The increased destabilization of the L-form with the increase in pH was interpreted to be due to mutual Coulombic repulsion of carboxylate side chains (pK approximately 6) and due to the disruption of salt bridge(s) between ionized carboxylates and protonated amino groups (pK approximately 10). Estimation of solvent accessibility of W residues by fluorescence quenching, and measurement of decay kinetics of fluorescence intensity and anisotropy strongly support the above model. Polylysine interacted stoichiometrically with the L-form of ferredoxin resulting in nativelike structure. In conclusion, our studies show that high concentration of salt stabilizes the haloarchaeal ferredoxin in two ways: (i) neutralization of Coulombic repulsion among carboxyl groups of the acidic residues, and (ii) salting out of hydrophobic residues leading to their burial and stronger interaction.     

Purification and characterization of cytochrome O from Azotobacter vinelandii.

The membrane-bound cytochrome O has been solubilized from the Azotobacter vinelandii electron transport particle and further purified by use of conventional chromatographic procedures. The spectral characteristics as well as the other properties noted for purified cytochrome O are reported herein.     

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.     

Purification and characterization of cytochrome o from Azotobacter vinelandii

The membrane-bound cytochrome o has been solubilized from the Azotobacter vinelandii electron transport particle and further purified by use of conventional chromatographic procedures. The spectral characteristics as well as the other properties noted for purified cytochrome o are reported herein.     

Amino acid sequence of [2Fe-2S] ferredoxin from Clostridium pasteurianum

The complete amino acid sequence of the [2Fe-2S] ferredoxin from the saccharolytic anaerobe Clostridium pasteurianum has been determined by automated Edman degradation of the whole protein and of peptides obtained by tryptic and by staphylococcal protease digestion. The polypeptide chain consists of 102 amino acids, including 5 cysteine residues in positions 11, 14, 24, 56, and 60. The sequence has been analyzed for hydrophilicity and for secondary structure predictions. In its native state the protein is a dimer, each subunit containing one [2Fe-2S] cluster, and it has a molecular weight of 23,174, including the four iron and inorganic sulfur atoms. The extinction coefficient of the native protein is 19,400 M-1 cm-1 at 463 nm. The positions of the cysteine residues, four of which are most probably the ligands of the [2Fe-2S] cluster, on the polypeptide chain of this protein are very different from those found in other [2Fe-2S] proteins, and in other ferredoxins in general. In addition, whole sequence comparisons of the [2Fe-2S] ferredoxin from C. pasteurianum with a number of other ferredoxins did not reveal any significant homologies. The likely occurrence of several phylogenetically unrelated ferredoxin families is discussed in the light of these observations.     

Mechanisms of redox-coupled proton transfer in proteins: role of the proximal proline in reactions of the [3Fe-4S] cluster in Azotobacter vinelandii ferredoxin I

The 7Fe ferredoxin from Azotobacter vinelandii (AvFdI) contains a [3Fe-4S](+/0) cluster that binds a single proton in its reduced level. Although the cluster is buried, and therefore inaccessible to solvent, proton transfer from solvent to the cluster is fast. The kinetics and energetics of the coupled electron-proton transfer reaction at the cluster have been analyzed in detail by protein-film voltammetry, to reveal that proton transfer is mediated by the mobile carboxylate of an adjacent surface residue, aspartate-15, the pK of which is sensitive to the charge on the cluster. This paper examines the role of a nearby proline residue, proline-50, in proton transfer and its coupling to electron transfer. In the P50A and P50G mutants, a water molecule has entered the cluster binding region; it is hydrogen bonded to the backbone amide of residue-50 and to the Asp-15 carboxylate, and it is approximately 4 A from the closest sulfur atom of the cluster. Despite the water molecule linking the cluster more directly to the solvent, proton transfer is not accelerated. A detailed analysis reveals that Asp-15 remains a central part of the mechanism. However, the electrostatic coupling between cluster and carboxylate is almost completely quenched, so that cluster reduction no longer induces such a favorable shift in the carboxylate pK, and protonation of the base no longer induces a significant shift in the pK of the cluster. The electrostatic coupling is crucial for maintaining the efficiency of proton transfer both to and from the cluster, over a range of pH values.     

In vitro activation of apo-aconitase using a [4Fe-4S] cluster-loaded form of the IscU [Fe-S] cluster scaffolding protein

Genetic experiments have established that IscU is involved in maturation of [Fe-S] proteins that require either [2Fe-2S] or [4Fe-4S] clusters for their biological activities. Biochemical studies have also shown that one [2Fe-2S] cluster can be assembled in vitro within each subunit of the IscU homodimer and that these clusters can be reductively coupled to form a single [4Fe-4S] cluster. In the present work, it is shown that the [4Fe-4S] cluster-loaded form of A. vinelandii IscU, but not the [2Fe-2S] cluster-loaded form, can be used for intact cluster transfer to an apo form of A. vinelandii aconitase A, a member of the monomeric dehydratase family of proteins that requires a [4Fe-4S] cluster for enzymatic activity. The rate of [4Fe-4S] cluster transfer from IscU to apo-aconitase A was not affected by the presence of the HscA/HscB co-chaperone system and MgATP. However, an altered form of a [4Fe-4S] cluster-containing IscU, having the highly conserved aspartate-39 residue substituted with alanine, is an effective inhibitor of wild-type [4Fe-4S] cluster-loaded IscU-directed activation of apo-aconitase A. In contrast, neither the clusterless form of IscU nor the [2Fe-2S] cluster-loaded form of IscU is an effective inhibitor of IscU-directed apo-aconitase A activation. These results are interpreted to indicate that the [2Fe-2S] and [4Fe-4S] cluster-loaded forms of IscU adopt different conformations that provide specificity with respect to the maturation of [2Fe-2S] and [4Fe-4S] centers in proteins.     

Exceptional stability of a [2Fe-2S] ferredoxin from hyperthermophilic bacterium Aquifex aeolicus

Aquifex aeolicus is the only hyperthermophile that is known to contain a plant- and mammalian-type [2Fe-2S] ferredoxin (Aae Fd1). This unique protein contains two cysteines, in addition to the four that act as ligands of the [2Fe-2S] cluster, which form a disulfide bridge. We have investigated the stability of Aae Fd1 with (wild-type) and without (C87A variant) the disulfide bond, with respect to pH, thermal and chemical perturbation, and compared the results to those for the mesophilic [2Fe-2S] ferredoxin from spinach. Unfolding reactions of all three proteins are irreversible due to cluster decomposition in the unfolded state. Wild-type and C87A Aae Fd1 proteins are extremely stable: unfolding at 20 degrees C requires high concentrations of the chemical denaturant and long incubation times. Moreover, their thermal-unfolding midpoints are 40-50 degrees higher than that for spinach ferredoxin (pH 7). The stability of the Aae Fd1 protein is significantly lower at pH 2.5 than pH 7 and 10, suggesting that ionic interactions play a role in structural integrity. Interestingly, the iron-sulfur cluster in C87A Aae Fd1 rearranges into a transient species with absorption bands at 520 and 610 nm, presumably a linear three-iron cluster, in the high-pH unfolded state.