Vertebrate-type and plant-type ferredoxins: crystal structure comparison and electron transfer pathway modelling

Crystallographic analysis of a fully functional, truncated bovine adrenodoxin, Adx(4-108), has revealed the structure of a vertebrate-type [2Fe-2S] ferredoxin at high resolution. Adrenodoxin is involved in steroid hormone biosythesis in adrenal gland mitochondria by transferring electrons from adrenodoxin reductase to different cytochromes P450. Plant-type [2Fe-2S] ferredoxins interact with photosystem I and a diverse set of reductases.A systematic structural comparison of Adx(4-108) with plant-type ferredoxins which share about 20 % sequence identity yields these results. (1) The ferredoxins of both types are partitioned into a large, strictly conserved core domain bearing the [2Fe-2S] cluster and a smaller interaction domain which is structurally different for both subfamilies. (2) In both types, residues involved in interactions with reductase are located at similar positions on the molecular surface and coupled to the [2Fe-2S] cluster via structurally equivalent hydrogen bonds. (3) The accessibility of the [2Fe-2S] cluster differs between Adx(4-108) and the plant-type ferredoxins where a solvent funnel leads from the surface to the cluster. (4) All ferredoxins are negative monopoles with a clear charge separation into two compartments, and all resulting dipoles but one point into a narrow cone located in between the interaction domain and the [2Fe-2S] cluster, possibly controlling predocking movements during interactions with redox partners. (5) Model calculations suggest that FE1 is the origin of electron transfer pathways to the surface in all analyzed [2Fe-2S] ferredoxins and that additional transfer probability for electrons tunneling from the more buried FE2 to the cysteine residue in position 92 of Adx is present in some.     

Deletions in the loop surrounding the iron-sulfur cluster of adrenodoxin severely affect the interactions with its native redox partners adrenodoxin reductase and cytochrome P450(scc) (CYP11A1)

The redox active iron-sulfur center of bovine adrenodoxin is coordinated by four cysteine residues in positions 46, 52, 55 and 92 and is covered by a loop containing the residues Glu-47, Gly-48, Thr-49, Leu-50 and Ala-51. In plant-type [2Fe-2S] ferredoxins, the corresponding loop consists of only four amino acids. The loop is positioned at the surface of the proteins and forms a boundary separating the [2Fe-2S] cluster from solvent. In order to analyze the biological function of the five amino acids of the loop in adrenodoxin (Adx) for this electron transfer protein each residue was deleted by site-directed mutagenesis. The resulting five recombinant Adx variants show dramatic differences among each other regarding their spectroscopic characteristics and functional properties. The redox potential is affected differently depending on the position of the conducted deletion. In contrast, all mutations in the protein loop influence the binding to the redox partners adrenodoxin reductase (AdR) and cytochrome P450(scc) (CYP11A1) indicating the importance of this loop for the physiological function of this iron--sulfur protein.     

The loop region covering the iron-sulfur cluster in bovine adrenodoxin comprises a new interaction site for redox partners

The amino acid in position 49 in bovine adrenodoxin is conserved among vertebrate [2Fe-2S] ferredoxins as hydroxyl function. A corresponding residue is missing in the cluster-coordinating loop of plant-type [2Fe-2S] ferredoxins. To probe the function of Thr-49 in a vertebrate ferredoxin, replacement mutants T49A, T49S, T49L, and T49Y, and a deletion mutant, T49Delta, were generated and expressed in Escherichia coli. CD spectra of purified proteins indicate changes of the [2Fe-2S] center geometry only for mutant T49Delta, whereas NMR studies reveal no transduction of structural changes to the interaction domain. The redox potential of T49Delta (-370 mV) is lowered by approximately 100 mV compared with wild type adrenodoxin and reaches the potential range of plant-type ferredoxins (-305 to -455 mV). Substitution mutants show moderate changes in the binding affinity to the redox partners. In contrast, the binding affinity of T49Delta to adrenodoxin reductase and cytochrome P-450 11A1 (CYP11A1) is dramatically reduced. These results led to the conclusion that Thr-49 modulates the redox potential in adrenodoxin and that the cluster-binding loop around Thr-49 represents a new interaction region with the redox partners adrenodoxin reductase and CYP11A1. In addition, variations of the apparent rate constants of all mutants for CYP11A1 reduction indicate the participation of residue 49 in the electron transfer pathway between adrenodoxin and CYP11A1.     

Purification and some properties of a 2Fe ferredoxin in Pseudomonas ovalis

A [2Fe-2S] ferredoxin was found in Pseudomonas ovalis which was grown in a medium supplemented with glucose and ammonium sulfate. The molecular weight of the 2Fe ferredoxin was estimated to be 13,000. It contained 2.2 gramatoms of non-heme iron and 2.3 gramatoms of acid-labile sulfur per mole protein. The absorption and circular dichroism spectra were characteristic of those of [2Fe-2S] type ferredoxins, especially adrenodoxin and putidaredoxin. The electron paramagnetic resonance spectrum of the reduced protein showed an axial symmetry (g = 2.020, g = 1.939). The amino acid composition was determined.     

Crystallization and preliminary analysis of oxidized, recombinant, heterocyst [2Fe-2S] ferredoxin from Anabaena 7120.

The [2Fe-2S] ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase. Crystals of recombinant heterocyst ferredoxin, coded for by the fdx H gene from Anabaena 7120 and overproduced in Escherichia coli, have been grown from ammonium sulfate solutions and are suitable for high resolution X-ray crystallographic analysis. They belong to the hexagonal space group P6(1) or P6(5) with unit cell dimensions of a = b = 44.2 A and c = 80.6 A. The crystals contain one molecule per asymmetric unit and diffract to a nominal resolution of 1.6 A. The molecular structure of this heterocyst ferredoxin is of special interest in that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different and, based on amino acid sequence alignments, three of these positions are located in the metal-cluster binding loop. Consequently, a high-resolution X-ray analysis of this [2Fe-2S] ferredoxin, and subsequent three-dimensional comparisons with other known ferredoxin models, will provide new insight into structure/function relationships for this class of redox proteins.     

Crystal structure determination at 1.4 A resolution of ferredoxin from the green alga Chlorella fusca.

BACKGROUND: [2Fe-2S] ferredoxins, also called plant-type ferredoxins, are low-potential redox proteins that are widely distributed in biological systems. In photosynthesis, the plant-type ferredoxins function as the central molecule for distributing electrons from the photolysis of water to a number of ferredox-independent enzymes, as well as to cyclic photophosphorylation electron transfer. This paper reports only the second structure of a [2Fe-2S] ferredoxin from a eukaryotic organism in its native form. RESULTS: Ferredoxin from the green algae Chlorella fusca has been purified, characterised, crystallised and its structure determined to 1.4 A resolution - the highest resolution structure published to date for a plant-type ferredoxin. The structure has the general features of the plant-type ferredoxins already described, with conformational differences corresponding to regions of higher mobility. Immunological data indicate that a serine residue within the protein is partially phosphorylated. A slightly electropositive shift in the measured redox potential value, -325 mV, is observed in comparison with other ferredoxins. CONCLUSIONS: This high-resolution structure provides a detailed picture of the hydrogen-bonding pattern around the [2Fe-2S] cluster of a plant-type ferredoxin; for the first time, it was possible to obtain reliable error estimates for the geometrical parameters. The presence of phosphoserine in the protein indicates a possible mechanism for the regulation of the distribution of reducing power from the photosynthetic electron-transfer chain.     

The crystal structure of Trichomonas vaginalis ferredoxin provides insight into metronidazole activation

Crystallographic studies revealing the three-dimensional structure of the oxidized form of the [2Fe-2S] ferredoxin from Trichomonas vaginalis (TvFd) are presented. TvFd, a member of the hydrogenosomal class of ferredoxins, possesses a unique combination of redox and spectroscopic properties, and is believed to be the biological molecule that activates the drug metronidazole reductively in the treatment of trichomoniasis. It is the first hydrogenosomal ferredoxin to have its structure determined. The structure of TvFd reveals a monomeric, 93 residue protein with a fold similar to that of other known [2Fe-2S] ferredoxins. It contains nine hydrogen bonds to the sulfur atoms of the cluster, which is more than the number predicted on the basis of the spectroscopic data. The TvFd structure contains a large dipole moment like adrenodoxin, and appears to have a similar interaction domain. Our analysis demonstrates that TvFd has a unique cavity near the iron-sulfur cluster that exposes one of the inorganic sulfur atoms of the cluster to solvent. This cavity is not seen in any other [2Fe-2S] ferredoxin with known structure, and is hypothesized to be responsible for the high rate of metronidazole reduction by TvFd.     

Direct expression of mature bovine adrenodoxin in Escherichia coli.

Site-directed mutagenesis was utilized to enable direct expression of the mature form of bovine adrenodoxin cDNA using the pKK223-3 expression vector in Escherichia coli. Expression was under control of the "tac" promoter and resulted in a direct expression of soluble mature bovine adrenodoxin (greater than 15 mg per liter). Chromatographic behavior of recombinant adrenodoxin did not differ from that reported for mature native adrenodoxin. The purified recombinant protein was identical to native mitochondrial adrenodoxin on the basis of molecular weight, NH2 terminal sequencing and immunoreactivity. E. coli lysates were brown in color, and the purified protein possessed a visible absorbance spectra identical to native bovine adrenodoxin consistent with incorporation of a [2Fe-2S] cluster in vivo. Recombinant bovine adrenodoxin was active in cholesterol side-chain cleavage when reconstituted with adrenodoxin reductase and cytochrome P450scc and exhibited kinetics reported for native bovine adrenodoxin. The presence of the adrenodoxin amino terminal presequence does not appear to be essential for correct folding of mature recombinant adrenodoxin in E. coli. This expression system should prove useful for overexpression of adrenodoxin mutants in future structure/function studies. The approach described herein can potentially be used to directly express the mature form of any protein in bacteria.     

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.     

Low-potential iron-sulfur centers in photosystem I: an X-ray absorption spectroscopy study

We have measured the X-ray absorption spectra of Fe in photosystem I (PS I) preparations from spinach and a thermophilic cyanobacterium, Synechococcus sp., to characterize structures of the Fe complexes that function as electron acceptors in PS I. These acceptors include centers A and B, which are probably typical [4Fe-4S] ferredoxins, and X. The structure of X is not known, but its electron paramagnetic resonance (EPR) spectrum has generated the suggestions that it is either a [2Fe-2S] or [4Fe-4S] ferredoxin or an Fe-quinone species. The iron X-ray absorption K-edge and iron extended X-ray absorption fine structure (EXAFS) spectra reveal that essentially all of the 11-14 Fe atoms present in the reaction center are present in the form of Fe-S centers and that not more than 1 atom out of 12 could be octahedral or oxygen-coordinated Fe. This suggests that, besides A and B, additional Fe-S clusters are present which are likely to be X. Our EXAFS spectra cannot be simulated adequately by a mixture of [4Fe-4S] ferredoxins with typical bond lengths and disorder parameters because the amplitude of Fe backscattering is small; however, excellent simulations of the data are consistent with a mixture of [2Fe-2S] ferredoxins and [4Fe-4S] ferredoxins, or with unusually distorted [4Fe-4S] clusters. We presume that the [2Fe-2S] or distorted [4Fe-4S] centers are X. The X-ray absorption spectra of PS I preparations from Synechococcus and spinach are essentially indistinguishable.     

Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus.

We have purified and characterized two ferredoxins, designated Fd-1 and Fd-2, from the soluble protein fraction of sulfonylurea herbicide induced Streptomyces griseolus. These cells have previously been shown to contain two inducible cytochromes P-450, P-450SU1 (CYP105A1) and P-450SU2 (CYP105B1), responsible for herbicide metabolism [O'Keefe, D. P., Romesser, J. A., & Leto, K. J. (1988) Arch. Microbiol. 149, 406-412]. Although Fd-2 is more effective, either ferredoxin can restore sulfonylurea monooxygenase activity to an aerobic mixture of NADPH, spinach ferredoxin:NADP oxidoreductase, purified cytochrome P-450SU1, and herbicide substrate. The gene for Fd-1 is located in the genome just downstream of the gene for cytochrome P-450SU1; the gene for Fd-2 follows the gene for P-450SU2. The deduced amino acid sequences of the two ferredoxins show that, if monomeric, each has a molecular mass of approximately 7 kDa, and alignment of the two sequences demonstrates that they are approximately 52% positionally identical. The spectroscopic properties and iron and acid-labile sulfide contents of both ferredoxins suggest that, as isolated, each contains a single [3Fe-4S] cluster. The presence of only three cysteines in Fd-1 and comparisons with three [4Fe-4S] ferredoxins with high sequence similarity suggest that both Fd-1 and Fd-2 have an alanine in the position where these [4Fe-4S] proteins have a fourth cysteine ligand to the cluster. Transformation of Streptomyces lividans, a strain unable to metabolize sulfonylureas, with DNA encoding both P-450SU1 and Fd-1 results in cells capable of herbicide metabolism. S. lividans transformants encoding only cytochrome P-450SU1 do not metabolize herbicide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adrenodoxin reductase-adrenodoxin complex structure suggests electron transfer path in steroid biosynthesis

The steroid hydroxylating system of adrenal cortex mitochondria consists of the membrane-attached NADPH-dependent adrenodoxin reductase (AR), the soluble one-electron transport protein adrenodoxin (Adx), and a membrane-integrated cytochrome P450 of the CYP11 family. In the 2.3-A resolution crystal structure of the Adx.AR complex, 580 A(2) of partly polar surface are buried. Main interaction sites are centered around Asp(79), Asp(76), Asp(72), and Asp(39) of Adx and around Arg(211), Arg(240), Arg(244), and Lys(27) of AR, respectively. In particular, the region around Asp(39) defines a new protein interaction site for Adx, similar to those found in plant and bacterial ferredoxins. Additional contacts involve the electron transfer region between the redox centers of AR and Adx and C-terminal residues of Adx. The Adx residues Asp(113) to Arg(115) adopt 3(10)-helical conformation and engage in loose intermolecular contacts within a deep cleft of AR. Complex formation is accompanied by a slight domain rearrangement in AR. The [2Fe-2S] cluster of Adx and the isoalloxazine rings of FAD of AR are 10 A apart suggesting a possible electron transfer route between these redox centers. The AR.Adx complex represents the first structure of a biologically relevant complex between a ferredoxin and its reductase.     

Redox chemistry of the Schizosaccharomyces pombe ferredoxin electron-transfer domain and influence of Cys to Ser substitutions

Schizosaccharomyces pombe (Sp) ferredoxin contains a C-terminal electron transfer protein ferredoxin domain (etp^Fd) that is homologous to adrenodoxin. The ferredoxin has been characterized by spectroelectrochemical methods, and Mössbauer, UV-Vis and circular dichroism spectroscopies. The Mössbauer spectrum is consistent with a standard diferric [2Fe-2S]²⁺ cluster. While showing sequence homology to vertebrate ferredoxins, the E°' and the reduction thermodynamics for etp^Fd (− 0.392 V) are similar to plant-type ferredoxins. Relatively stable Cys to Ser derivatives were made for each of the four bound Cys residues and variations in the visible spectrum in the 380-450 nm range were observed that are characteristic of oxygen ligated clusters, including members of the [2Fe-2S] cluster IscU/ISU scaffold proteins. Circular dichroism spectra were similar and consistent with no significant structural change accompanying these mutations. All derivatives were active in an NADPH-Fd reductase cytochrome c assay. The binding affinity of Fd to the reductase was similar, however, V_max reflecting rate limiting electron transfer was found to decrease ~ 13-fold. The data are consistent with relatively minor perturbations of both the electronic properties of the cluster following substitution of the Fe-bond S atom with O, and the electronic coupling of the cluster to the protein.     

A new electron transport mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin

The adrenal ferredoxin (adrenodoxin, Adx) is an acidic 14.4-kDa [2Fe-2S] ferredoxin that belongs to the vertebrate ferredoxin family. It is involved in the electron transfer from the flavoenzyme NADPH-adrenodoxin-reductase to cytochromes P-450(scc) and P-450(11)(beta). The interaction between the redox partners during electron transport has not yet been fully established. Determining the tertiary structure of an electron-transfer protein may be very helpful in understanding the transport mechanism. In the present work, we report a structural study on the oxidized and reduced forms of bovine adrenodoxin (bAdx) in solution using high-resolution NMR spectroscopy. The protein was produced in Escherichia coli and singly or doubly labeled with (15)N or (13)C/(15)N, respectively. Approximately 70 and 75% of the (15)N, (13)C, and (1)H resonances could be assigned for the reduced and the oxidized bAdx, respectively. The secondary and tertiary structures of the reduced and oxidized states were determined using NOE distance information. (1)H(N)-T(1) relaxation times of certain residues were used to obtain additional distance constraints to the [2Fe-2S] cluster. The results suggest that the solution structure of oxidized Adx is quite similar to the X-ray structure. However, structural changes occur upon reduction of the [2Fe-2S] cluster, as indicated by NMR measurements. It could be shown that these conformational changes, especially in the C-terminal region, cause the dissociation of the Adx dimer upon reduction. A new electron transport mechanism proceeding via a modified shuttle mechanism, with both monomers and dimers acting as electron carriers, is proposed.     

Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan-modified NADPH-adrenodoxin reductase

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition.     

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.     

Kidney and adrenal mitochondria contain two forms of NADPH-adrenodoxin reductase-dependent iron-sulfur proteins. Isolation of the two porcine renal ferredoxins

Two NADPH-adrenodoxin reductase-dependent iron-sulfur proteins were detected in both porcine kidney and bovine adrenal mitochondria by using high resolution polyacrylamide electrophoresis. Adrenodoxin (Mr = 12,000) constituted the major ferredoxin activity in adrenal mitochondria and a similarly sized protein (Mr = 11,500) was isolated as the major renal ferredoxin activity. A second, higher molecular weight ferredoxin was observed in both adrenal (Mr = 13,300) and kidney (Mr = 13,000) mitochondria. The two renal ferredoxins were isolated by the use of ion exchange, gel exclusion, and preparative electrophoretic techniques. An absorption spectrum typical of [2Fe-2S] ferredoxins was obtained for each protein; however, the larger renal molecule had an unusually high 276 nm absorbance. Immunologic studies revealed a significant degree of antigenic commonality between the two renal proteins as well as specific cross-reactivity of adrenodoxin with antiserum raised against the renal proteins. A possible precursor-product relationship between the paired renal and adrenal ferredoxins is discussed.     

Adrenodoxin-cytochrome P450scc interaction as revealed by EPR spectroscopy: comparison with the putidaredoxin-cytochrome P450cam system.

The cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc comprises three consecutive monooxygenase reactions (22R-hydroxylation, 20S-hydroxylation, and C(20)-C(22) bond scission) that produces pregnenolone. The electron equivalents necessary for the oxygen activation are supplied from a 2Fe-2S type ferredoxin, adrenodoxin. We found that 1:1 stoichiometric binding of oxidized adrenodoxin to oxidized cytochrome P450scc complexed with cholesterol or 25-hydroxycholesterol caused shifts of the high-spin EPR signals of the heme moiety at 5 K. Such shifts were not observed for the low-spin EPR signals. Ligation of CO or NO to the reduced heme of cytochrome P450scc complexed with reduced adrenodoxin and various steroid substrates did not cause any change in the axial EPR spectrum of the reduced iron-sulfur center at 77 K. These results are in remarkable contrast to those obtained for the cytochrome P450cam-d-camphor-putidaredoxin ternary complex, suggesting that the mode of cross talk between adrenodoxin and cytochrome P450scc is very different from that in the Pseudomonas system. The difference may be primarily due to the location of the charged amino acid residues of the ferredoxins important for the interaction with the partner cytochrome P450.     

Electron transfer may occur in the chlorosome envelope: the CsmI and CsmJ proteins of chlorosomes are 2Fe-2S ferredoxins

Chlorosomes of the green sulfur bacterium Chlorobium tepidum have previously been shown to contain at least 10 polypeptides [Chung, S., Frank, G., Zuber, H., and Bryant, D. A. (1994) Photosynth. Res. 41, 261-275]. Based upon the N-terminal amino acid sequences determined for two of these proteins, the corresponding genes were isolated using degenerate oligonucleotide hybridization probes. The csmI and csmJ genes encode proteins of 244 and 225 amino acids, respectively. A third gene, denoted csmX, that predicts a protein of 221 amino acids with strong sequence similarity to CsmI and CsmJ, was found to be encoded immediately upstream from the csmJ gene. All three proteins have strong sequence similarity in their amino-terminal domains to [2Fe-2S] ferredoxins of the adrenodoxin/putidaredoxin subfamily of ferredoxins. CsmI and CsmJ were overproduced in Escherichia coli, and both proteins were shown by EPR spectroscopy to contain iron-sulfur clusters. The g-tensor and relaxation properties are consistent with their assignment as [2Fe-2S] clusters. Isolated chlorosomes were also shown to contain [2Fe-2S] clusters whose properties were similar to those of the recombinant CsmI and CsmJ proteins. Redox titration of isolated chlorosomes showed these clusters to have potentials of about -201 and +92 mV vs SHE. The former potential is similar to that measured by redox titration of the clusters in inclusion bodies of CsmJ. Possible roles for these iron-sulfur proteins in electron transport and light harvesting are discussed.     

Crystal structure of Escherichia coli Fdx, an adrenodoxin-type ferredoxin involved in the assembly of iron-sulfur clusters

Escherichia coli ferredoxin (Fdx) is an adrenodoxin-type [2Fe-2S] ferredoxin. Recent genetic analyses show that it has an essential role in the maturation of various iron-sulfur (Fe-S) proteins. Fdx probably functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. Its crystal structure was determined by the multiple-wavelength anomalous dispersion method using the iron atoms in the [2Fe-2S] cluster of the protein and then refined to R and R(free) values of 0.255 and 0.278, respectively, at 1.7 A resolution. The structure of Fdx is similar to the structures of bovine adrenodoxin (Adx) and Pseudomonas putida putidaredoxin (Pdx) whose respective root-mean-square deviations of the corresponding Calpha atoms are 1.8 and 2.2 A. This analysis also revealed the structure of the C-terminal residues protruding into the solvent, which is missing in Adx and Pdx. The [2Fe-2S] cluster is located at the edge of the molecule and bonds with the Sgamma atoms of Cys42, Cys48, Cys51, and Cys87. Electrostatic potential analysis showed that the surface of Fdx has two negatively charged areas separated by a hydrophobic lane. One is conserved on the surface of Adx which is an area of interaction with adrenodoxin reductase. Cys46 is located on the molecular surface in the vicinity of the [2Fe-2S] cluster, an indication that it may be involved in Fe-S cluster formation.