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1.
Ex novo enzymic synthesis of the two 4Fe-4S clusters of Clostridium pasteurianum ferredoxin has been achieved by incubation of the apoprotein with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, DL-dihydrolipoate and ferric ammonium citrate. This enzymic reconstitution procedure was compared to a chemical one, in which the enzyme was replaced by sodium sulfide. A further comparison was made with the results previously obtained in the enzymic synthesis of the 2Fe-2S cluster of spinach ferredoxin, allowing the following conclusions to be drawn. The nature of the cluster to be inserted into the reconstituted iron-sulfur protein is determined by the apoprotein itself. The refolding of the structure of the iron-sulfur proteins around the newly inserted cluster is the rate-limiting step in both chemical and enzymic reconstitution. Rhodanese appears to play a role in the recovery of the native architecture of the reconstituted iron-sulfur protein(s). The extension to the 4Fe-4S centers of the rhodanese-based biosynthetic system allows this enzymic route to be proposed as a general way to the in vivo synthesis of iron-sulfur structures.  相似文献   

2.
The ferredoxin from Clostridium pasteurianum, which contains two [4Fe-4S] clusters, was investigated in its oxidized and reduced states by two-dimensional (2D) (1)H-(1)H nuclear Overhauser enhancement spectroscopy (NOESY). Comparison of the data from the oxidized ferredoxin with those published previously revealed the same NOE connectivities. No previous (1)H-(1)H NOESY study of the fully reduced ferredoxin has previously been published. However, it was possible to compare our results with those of a 2D exchange spectroscopy investigation of half-reduced C. pasteurianum ferredoxin. The present results with reduced C. pasteurianum ferredoxin confirm many of the (1)H peaks and NOE interactions reported earlier, revise others, and locate resonances previously undetected. When the ferredoxin was slightly exposed to oxygen, several of the hyperfine shifted resonances were irreversibly influenced. A resonance at 34 ppm in the (1)H NMR spectra of both redox states is indicative of oxygen exposure. These results indicate the importance of keeping the ferredoxin strictly anaerobic during purification and solvent exchange.  相似文献   

3.
The ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated through 1H-NMR spectroscopy in the reduced and partially oxidized states. The 1H-NMR spectrum of fully reduced ferredoxin, obtained by addition of stoichiometric amounts of dithionite, has been characterized. One- and two-dimensional NMR saturation transfer experiments on partially reduced samples have allowed the isotropically shifted signals of the reduced form to be correlated to those of the oxidized form, for which the complete assignment of the beta-CH2 cysteinyl residues is available. In addition, observation of the 1H-NMR signals of the intermediate species with characteristic chemical shift values for each cluster allowed us to assign all the Cys beta-CH2 signals to cluster I or cluster II and to calculate the difference in redox potential between them. Starting from these results, reanalysis of the 1H-NMR features of the two clusters in the oxidized form showed that they are strikingly similar, supporting the idea of a high degree of internal symmetry between them, in agreement with crystallographic results on an homologous ferredoxin. On the other hand, the 1H-NMR properties of the two clusters in the reduced form deviate considerably from each other, suggesting that reduction of the clusters brings about different structural changes and loss of internal symmetry. A theoretical approach is reported to account for the isotropic shifts and the temperature dependence of the NMR signals of the reduced protein.  相似文献   

4.
The ferredoxin from Clostridium pasteurianum contains two Fe4S4 clusters. In this paper we determine their oxidation-reduction midpoint potentials; we find them to be essentially identical (within 10 mV) and to have pH-independent Em values of -412 +/- 11 mV from pH 6.3 to 10.0.  相似文献   

5.
Amino acid sequence of [2Fe-2S] ferredoxin from Clostridium pasteurianum   总被引:4,自引:0,他引:4  
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.  相似文献   

6.
The [2Fe-2S] ferredoxin from Clostridium pasteurianum had previously been shown to interact specifically with the nitrogenase MoFe protein, and electrostatic forces were found to be important contributors to the interaction. This phenomenon has now been analyzed in detail by using ferredoxin variants in which charge inversions or cancellations were introduced on all charged residues. The mutated forms of the ferredoxin were covalently cross-linked to the MoFe protein. The reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their nitrogenase activity was measured. The latter displayed a consistent inverse correlation with the amount of cross-linked MoFe protein. The data allowed an unambiguous identification of the ferredoxin residues (glutamates 31, 34, 38, 39, 84, 85) that are involved in the interaction with the MoFe protein. Furthermore, whereas the wild-type ferredoxin yielded approximately equal amounts of cross-linked products with the alpha and beta subunits of the MoFe protein, some of its molecular variants displayed a differential decrease of reactivity towards one or the other of these subunits. The positions on the ferredoxin molecule of the residues interacting with the MoFe protein were determined using the recently elucidated crystal structure of the homologous [2Fe-2S] ferredoxin from Aquifex aeolicus.  相似文献   

7.
J M Moulis  J Meyer 《Biochemistry》1982,21(19):4762-4771
The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from Clostridium pasteurianum have been replaced by selenium. The substitution is readily carried out by incubating the apoferredoxin with excess amounts of Fe3+, selenite, and dithiothreitol under anaerobic conditions. The UV-visible absorption spectrum of the Se-substituted ferredoxin, the core extrusion of its active sites, and analyses of its iron and selenium contents show that it contains two [4Fe-4Se] clusters. The Se-substituted ferredoxin is considerably less resistant to oxygen or to acidic and alkaline pH than the native ferredoxin: the half-lives of the former are 20-500 times shorter than those of the latter. The native ferredoxin and the Se-substituted ferredoxin display similar kinetic properties when used as electron donors to the hydrogenase from C. pasteurianum. It is of note, however, that the Km and Vmax values are lower for the 2[4Fe-4Se] ferredoxin than for the 2[4Fe-4S] ferredoxin. Reductive and oxidative titrations with dithionite and with thionine, respectively, show that both ferredoxins are two-electron carriers. The redox potentials of the ferredoxins have been measured by equilibrating them with the H2/H+ couple via hydrogenase: values of -423 and -417 mV have been found for the 2[4Fe-4S] ferredoxin and 2[4Fe-4Se] ferredoxin, respectively. Ferredoxins containing both chalcogenides in their [4Fe-4X] (X = S, Se) clusters have been prepared by reconstitution reactions involving mixtures of sulfide and selenide: the latter experiments show that sulfide and selenide are equally reactive in the incorporation of [4Fe-4X] (X = S, Se) sites into ferredoxin. The present report, together with former studies, establishes the general feasibility of the Se/S substitution in [2Fe-2S] and in [4Fe-4S] clusters of proteins and of synthetic analogues.  相似文献   

8.
A gene encoding the exact sequence of Clostridium pasteurianum 2[4Fe-4S] ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli. The synthetic gene is efficiently expressed in E. coli and its product has been purified and characterized. The N-terminal sequence is identical to that of the protein isolated from C. pasteurianum and the recombinant ferredoxin contains the exact amount of [4Fe-4S] clusters (2 per monomer) expected for homogeneous holoferredoxin. It displays reduction potential and kinetic parameters as electron donor to C. pasteurianum hydrogenase I identical to those determined for the native ferredoxin. All of these properties demonstrate that the 2[4Fe-4S] ferredoxin expressed in E. coli is identical to the parent clostridial protein.  相似文献   

9.
Oxidized ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated using 2D 1H NMR spectroscopy at 600 MHz. 2D NMR experiments allowed complete assignment of the sixteen isotropically shifted signals corresponding to the beta-CH2 protons of the eight metal coordinated cysteines. Geminal connectivities of Cys beta-CH2 protons were identified through magnitude COSY experiments and confirmed through 2D NOESY experiments. A few additional signals could be assigned to the corresponding alpha-CH protons. The importance of 2D experiments to achieve firm assignments of isotropically shifted signals in paramagnetic metalloproteins is stressed.  相似文献   

10.
The [2Fe-2S] ferredoxin ("Red paramagnetic protein", RPP) from C. pasteurianum has been found to be composed of two identical subunits of 10,000 +/- 2 000 daltons, each containing a [2Fe-2S] cluster. Resonance Raman (RR) spectra of RPP have been obtained at 23 degrees K, and compared to those of spinach ferredoxin (Sp Fd). Ten modes of the [2Fe-2S] chromophore were observed in the 100-450 cm-1 range. Assignments of non fundamental modes in the 500-900 cm-1 range allowed correlations between fundamental stretching modes of RPP and Sp Fd. Although assuming a [2Fe-2S] structure, the chromophore of RPP differs from that of Sp Fd by its conformation and by a slight weakening of Fe-S bonds, involving both the inorganic core and the cysteine ligands.  相似文献   

11.
 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  相似文献   

12.
The effect of deuterium substitution of exchangeable hydrogen atoms on the reduction potential of Clostridium pasteurianum 2(4Fe-4S) ferredoxin has been studied. The studies were conducted to determine if NH ?S hydrogen bonds to the iron–sulfur cluster are dominant in the mechanism of influence of the protein on cluster reduction potential, as has been proposed [Carter, C. W. (1977) J. Biol. Chem. 252 , 7802–7811]. Deuteration of the slowly exchangeable hydrogen atoms, however, yields essentially no shift in the reduction potential (?0.2 ± 0.8 mV), suggesting that NH ?S bonds are not important modifiers of cluster reduction potential in this protein.  相似文献   

13.
Azotobacter vinelandii (4Fe-4S)2 ferredoxin I (Fd I) is an electron transfer protein with Mr equals 14,500 and Eo equals -420 mv. It exhibits and EPR signal of g equals 2.01 in its isolated form. This resonance is almost identical with the signal that originates from a "super-oxidized" state of the 4Fe-4S cluster of potassium ferricyanide-treated Clostridium ferredoxin. A cluster that exhibits this EPR signal at g equals 2.01 is in the same formal oxidation state as the cluster in oxidized Chromatium High-Potential-Iron-Protein (HiPIP). On photoreduction of Fd I with spinach chloroplast fragments, the resonance at g equals 2.01 vanishes and no EPR signal is observed. This EPR behavior is analogous to that of reduced HiPIP, which also fails to exhibit an EPR spectrum. These characteristics suggest that a cluster in A. vinelandii Fd I functions between the same pair of states on reduction as does the cluster in HiPIP, but with a midpoint reduction potential of -420 mv in contrast to the value of +350 mv characteristic of HiPIP. Quantitative EPR and stoichoimetry studies showed that only one 4Fe-4S cluster in this (4Fe-4S)2 ferredoxin is reduced. Oxidation of Fd I with potassium ferricyanide results in the uptake of 1 electron/mol as determined by quantitative EPR spectroscopy. This indicates that a cluster in Fd I shows no electron paramagnetic resonance in the isolated form of the protein accepts an electron on oxidation, as indicated by the EPR spectrum, and becomes paramagnetic. The EPR behavior of this oxidizable cluster indicates that it also functions between the same pair of oxidation states as does the Fe-S cluster in HiPIP. The midpoint reduction potential of this cluster is approximately +340 mv. A. vinelandii Fd I is the first example of an iron-sulfur protein which contains both a high potential cluster (approximately +340 mv) and a low potential cluster (-420 mv). Both Fe-S clusters appear to function between the same pair of oxidation states as the single Fe-S cluster in Chromatium HiPIP, although the midpoint reduction potentials of the two clusters are approximately 760 mv different.  相似文献   

14.
The primary structure of a ferredoxin isolated from D. desulfuricans Norway strain, which we called ferredoxin II (Fd II) has been elucidated. This ferredoxin is a dimer constituted of two identical subunits of molecular weight 6000. In ferredoxin II two (4 Fe-4 S) centers are present per subunit instead of one (Fe-S) center as is the case for the other ferredoxins isolated from Desulfovibrio and for Fd I from the same organism. The comparison of amino-acid sequences shows that ferredoxin II presents more homologies with clostridial type ferredoxin than with the ferredoxins from D. gigas and D. africanus.  相似文献   

15.
16.
The electron transfer system of the biphenyl dioxygenase BphA, which is derived from Acidovorax sp. (formally Pseudomonas sp.) strain KKS102, is composed of an FAD-containing NADH-ferredoxin reductase (BphA4) and a Rieske-type [2Fe-2S] ferredoxin (BphA3). Biochemical studies have suggested that the whole electron transfer process from NADH to BphA3 comprises three consecutive elementary electron-transfer reactions, in which BphA3 and BphA4 interact transiently in a redox-dependent manner. Initially, BphA4 receives two electrons from NADH. The reduced BphA4 then delivers one electron each to the [2Fe-2S] cluster of the two BphA3 molecules through redox-dependent transient interactions. The reduced BphA3 transports the electron to BphA1A2, a terminal oxygenase, to support the activation of dioxygen for biphenyl dihydroxylation. In order to elucidate the molecular mechanisms of the sequential reaction and the redox-dependent interaction between BphA3 and BphA4, we determined the crystal structures of the productive BphA3-BphA4 complex, and of free BphA3 and BphA4 in all the redox states occurring in the catalytic cycle. The crystal structures of these reaction intermediates demonstrated that each elementary electron transfer induces a series of redox-dependent conformational changes in BphA3 and BphA4, which regulate the interaction between them. In addition, the conformational changes induced by the preceding electron transfer seem to induce the next electron transfer. The interplay of electron transfer and induced conformational changes seems to be critical to the sequential electron-transfer reaction from NADH to BphA3.  相似文献   

17.
18.
The prototype ferredoxin maquette, FdM, is a 16-amino acid peptide which efficiently incorporates a single [4Fe-4S]2+/+ cluster with spectroscopic and electrochemical properties that are typical of natural bacterial ferredoxins. Using this synthetic protein scaffold, we have investigated the role of the nonliganding amino acids in the assembly of the iron-sulfur cluster. In a stepwise fashion, we truncated FdM to a seven-amino acid peptide, FdM-7, which incorporates a cluster spectroscopically identical to FdM but in lower yield, 29% relative to FdM. FdM-7 consists solely of the. CIACGAC. consensus ferredoxin core motif observed in natural protein sequences. Initially, all of the nonliganding amino acids were substituted for either glycine, FdM-7-PolyGly (.CGGCGGC.), or alanine, FdM-7-PolyAla (.CAACAAC.), on the basis of analysis of natural ferredoxin sequences. Both FdM-7-PolyGly and FdM-7-PolyAla incorporated little [4Fe-4S]2+/+ cluster, 6 and 7%, respectively. A systematic study of the incorporation of a single isoleucine into each of the four nonliganding positions indicated that placement either in the second or in the sixth core motif positions,.CIGCGGC. or.CGGCGIC., restored the iron-sulfur cluster binding capacity of the peptides to the level of FdM-7. Incorporation of an isoleucine into the fifth position,.CGGCIGC., which in natural ferredoxins is predominantly occupied by a glycine, resulted in a loss of [4Fe-4S] affinity. The substitution of leucine, tryptophan, and arginine into the second core motif position illustrated the stabilization of the [4Fe-4S] cluster by bulky hydrophobic amino acids. Furthermore, the incorporation of a single isoleucine into the second core motif position in a 16-amino acid ferredoxin maquette resulted in a 5-fold increase in the level of [4Fe-4S] cluster binding relative to that of the glycine variant. The protein design rules derived from this study are fully consistent with those derived from natural ferredoxin sequence analysis, suggesting they are applicable to both the de novo design and structure-based redesign of natural proteins.  相似文献   

19.
Proton magnetic resonance spectra at 500 MHz are reported for the oxidized and reduced forms of the 2[4Fe-4S]-ferredoxin from Clostridium pasteurianum. The reduced protein showed additional peaks in the 10–60 ppm region, which were previously unobserved, and there were significant differences between oxidized and reduced states in the whole region. The electron exchange rate in partially reduced ferredoxin is slow on the n.m.r. time scale when reduced with sodium dithionite, but fast when zinc reduced methyl viologen is used as reducing agent. We explain the difference between fast and slow exchange as being due to the different chemical properties of the two reducing agents.  相似文献   

20.
Knowing the manner of protein-protein interactions is vital for understanding biological events. The plant-type [2Fe-2S] ferredoxin (Fd), a well-known small iron-sulfur protein with low redox potential, partitions electrons to a variety of Fd-dependent enzymes via specific protein-protein interactions. Here we have refined the crystal structure of a recombinant plant-type Fd I from the blue green alga Aphanothece sacrum (AsFd-I) at 1.46 Å resolution on the basis of the synchrotron radiation data. Incorporating the revised amino-acid sequence, our analysis corrects the 3D structure previously reported; we identified the short α-helix (67-71) near the active center, which is conserved in other plant-type [2Fe-2S] Fds. Although the 3D structures of the four molecules in the asymmetric unit are similar to each other, detailed comparison of the four structures revealed the segments whose conformations are variable. Structural comparison between the Fds from different sources showed that the distribution of the variable segments in AsFd-I is highly conserved in other Fds, suggesting the presence of intrinsically flexible regions in the plant-type [2Fe-2S] Fd. A few structures of the complexes with Fd-dependent enzymes clearly demonstrate that the protein-protein interactions are achieved through these variable regions in Fd. The results described here will provide a guide for interpreting the biochemical and mutational studies that aim at the manner of interactions with Fd-dependent enzymes.  相似文献   

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