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PubMed1998

Amino acid sequence of ferredoxin from Physalis alkekengi var. francheti.

The complete amino acid sequence of [2Fe-2S] ferredoxin from Physalis alkekengi var. francheti has been determined by automated Edman degradation of the entire Cm-protein and of the peptides obtained by trypsin and endoproteinase Asp-N digestions. This ferredoxin exhibited ten, ten, and nine differences respectively in the amino acid sequence, when compared with the ferredoxins of Datura stramonium, D. metel, and D. arborea, but 21-28 differences for other angiosperms, and 34-37 differences for fern and horsetails. These results are in harmony with the taxonomic position for these plants.

Amino Acid Sequence↗
PubMed1979

Oxidation-reduction potentials of the hemes in cytochrome C3 from Desulfovibrio gigas in the presence and absence of ferredoxin by EPR spectroscopy.

1. Ferricytochrome c3 from D. gigas exhibits two low-spin ferric heme EPR resonances with gz-values at 2.959 and 2.853. Ferrocytochrome c3 is diamagnetic based on the absence of any EPR signals. 2. EPR potentiometric titrations result in the resolution of the two low-spin ferric heme resonances into two additional heme components representing in total the four hemes of the cytochrome, with EM values of -235 mV and -315 mV at heme resonance I and EM values of -235 mV and -306 mV at heme resonance II. 3. EPR spectroscopy has detected a significant diminution of intensity (approx. 60 p. 100) in the gx amplitude of ferricytochrome c3 in the presence of D. gigas ferredoxin II. The presence of ferredoxin II also causes a more negative shift in the EM of the second components of the signals at heme resonances I and II of cytochrome C3. Both observations suggest that an interaction has occurred between cytochrome C3 and ferredoxin II. 4. The results presented suggest that the heme ligand environment of ferricytochrome c3 from D. gigas is less perturbed and/or less asymmetric than environment for ferricytochrome c3 from D. vulgaris whose EPR behavior indicates the non-equivalence of all four hemes.

Cytochrome c Group↗
PubMed2000

A cluster exposed: structure of the Rieske ferredoxin from biphenyl dioxygenase and the redox properties of Rieske Fe-S proteins.

BACKGROUND: Ring-hydroxylating dioxygenases are multicomponent systems that initiate biodegradation of aromatic compounds. Many dioxygenase systems include Rieske-type ferredoxins with amino acid sequences and redox properties remarkably different from the Rieske proteins of proton-translocating respiratory and photosynthetic complexes. In the latter, the [Fe2S2] clusters lie near the protein surface, operate at potentials above +300 mV at pH 7, and express pH- and ionic strength-dependent redox behavior. The reduction potentials of the dioxygenase ferredoxins are approximately 150 mV and are pH-independent. These distinctions were predicted to arise from differences in the exposure of the cluster and/or interactions of the histidine ligands. RESULTS: The crystal structure of BphF, the Rieske-type ferredoxin associated with biphenyl dioxygenase, was determined by multiwavelength anomalous diffraction and refined at 1.6 A resolution. The structure of BphF was compared with other Rieske proteins at several levels. BphF has the same two-domain fold as other Rieske proteins, but it lacks all insertions that give the others unique structural features. The BphF Fe-S cluster and its histidine ligands are exposed. However, the cluster has a significantly different environment in that five fewer polar groups interact strongly with the cluster sulfide or the cysteinyl ligands. CONCLUSIONS: BphF has structural features consistent with a minimal and perhaps archetypical Rieske protein. Variations in redox potentials among Rieske clusters appear to be largely the result of local electrostatic interactions with protein partial charges. Moreover, it appears that the redox-linked ionizations of the Rieske proteins from proton-translocating complexes are also promoted by these electrostatic interactions.

Amino Acid Sequence↗
PubMed2002

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.

Bacteria↗
PubMed2002

A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus.

A [3Fe-4S](1+/0) ferredoxin was isolated from the thermohalophilic and strict aerobic bacterium Rhodothermus marinus. It is a small protein, with an apparent molecular mass of 9 kDa. Its N-terminal amino acid sequence reveals the capability of binding two tetranuclear clusters. However, upon purification, it contains a single [3Fe-4S](1+/0), with an unusually low reduction potential of -650 mV, determined by cyclic voltammetry at pH 7.6. [1H]NMR spectroscopy shows that the protein contains a single, homogeneous, trinuclear centre. When purified under anaerobic conditions, the EPR [3Fe-4S](1+/0) centre signal is also observed. However, it can now be reduced by dithionite and a new signal attributed to a [4Fe-4S](2+/1+) cluster develops. This can also be observed upon reconstitution of the prosthetic groups. The function of this ferredoxin in R. marinus is still unknown but it is very sensitive to oxygen, an unexpected characteristic for a protein from an aerobic organism. The thermodynamic stability of the R. marinus ferredoxin was also investigated and was shown to be high. Thermal and chemical unfolding reactions appear as single, cooperative transitions. The midpoint (T(m)) for thermally induced unfolding is 102+/-2 degrees C (pH 7). Unfolding induced by the chemical denaturant guanidine hydrochloride (GuHCl) shows a transition midpoint at 5.0 M GuHCl (pH 7.0, 20 degrees C). The iron-sulfur cluster degrades upon polypeptide unfolding, resulting in an irreversible denaturation process.

Amino Acid Sequence↗
PubMed1995

1H NMR investigation of the paramagnetic cluster environment in Pyrococcus furiosus three-iron ferredoxin: sequence-specific assignment of ligated cysteines independent of tertiary structure.

One- and two-dimensional 1H NMR data tailored to detect paramagnetically relaxed protons near the S = 1/2, three-iron-sulfur cluster of the ferredoxin from the hyperthermophile Pyrococcus furiosus are analyzed to sequence specifically assign the hyperfine shifted ligated cysteine signals, to determine the nature of the secondary structural elements on which these cysteines reside, and to define the tertiary contacts of the cluster with the remainder of the previously characterized secondary structure remote from the cluster [Teng, Q., Zhou, Z.-H., Busse, S.C., Howard, J.B., Adams, M. W. W., & La Mar, G. N. (1994) Biochemistry 33, 6316-6326]. Inspection of the geometry of the cluster ligating cysteines in the six structurally characterized cubane ferredoxin (Fd) clusters reveals a pattern of distances from the cluster iron(s) that indicate that each Cys will exhibit one backbone proton that will allow the detection of dipolar connectivities to the backbone of adjacent residues. It is expected that the first and last of the Cys in the cluster consensus binding sequence will exhibit weakly relaxed peptide NH and strongly relaxed C alpha H signals, while the two central Cys in that sequence will exhibit strongly relaxed peptide NH but weakly relaxed C alpha H peaks. These dipolar contacts are clearly observed for the three ligated Cys in 3Fe P. furiosus Fd, providing the first sequence specific assignment of ligated cysteines which do not explicitly require knowledge of the tertiary structure of the protein. This approach is proposed to have very general application to cubane ferredoxins. A combination of steady-state NOEs and short mixing time NOESY experiments demonstrate that Cys17 is on a short helix through Leu20 and that Cys56 likely initiates a type I turn, as observed in the crystal structure of the 3Fe Fd for Desulfovibrio gigas [Kissinger, C. R., Sieker, L. C., Adman, E. T., & Jensen, L. H. (1991) J. Mol. Biol. 219, 693-715]. The observed relaxation rates of resolved or partially resolved signals are shown to correlate with their proximity to the various iron in the cluster, as determined for the homologous residues in D. gigas Fd, providing additional qualitative information on tertiary contacts of the cluster.

Amino Acid Sequence↗
PubMed1993

Effect of replacing conserved proline residues on the EPR and NMR properties of Clostridium pasteurianum 2[4Fe-4S] ferredoxin.

Most of [4Fe-4S] proteins bind their metallic center by four cysteine residues, three clustered in a single stretch of seven amino acids and a remote fourth generally followed by a proline residue. Two such prolines in Clostridium pasteurianum 2[4Fe-4S] ferredoxin have been substituted by different amino acids and the resulting molecular variants studied with EPR and NMR spectroscopies. The isolated EPR contributions of the [4Fe-4S]+ clusters do not change much in all variants. The exact positions or the number of features composing the fully reduced EPR spectra built by the two interacting [4Fe-4S]+ S = 1/2 systems vary slightly but, in none of the proteins in which either proline 19 or 48 were substituted, do they indicate a major difference either in the folding of the ferredoxin or in the electronic structure of its clusters. A subset of paramagnetically shifted NMR signals is significantly affected by these replacements at both redox levels. The corresponding protons belong to two cysteines liganding the cluster close to the substitution. These data, combined with the presently available three-dimensional information, form the basis for partial assignments of the most shifted resonances in the NMR spectra of such proteins. The positions of intermediate lines in the NMR spectra of semireduced ferredoxins depend on the difference between the redox potentials of the two clusters; this difference is sensitive to the substitutions of either conserved proline residue by lysine.

Amino Acid Sequence↗
PubMed1991

A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties.

The entire polypeptide of Clostridium pasteurianum ferredoxin (Fd) with a site-substituted tyrosine-2----histidine-2 was synthesized using standard t-Boc procedures, reconstituted to the 2[4Fe-4S] holoprotein, and compared to synthetic C. pasteurianum and native Fds. Although histidine-2 is commonly found in thermostable clostridial Fds, the histidine-2 substitution into synthetic C. pasteurianum Fd did not significantly increase its thermostability. The reduction potential of synthetic histidine-2 Fd was -343 and -394 mV at pH 6.4 and 8.7, respectively, versus standard hydrogen electrode. Similarly, Clostridium thermosaccharolyticum Fd which naturally contains histidine-2 was previously determined to have a pH-dependent reduction potential [Smith, E.T., & Feinberg, B.A. (1990) J. Biol. Chem. 265, 14371-14376]. An electrostatic model was used to calculate the observed change in reduction potential with pH for a homologous ferredoxin with a known X-ray crystal structure containing a hypothetical histidine-2. In contrast, the reduction potential of both native C. pasteurianum Fd and synthetic Fd with the C. pasteurianum sequence was -400 mV versus standard hydrogen electrode and was pH-independent [Smith, E.T., Feinberg, B.A., Richards, J.H., & Tomich, J.M. (1991) J. Am. Chem. Soc. 113, 688-689]. On the basis of the above results, we conclude that the observed pH-dependent reduction potential for both synthetic and native ferredoxins that contain histidine-2 is attributable to the electrostatic interaction between histidine-2 and iron-sulfur cluster II which is approximately 6 A away.

Amino Acid Sequence↗
PubMed2001

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.

Circular Dichroism↗
PubMed1992

Proton NMR investigation of the oxidized three-iron clusters in the ferredoxins from the hyperthermophilic archae Pyrococcus furiosus and Thermococcus litoralis.

The 3Fe forms of ferredoxins (Fd) from the hyperthermophilic archaebacteria Pyrococcus furiosus (Pf) and Thermococcus litoralis (Tl) have been investigated by 1H NMR. A combination of one-dimensional nuclear Overhauser and two-dimensional NOESY and bond correlation spectroscopy provides the assignment of the aromatic residues, one conserved valine, and the location of the signals for each of the three cysteines coordinated to the clusters. Dipolar contacts between the Trp 2 and Tyr 46 in Pf Fd and from an invariant phenylalanine to an invariant valine and a cluster cysteine in both Fd confirm a folding pattern for these proteins that is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibro gigas. The sequence-specific assignment of the buried cysteine near the invariant phenylalanine has been made. The temperature dependence of the contact-shifted cysteinyl residues reveals a distinct 2:1 asymmetry in the magnetic coupling among the three high-spin ferric ions, in that one cysteine exhibits Curie behavior, while the other two cysteines display anti-Curie behavior. These magnetic properties are rationalized qualitatively on the basis of a magnetic coupling scheme where two iron couple to yield an intermediate spin of 2 which couples to the remaining S = 5/2 iron to yield the total cluster spin 1/2. This magnetic asymmetry appears to be a characteristic feature of oxidized 3 Fe clusters. Pf Fd also undergoes a dynamic equilibrium between two alternate forms that differ slightly in the environment of two of the coordinated cysteines. Analysis of the pattern of the contact shifts for the three cysteines in the two ferredoxins suggests that the cysteine coordinated to the unique iron does not have the same sequence origin.

Amino Acid Sequence↗
PubMed1991

Crystallization and structure determination to 2.5-A resolution of the oxidized [2Fe-2S] ferredoxin isolated from Anabaena 7120.

The molecular structure of the oxidized form of the [2Fe-2S] ferredoxin isolated from the cyanobacterium Anabaena species strain PCC 7120 has been determined by X-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R factor of 18.7%. Crystals used in this investigation belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 37.42 A, b = 38.12 A, and c = 147.12 A and two molecules in the asymmetric unit. The three-dimensional structure of this ferredoxin was solved by a method that combined X-ray data from one isomorphous heavy-atom derivative with noncrystallographic symmetry averaging and solvent flattening. As in other plant-type [2Fe-2S] ferredoxins, the iron-sulfur cluster is located toward the outer edge of the molecule, and the irons are tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteine residues. The main secondary structural elements include four strands of beta-pleated sheet and three alpha-helical regions.

Crystallization↗
PubMed1994

Mutated forms of the [2Fe-2S] ferredoxin from Clostridium pasteurianum with noncysteinyl ligands to the iron-sulfur cluster.

The [2Fe-2S] ferredoxin from Clostridium pasteurianum is unique among ferredoxins, both by its sequence and by the distribution of its cysteine residues (in positions 11, 14, 24, 56, 60). Thus, no homologous sequences are available to infer, by comparison, the identity of the ligands of the iron-sulfur cluster. Therefore, in order to obtain information on the latter point, a combination of site-directed mutagenesis and UV-vis, EPR, and resonance Raman spectroscopy has been implemented. All of the cysteine residues have individually been replaced by serine and two of them by alanine. Cysteine 14 could be replaced by either serine or alanine without any modification of the spectroscopic properties of the protein and was therefore dismissed as a ligand of the [2Fe-2S] cluster. The C56S, and C60S-mutated proteins were both found to display UV-vis, EPR, and resonance Raman spectra consistent with serine-coordinated [2Fe-2S] clusters. The C11S-mutated protein was considerably less stable than the wild type ferredoxin. This observation, together with the hypsochromic shifts of UV-visible absorption features upon cysteine 11-->serine mutation, suggested cysteine 11 to be a ligand of the [2Fe-2S] cluster. Cysteine 24 could be replaced by either serine or alanine without decreasing the stability of the protein and without dramatically changing its spectroscopic properties. Thus, either cysteine 24 is not a ligand of the [2Fe-2S] cluster or it is replaced by another ligand in the C24A mutated protein. A [2Fe-2S] cluster was also assembled in the C14A/C24A doubly mutated protein, i.e., in a polypeptide chain containing only three cysteine residues.2+ off

Alanine↗
PubMed1975

Modification of the automated sequence determination as applied to the sequence determination of the Spirulina maxima ferredoxin.

The amino acid sequence of the Spirulina maxima ferredoxin was shown to be: H2N-Ala-Thr-Tyr-Lys-Val-Thr-Leu-Ile-Ser-Glu-Ala-Glu-Gly-Ile-Asn-Glu-Thr-Ile-Asp-Cys-Asp-Asp-Asp-Thr-Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Lys-Ile-Thr-Ser-Gly-Ser-Ile-Asp-Gln-Ser-Asp-Gln-Ser-Phe-Leu-Asp-Asp-asp-Gln-Ile-Gln-Ala-Gly-Tyr-Val-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Thr-Ile-Gln-Thr-His-Gln-Glu-Glu-Gly-Leu-Tyr-COOH. The S. maxima ferredoxin is the first procaryote ferredoxin of the plant-algal type to be reported. A modification of the automated sequence determination of a peptide, which was extracted by the organic solvents used to remove excess reagents and the amino acid thiazoline, was utilized to complete the sequence of a 36 residue tryptic peptide.

Amino Acid Sequence↗
PubMed2002

NMR structure of the [2Fe-2S] ferredoxin domain from soluble methane monooxygenase reductase and interaction with its hydroxylase.

The soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) is a multicomponent enzyme system required for the conversion of methane to methanol. It comprises a hydroxylase, a regulatory protein, and a reductase. The reductase contains two domains: an NADH-binding and FAD-containing flavin domain and a ferredoxin (Fd) domain carrying a [2Fe-2S] cofactor. Here, we report the solution structure of the reduced form of the 98-amino acid Fd domain (Blazyk, J. L., and Lippard, S. J. Unpublished results) determined by nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics calculations. The structure consists of six beta strands arranged into two beta sheets as well as three alpha helices. Two of these helices form a helix-proline-helix motif, unprecedented among [2Fe-2S] proteins. The [2Fe-2S] cluster is coordinated by the sulfur atoms of cysteine residues 42, 47, 50, and 82. The 10.9 kDa ferredoxin domain of the reductase protein transfers electrons to carboxylate-bridged diiron centers in the 251 kDa hydroxylase component of sMMO. The binding of the Fd domain with the hydroxylase was investigated by NMR spectroscopy. The hydroxylase binding surface on the ferredoxin protein has a polar center surrounded by patches of hydrophobic residues. This arrangement of amino acids differs from that by which previously studied [2Fe-2S] proteins interact with their electron-transfer partners. The critical residues on the Fd domain involved in this binding interaction map well onto the universally conserved residues of sMMO enzymes from different species. We propose that the [2Fe-2S] domains in these other sMMO systems have a fold very similar to the one found here for M. capsulatus (Bath) MMOR-Fd.

Amino Acid Sequence↗
PubMed2002

Comparison of functional domains in vertebrate-type ferredoxins.

The vertebrate-type Cys(4)Fe(2)S(2) ferredoxins are a class of small acidic proteins that typically act as electron shuttles between NAD(P)H-dependent reductases and monoxygenases, particularly cytochromes P450. Nuclear magnetic resonance assignments and detailed analysis of nuclear Overhauser effects permit the direct comparison of the functional C-terminal domains of three vertebrate-type ferredoxins, the mammalian adrenodoxin (Adx) and the bacterial ferredoxins putidaredoxin (Pdx) and terpredoxin (Tdx). In particular, homologous hydrogen-bonding networks involving a conserved basic residue (His 49 in Pdx, His 56 in Adx, Arg 49 in Tdx) are detailed. This hydrogen bond network appears to play a role in the mechanical transmission of redox-dependent conformational and dynamic changes from the iron-sulfur binding loop to the C-terminal domain. Hydrogen/deuterium exchange measurements have been made in Adx as a function of oxidation state for comparison with previous studies of Pdx and Tdx. The results of these measurements highlight the importance of the conserved basic residue in the linkage between oxidation state and protein dynamics. Finally, a series of mutations have been made in the C-terminal domain of Pdx, including one, Y51F, that disrupts the proposed hydrogen-bonding network without perturbing steric and hydrophobic interactions in the functional domain. Although the mutant is considerably destabilized with respect to wild-type Pdx, relatively unperturbed chemical shifts for residues near the site of the mutation and NOEs between water and Phe 51 suggest that the network is reconstituted with a solvent water in place of the tyrosine hydroxyl group in this mutant.

Adrenodoxin↗
PubMed2006

Reduction of the pea ferredoxin-NADP(H) reductase catalytic efficiency by the structuring of a carboxyl-terminal artificial metal binding site.

Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, and adrenodoxin) to redox-based metabolisms in plastids, mitochondria, and bacteria. The FNRs from plants and most eubacteria constitute a unique family, the plant-type ferredoxin-NADP(H) reductases. Plastidic FNRs are quite efficient at sustaining the demands of the photosynthetic process. At variance, FNRs from organisms with heterotrophic metabolisms or anoxygenic photosynthesis display turnover numbers that are 20-100-fold lower than those of their plastidic and cyanobacterial counterparts. To gain insight into the FNR structural features that modulate enzyme catalytic efficiency, we constructed a recombinant FNR in which the carboxyl-terminal amino acid (Tyr308) is followed by an artificial metal binding site of nine amino acids, including four histidine residues. This added structure binds Zn2+ or Co2+ and, as a consequence, significantly reduces the catalytic efficiency of the enzyme by decreasing its kcat. The Km for NADPH and the Kd for NADP+ were increased 2 and 3 times, respectively, by the addition of the amino acid extension in the absence of Zn2+. Nevertheless, the structuring of the metal binding site did not change the Km for NADPH or the Kd for NADP+ of the FNR-tail enzyme. Our results provide experimental evidence which indicates that mobility of the carboxyl-terminal backbone region of the FNR, mainly Tyr308, is essential for obtaining an FNR enzyme with high catalytic efficiency.

Amino Acid Sequence↗
PubMed1997

An NMR study of the 7Fe-8S ferredoxin from Rhodopseudomonas palustris and reinterpretation of data on similar systems.

The oxidized 7Fe-8S ferredoxin from Rhodopseudomonas palustris is shown to possess a unique 1H NMR spectrum displaying at least one hyperfine-shifted beta-CH2 signal for each cysteine bound to the [3Fe-4S] cluster. COSY and TOCSY spectra and 1- and 2-dimensional NOE experiments, in conjunction with a thorough reexamination of the 1H NMR data on similar systems, permitted the sequential assignment of all of the cysteine beta-CH2 protons even in the absence of the amino acid sequence. The sequential assignment stems on the homology of the hyperfine shift pattern with those of other sequenced 7Fe-8S ferredoxins, which points to a substantial homology in tertiary structure. From the assignment, an analysis of the antiferromagnetic coupling in the [3Fe-4S] system was performed on the basis of a general model of exchange coupling. The NMR signal patterns of [3Fe-4S] clusters in both 3Fe-4S and 7Fe-8S ferredoxins have been discussed, and some correlations are proposed between signal patterns and the primary sequence.

Cysteine↗
PubMed1999

Unusual NMR, EPR, and Mössbauer properties of Chromatium vinosum 2[4Fe-4S] ferredoxin.

The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities. Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguously assigned here through NMR, EPR, and Mössbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E degrees ' of -460 mV. Its S = 1/2 EPR signal was fast relaxing and severely broadened by g-strain, and its Mössbauer spectra were broad and unresolved. These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal alpha-helix. Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an approximately 60 mV positive shift of the potential. At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K. Contact-shifted NMR resonances of beta-CH2 protons were detected in all combinations of redox states. These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII.CysIVP sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.

Bacterial Proteins↗