The stoicheiometry of electron transfer by bacterial and plant ferredoxins

1. The number of electrons carried by ferredoxins from spinach, the blue-green alga Anacystis nidulans, the anaerobic bacterium Clostridium welchii and the photosynthetic bacterium Chromatium was determined. 2. Ferredoxins were reduced by illuminated chloroplasts, and the stoicheiometry of the reoxidation in the dark of the ferredoxins by NADP and benzyl viologen was measured. 3. Spinach and A. nidulans ferredoxins were found to be one-electron carriers, and Cl. welchii and Chromatium ferredoxins were two-electron carriers.     

Thioredoxin from Rhodospirillum rubrum: primary structure and relation to thioredoxins from other photosynthetic bacteria.

Thioredoxin was isolated from a photosynthetic purple nonsulfur bacterium, Rhodospirillum rubrum, and its primary structure was determined by high-performance tandem mass spectrometry. The sequence identity of R. rubrum thioredoxin to Escherichia coli thioredoxin was intermediate to those of the Chlorobium thiosulfatophilum and Chromatium vinosum proteins. The results indicate that R. rubrum has an NADP-thioredoxin system similar to that of other photosynthetic purple bacteria.     

Thioredoxin system of the photosynthetic anaerobe Chromatium vinosum

Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles aerobic bacterial cells in that it has an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. Both protein components were purified to homogeneity, and some of their properties were determined. Chromatium vinosum thioredoxin was slightly larger than other bacterial thioredoxins (13 versus 12 kilodaltons) but was similar in its specificity (ability to activate chloroplast NADP-malate dehydrogenase more effectively than chloroplast fructose-1,6-bisphosphatase) and immunological properties. As in other bacteria, Chromatium vinosum NADP-thioredoxin reductase was an arsenite-sensitive flavoprotein composed of two 33.5-kilodalton subunits, that required thioredoxin for the NADPH-linked reduction of 5,5'-dithiobis(2-nitrobenzoic acid). Chromatium vinosum NADP-thioredoxin reductase very effectively reduced several different bacterial-type thioredoxins (Escherichia coli, Chlorobium thiosulfatophilum (this name has not been approved by the International Committee of Systematic Bacteriology), Rhizobium meliloti) but not others (Clostridium pasteurianum, spinach chloroplast thioredoxin m). The results show that Chromatium vinosum contains an NADP-thioredoxin system typical of evolutionarily more advanced microorganisms.     

Redox potentials of flavocytochromes c from the phototrophic bacteria, Chromatium vinosum and Chlorobium thiosulfatophilum.

The redox potentials of flavocytochromes c (FC) from Chromatium vinosum and Chlorobium thiosulfatophilum have been studied as a function of pH. Chlorobium FC has a single heme which has a redox potential of +98 mV at pH 7 (N = 1) that is independent of pH between 6 and 8. The average two-electron redox potential of the flavin extrapolated to pH 7 is +28 mV and decreases 35 mV/pH between pH 6 and 7. The anionic form of the flavin semiquinone is stabilized above pH 6. The redox potential of Chromatium FC is markedly lower than for Chlorobium. The two hemes in Chromatium FC appear to have a redox potential of 15 mV at pH 7 (N = 1), although they reside in very different structural environments. The hemes of Chromatium FC have a pH-dependent redox potential, which can be fit in the simplest case by a single ionization with pK = 7.05. The flavin in Chromatium FC has an average two-electron redox potential of -26 mV at pH 7 and decreases 30 mV/pH between pH 6 and 8. As with Chlorobium, the anionic form of the flavin semiquinone of Chromatium FC is stabilized above pH 6. The unusually high redox potential of the flavin, a stabilized anion radical, and sulfite binding to the flavin in both Chlorobium and Chromatium FCs are characteristics shared by the flavoprotein oxidases. By analogy with glycolate oxidase and lactate dehydrogenase for which there are three-dimensional structures, the properties of the FCs are likely to be due to a positively charged amino acid side chain in the vicinity of the N1 nitrogen of the flavin.     

Covalent structure of the diheme cytochrome subunit and amino-terminal sequence of the flavoprotein subunit of flavocytochrome c from Chromatium vinosum.

The complete sequence of the 21-kDa cytochrome subunit of the flavocytochrome c (FC) from the purple phototrophic bacterium Chromatium vinosum has been determined to be as follows: EPTAEMLTNNCAGCHG THGNSVGPASPSIAQMDPMVFVEVMEGFKSGEIAS TIMGRIAKGYSTADFEKMAGYFKQQTYQPAKQSF DTALADTGAKLHDKYCEKCHVEGGKPLADEEDY HILAGQWTPYLQYAMSDFREERRPMEKKMASKL RELLKAEGDAGLDALFAFYASQQ. The sequence is the first example of a diheme cytochrome in a flavocytochrome complex. Although the locations of the heme binding sites and the heme ligands suggest that the cytochrome subunit is the result of gene doubling of a type I cytochrome c, as found with Azotobacter cytochrome c4, the extremely low similarity of only 7% between the two halves of the Chromatium FC heme subunit rather suggests that gene fusion is at the evolutionary origin of this cytochrome. The two halves also require a single residue internal deletion for alignment. The first half of the Chromatium FC heme subunit is 39% similar to the monoheme subunit of the FC from the green phototrophic bacterium Chlorobium thiosulfatophilum, but the second half is only 9% similar to the Chlorobium subunit. The N-terminal sequence of the Chromatium FC flavin subunit was determined up to residue 41 as AGRKVVVVGGGTGGATAAKYIKLADPSIEVTLIEP NTKYYT. It shows more similarity to the Chlorobium FC flavin subunit (60%) than do the two heme subunits. The N terminus of the flavin subunit is homologous to a number of flavoproteins, including succinate dehydrogenase, glutathione reductase, and monamine oxidase. There is no obvious homology to the Pseudomonas putida FC flavin subunit, which suggests that the two types of flavocytochrome c arose by convergent evolution. This is consistent with the dissimilar enzyme activities of FC as sulfide dehydrogenase in the phototrophic bacteria and as p-cresol methylhydroxylase in Pseudomonas. We also present a sequence "fingerprint" pattern for the recognition of FAD-binding proteins which is an extended version of the consensus sequence previously presented (Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107) for nucleotide binding sites.     

Crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum: evolutionary and mechanistic inferences for [3/4Fe-4S] ferredoxins.

The crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 A resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N-terminal domain coordinates the two [4Fe-4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six-residue insertion between two ligands of one cluster forming a two-turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH...S H-bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5-turn alpha-helix at the C-terminus that covers one side of the same cluster and is linked to the cluster-binding domain by a six-residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe-4S] ferredoxins from a common ancestral cluster-binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.     

The iron electron-nuclear double resonance (ENDOR) of 4-Fe clusters in iron-sulfur proteins from Chromatium and Clostridium pasteurianum.

Iron electron-nuclear double resonance (ENDOR) measurements were made of the 4-Fe clusters in oxidized Chromatium high-potential iron-sulfur protein, dithionite-reduced high-potential iron-sulfur protein in 80% dimethylsulphoxide, fully reduced Clostridium pasteurianum ferredoxin in aqueous solution and in 80% dimethylsulfoxide. The hyperfine couplings determined show that: i) the electron distribution in each case is nearly symmetric; ii) there are two types of iron in oxidized high potential iron-sulfur protein; iii) only one type of iron is observed in each fully reduced 4-Fe cluster; iv) the data also suggest a greater electron delocalization onto the ligands as compared to the 2-Fe ferredoxins.     

Hydrogenases of phototrophic microorganisms

This review surveys recent work done in the laboratory of the author and related laboratories on the properties and possible practical applications of hydrogenases of phototrophic microorganisms. Homogeneous hydrogenase preparations were obtained from purple non-sulfur (Rhodospirillum rubrum S1, Rhodobacter capsulatus B10) and purple sulfur (Chromatium vinosum D, Thiocapsa roseopersicina BBS) bacteria, and from the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum L; highly purified hydrogenase samples were prepared from the cyanobacterium Anabaena cylindrica and from the green alga Chlamydomonas reinhardii. It was shown that hydrogenases of R. capsulatus and T. roseopersicina contain Ni and Fe-S cluster. The cytochromes of the c or b type serve as native electron acceptors for the hydrogenases of the purple bacteria and cyanobacteria; rubredoxin or cytochrome c for the hydrogenase of the green sulfur bacterium; and ferredoxin for Ch. reinhardii hydrogenase. The hydrogenase of T. roseopersicina BBS reversibly activates H2 at Eh less than -290 mV (pH 7), whereas those from R. capsulatus and from C. limicola f. thiosulfatophilum exhibit their maximum activity at Eh greater than -300 mV and are thus favourable only for the H2 uptake. Hydrogenase synthesis in different phototrophs depends on pO2, H2 concentrations and organic substrates. Organic compounds, which serve as electron donors and carbon sources, repress hydrogenase synthesis in R. rubrum, R. capsulatus and in Ectothiorhodospira shaposhnikovii when present at high concentrations. The synthesis of T. roseopersicina hydrogenase is constitutive. H2 notably stimulates hydrogenase activity in R. capsulatus. The synthesis of hydrogenase in R. sphaeroides 2R occurs only in the presence of H2 and does not depend on the presence of organic compounds in the medium.     

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.     

Comparison of the properties of two hydrogenases from the photosynthetic bacterium Chromatium vinosum

Chromatium vinosum hydrogenases I and II were purified to specific activities of 9.6 and 28.0 units/mg protein, respectively. They have the same isoelectric point (pI = 4.1), and their visible spectra are typical of iron-sulfur proteins. Hydrogenase II in general was more stable than hydrogenase I. Both enzymes lost their activities slowly during storage in air, and this inactivation was more apparent in preparations of hydrogenase I. Bovine serum albumin helped to stabilize hydrogenase I against thermal and storage inactivation. The pH optima of H2-evolution activity of hydrogenases I and II were 7.4 and 5.4, respectively. Neither enzyme was able to evolve H2 from reduced ferredoxins as the sole electron carrier, but ferredoxins had an effect on the activity with methyl viologen as carrier to hydrogenase I. None of the natural compounds tested was able to serve as a physiological donor for H2 production. Hydrogenase I was more susceptible than hydrogenase II to inhibition by heavy metal ions and other enzyme inhibitors. Both enzymes were reversibly inhibited by CO with Ki values of 12 and 6 Torr for hydrogenase I and II, respectively. Hydrogenase I was more sensitive to denaturation by urea and guanidinium chloride while hydrogenase II was more susceptible to sodium dodecyl sulfate. Both enzymes were rapidly and irreversibly inactivated by dimethyl sulfoxide. Hydrogenase I evolved H2 from methyl viologen and ferredoxin photoreduced by chloroplasts. The enzymes differed in their iron and acid-labile sulfur contents.     

Purification and characterization of a fix ABCX-linked 2[4Fe-4S] ferredoxin from Azotobacter vinelandii

 Ferredoxins that contain 2[4Fe-4S]^2+/+ clusters can be divided into two classes. The "clostridial-type" ferredoxins have two CysXXCysXXCysXXXCysPro motifs. The "photosynthetic bacterial and nif-related" ferredoxins have one motif of that type and one more unusual CysXXCysX_7–9CysXXXCysPro motif. In Azotobacter vinelandii three gene sequences have been reported that contain the latter motif, but until now none of the gene products has been purified. Here we report the purification of a small anionic [Fe-S] protein with yields of ∼3 mg per 500 g cell paste. NH₂-terminal sequence analysis shows that this protein is the product of a previously sequenced A. vinelandii gene that is found upstream of fixA and is cotranscribed with fixABCX. That gene was originally named fixP, but since that gene designation is now commonly used for a very different cb-type cytochrome oxidase we have renamed the gene fixFd and its product Fix Fd. Its sequence places Fix Fd in the class of "photosynthetic bacterial and nif-related" 2[4Fe-4S]^2+/1+ ferredoxins that includes Chromatium vinosum ferredoxin. Studies of the purified protein by Fe analysis, absorption, CD and EPR spectroscopies and electrochemistry confirm this characterization; the reduction potentials of the two clusters are –440 mV vs SHE. The fact that A. vinelandii synthesizes three different proteins with the same sequence motif, each of which is likely to have a different function, shows that although sequence motifs may be used reliably to classify ferredoxins by cluster type they cannot yet be used reliably for classifying ferredoxins by function. Received: 31 January 1997 / Accepted: 9 June 1997     

Unusual spectroscopic and electrochemical properties of the 2[4Fe-4S] ferredoxin of Thauera aromatica

A reduced ferredoxin serves as the natural electron donor for key enzymes of the anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica. It contains two [4Fe-4S] clusters and belongs to the Chromatium vinosum type of ferredoxins (CvFd) which differ from the "clostridial" type by a six-amino acid insertion between two successive cysteines and a C-terminal alpha-helical amino acid extension. The electrochemical and electron paramagnetic resonance (EPR) spectroscopic properties of both [4Fe-4S] clusters from T. aromatica ferredoxin have been investigated using cyclic voltammetry and multifrequency EPR. Results obtained from cyclic voltammetry revealed the presence of two redox transitions at -431 and -587 mV versus SHE. X-band EPR spectra recorded at potentials where only one cluster was reduced (greater than -500 mV) indicated the presence of a spin mixture of S = (3)/(2) and (5)/(2) spin states of one reduced [4Fe-4S] cluster. No typical S = (1)/(2) EPR signals were observed. At lower potentials (less than -500 mV), the more negative [4Fe-4S] cluster displayed Q-, X-, and S-band EPR spectra at 20 K which were typical of a single S = (1)/(2) low-spin [4Fe-4S] cluster with a g(av) of 1.94. However, when the temperature was decreased stepwise to 4 K, a magnetic interaction between the two clusters gradually became observable as a temperature-dependent splitting of both the S = (1)/(2) and S = (5)/(2) EPR signals. At potentials where both clusters were reduced, additional low-field EPR signals were observed which can only be assigned to spin states with spins of >(5)/(2). The results that were obtained establish that the common typical amino acid sequence features of CvFd-type ferredoxins determine the unusual electrochemical properties of the [4Fe-4S] clusters. The observation of different spin states in T. aromatica ferredoxin is novel among CvFd-type ferredoxins.     

Oxidation-reduction properties of several low potential iron-sulfur proteins and of methylviologen.

Apparent oxidation-reduction potentials at pH 7.0 and 25 degrees C were determined using the H2-hydrogenase system with ferredoxins from the following sources: Clostridium pasteurianum, -403 mV; C tartarovorum, -424 mV; C. acidi-urici, -434 mV; Peptococcus aerogenes, -427 mV; Chromatium D, -482 mV (pH 8.0); B. polymyxa, Fd I, -377 mV, and Fd II, -422 mV; and spinach, -428 mV. The pH dependence of these values was variable, ranging from -2 to -24 mV/pH unit increase for different ferredoxins. Over the range of buffer concentrations between 0.05 and 0.2 M, the potentials did not vary significantly. The number of electrons transferred during reduction (as determined by integrations of EPR spectra and by dithionite titration) is 2 for the first five proteins, while potentiometric data for all the cases fit a Nernst equation for which n = 1. The E degrees' value for the redox indicator methylviologen at pH 7.4 was found to be -460 mV, according to both the H2-hydrogenase system and cyclic voltammetry, significantly different from the value previously reported at higher pH's. Additionally, the presence of C. pasteuranum ferredoxin appears to shift the E degrees value of methylviologen to even more negative values. An analysis of sources of error inherent with potential determinations with H2 and hydrogenase is presented. The electronic and EPR spectra of P. aerogenes ferredoxin, for which the x-ray structure has been published, are given here. It appears that the determination of potentials of ferredoxin and other low-potential porteins with the H2-hydrogenase system affords certain experimental advantages over alternative methods currently employed with these and similar substances.     

M?ssbauer effect and electron paramagnetic resonance studies on yeast aconitase.

M?ssbauer effect and electron paramagnetic resonance (EPR) were measured for yeast aconitase [EC 4.2.1.3] purified from the cells of Candida lipolytica (ATCC 200114). M?ssbauer spectra suggested that yeast aconitase nostly contained two high-spin Fe(III) ions in an antiferromagnetically coupled binuclear complex that resembled oxidized 2 Fe ferredoxins, together with a small amount of high-spin Fe(II). EPR spectra recorded no signal at 77degreesK, but showed a slightly asymmetric signal centered at g=2.0 at 4.2degreesK, presumably due to the small amount of Fe(II) Fe(III) pairs.     

In situ analysis of phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno (Switzerland)

Comparative sequence analysis of a 16S rRNA gene clone library from the chemocline of the meromictic Lake Cadagno (Switzerland) revealed the presence of a diverse number of phototrophic sulfur bacteria. Sequences resembled those of rRNA of type strains Chromatium okenii DSM169 and Amoebobacter purpureus DSM4197, as well as those of four bacteria forming a tight cluster with A. purpureus DSM4197 and Lamprocystis roseopersicina DSM229. In situ hybridization with fluorescent (Cy3 labeled) oligonucleotide probes indicated that all large-celled phototrophic sulfur bacteria in the chemocline of Lake Cadagno were represented by C. okenii DSM169, while small-celled phototrophic sulfur bacteria consisted of four major populations with different distribution profiles in the chemocline indicating different ecophysiological adaptations.     

Preparation and partial characterization of iron-sulfur, iron-selenium, and iron-tellurium complexes of bovine serum albumin.

An artificial Fe-S* protein was prepared by the reaction of bovine serum albumin with FeSO4 and Na2S or with a synthetic Fe-S*-1,4-butanenedithiol complex. These improved methods enabled us to characterize the derivatives from serum albumin. The Fe-S* albumin complex has about 20 iron ions and 14 labile sulfur atoms per molecule of the protein, whose absorption spectrum closely resembled that of 2Fe-2S* proteins. Its electron paramagnetic resonance spectrum exhibited signals different from those of ferredoxins. The addition of p-chloromercuriphenylsulfonate quenched the optical absorption in the visible region as well as the electron paramagnetic resonance signals. These properties of the albumin-iron complex are similar to those of iron-sulfur dithiothreitol and mercaptoethanol complexes, suggesting that the albumin-iron complex has one or more protein ligands besides sulfur lignads. Presumably, the oxygen atom of the tyrosine residue, or other hydroxyamino acids participates in the complex formation. In this context, the albumin polypeptide appears to be incapable of forming an iron-sulfur cluster identical to those of ferredoxins. Yet, from the albumin-iron derivative, the extrusion of the iron-sulfur core with benzenethiol provided products similar to those from ferredoxins. The iron-selenium and iron-tellurium derivatives of the bovine serum albumin were prepared and partially characterized by optical absorption and electron paramagnetic resonsnace spectroscopies. These results imply that both selenium and tellurium can be incorporated into the protein molecule as the respective labile components.     

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A₂₇₈/A₄₉₀ is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids.

The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile sulfur groups and either four (ferredoxin II) or six (ferredoxin I) cysteine residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A₃₈₅/A₂₈₃ absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively.

The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology.

The physiological activity of ferredoxins I and II appears to be similar as far as the electron transfer in the phosphoroclastic reaction is concerned.     

Adduct formation between sulfite and the flavin of phototrophic bacterial flavocytochromes c. Kinetics of sequential bleach, recolor, and rebleach of flavin as a function of pH.

The kinetics of sulfite adduct formation with the bound flavin in flavocytochromes c from the purple phototrophic bacterium Chromatium vinosum and the green phototrophic bacterium Chlorobium thiosulfatophilum have been investigated as a function of pH. Both species of flavocytochrome c rapidly react with sulfite to form a flavin sulfite adduct (k = 10(3)-10(5) M-1 s-1) which is bleached at 450-475 nm and has associated charge-transfer absorbance at 660 nm. The rate constant for adduct formation in flavocytochrome c is 2-4 orders of magnitude faster than for model flavins of comparable redox potential and is likely to be due to a basic residue near the N-1 position of the flavin, which not only raises the redox potential but also stabilizes the negatively charged adduct. There is a pK for adduct formation at 6.5, which suggests that the order of magnitude larger rate constant at pH 5 as compared to pH 10 in flavocytochrome c is due the influence of another positive charge, possibly a protonated histidine residue. The adduct is indefinitely stable at pH 5 but decomposes (the flavin recolors) in a first-order process accelerating above pH 6 (at pH 10, k = 0.1 s-1). The pK for recoloring is 8.5, which is suggestive of a cysteine sulfhydryl. On the basis of the observed pK and available chemical information, we believe that recoloring is due to a secondary effect of the reaction of sulfite with a protein cystine disulfide, which is adjacent to the flavin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unusual NMR, EPR, and M?ssbauer 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 M?ssbauer 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 M?ssbauer 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.