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Query: UNIPROT:O14944 (EPR)
 13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The biochemical and biophysical properties of the Ala45Ser mutant of the [2Fe-2S] ferredoxin from vegetative cells of the cyanobacterium Anabaena sp. 7120 are described. This novel protein, which incorporates the residue present in many higher plant ferredoxins into the analogous position of a typical cyanobacterial ferredoxin, was prepared to probe the origin of the characteristic spectrochemical and functional differences between the ferredoxins from these two sources. The variant protein was produced by site-directed mutagenesis and was expressed as the holoprotein in Escherichia coli. Although the UV-vis spectrum of the Ala45Ser mutant was indistinguishable from that of the wild-type (WT) protein, the circular dichroism (CD) spectrum of the mutant was distinct and similar in appearance to that of spinach ferredoxin, which possesses a Ser residue at the analogous position. The values of the principal g factors of the EPR spectrum of the dithionite-reduced mutant protein differed from those of the WT spectrum and resembled those of plant ferredoxins containing serine at position 45. Analysis of the mutant EPR spectrum according to the method of Blumberg indicated greater covalent interactions between the localized ferrous site of the cluster and the protein matrix relative to the WT protein. The resonance Raman spectrum of Ala45Ser Anabaena ferredoxin was distinct from the spectrum of the WT protein and showed exceptional similarity to the spectrum of higher plant ferredoxins, such as spinach ferredoxin. The mutant protein spectrum displayed considerably greater deuterium dependent isotope shifts for bands ascribed to terminal Fe-S stretching modes than did the WT spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The environment of [2Fe-2S] clusters in ferredoxins: the role of residue 45 probed by site-directed mutagenesis. 757 85

The 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 comprises two components with four redox active sites necessary for activity. We present an EPR characterization of the iron-sulfur centres in the purified reductase and oxygenase component of this novel enzyme system. The oxygenase component was identified as a Rieske [2Fe2S] protein on the basis of its characteristic EPR spectrum with gz,y,x = 2.01, 1.91, 1.76 and gav = 1.893. The reductase component, an iron-sulfur flavoprotein, contained a [2Fe2S] cluster with gz,y,x = 2.03, 1.94, 1.89 and the average g-value (gav) of 1.953, typical of a ferredoxin-type centre. In redox titrations at pH 7, the midpoint potentials were determined to be -180 mV +/- 30 mV and -100 mV +/- 10 mV for the reductase and oxygenase component, respectively. A detailed comparison to other multicomponent enzyme systems is presented pointing out the EPR and redox properties of the FeS centres involved.
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PMID:The 2Fe2S centres of the 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 studied by EPR spectroscopy. 757 19

We report the first purification and characterization of a pyruvate-ferredoxin oxidoreductase (POR) from a sulfate-reducing bacterium, Desulfovibrio africanus. The enzyme as isolated is highly stable in the presence of oxygen and exhibits a specific activity of 14 U/mg. D. africanus POR is a 256 kDa homodimer which contains thiamine pyrophosphate (TPP) and iron-sulfur clusters. EPR spectroscopic study of the enzyme indicates the presence of three [4Fe-4S]2+/1- centers/subunits. The midpoint potentials of the three centers are -390 mV, -515 mV and -540 mV. The catalytic mechanism of POR involves a free radical intermediate which disappears when coenzyme A is added. This behaviour is discussed in terms of an electron-transport chain from TPP to the acceptor. The enzyme activated by dithioerythritol shows an exceptionally high activity compared with other mesophile PORs and becomes very sensitive to oxygen in contrast to the enzyme before activation. The comparison of EPR spectra given by the as isolated and activated enzymes shows that neither the nature, nor the arrangement of FeS centers are affected by the activation process. D. africanus ferredoxins I and II are involved as the physiological electron carriers of the enzyme. POR was shown to be located in the cytoplasm by immunogold labelling.
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PMID:Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus. 761 53

Mutations of the conserved residue Glu-92 to lysine, glutamine, and alanine have been performed in the recombinant ferredoxin I of spinach leaves. The purified ferredoxin mutants were found twice as active with respect to wild-type protein in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase in the presence of ferredoxin. Cyclic voltammetry and EPR measurements showed that the mutations cause a change in the [2Fe-2S] cluster geometry, whose redox potential becomes approximately 80 mV less negative. These data point to a role of the Glu-92 side-chain in determining the low redox potential typical of the [2Fe-2S] cluster of chloroplast and cyanobacterial ferredoxins. Also a ferredoxin/ferredoxin-NADP+ reductase chimeric protein obtained by gene fusion was overproduced in Escherichia coli and purified. Fusion of the ferredoxin with its reductase causes only minor effects to the iron-sulfur cluster, as judged by cyclic voltammetry and EPR measurements.
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PMID:Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties. 762 9

The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one [3Fe-4S] cluster and one [4Fe-4S] cluster. This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin. The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe-S clusters under anaerobic conditions. A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms. The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH. The reconstituted ferredoxin was purified and shown to be distinct from the native [7Fe-8S] ferredoxin, based on several biochemical and spectroscopic criteria. In the oxidized state, EPR revealed the quasi-absence of [3Fe-4S] cluster. 1H-NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2[4Fe-4S] ferredoxin. This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur. Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe-S clusters, without formation of [7Fe-8S] form. Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron-transfer assay coupled to nitrogenase activity. The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins.
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PMID:Characterization of a 2[4Fe-4S] ferredoxin obtained by chemical insertion of the Fe-S clusters into the apoferredoxin II from Rhodobacter capsulatus. 763 51

The fdxN gene from Rhizobium meliloti encoding a bacterial-type ferredoxin (FdxN) was expressed in Escherichia coli under the control of the lac promoter. The fdxN gene product was purified under anaerobic conditions by ion-exchange chromatography and gel-filtration steps using an antiserum raised against an FdxN-LacZ fusion protein as a detection system. The purified ferredoxin was shown to be identical to the predicted R. meliloti FdxN protein in its amino acid composition and N-terminal amino acid sequence. Chemical determination of the iron content revealed 8.6 +/- 0.6 mol Fe/mol FdxN. The ultraviolet/visible absorption spectrum of the FdxN protein in the oxidized form exhibited maxima at 284 nm and 378 nm, with an A378/A284 ratio of 0.7. EPR spectroscopy revealed a rhombic signal when FdxN was partially reduced, and a broad signal indicative of spin-spin interaction when fully reduced, suggesting the presence of two Fe-S cluster/ferredoxin polypeptide. Our data suggest that FdxN contains two [4Fe-4S] clusters. Purified FdxN was able to mediate electron transport between illuminated chloroplasts and Rhodobacter capsulatus nitrogenase in vitro.
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PMID:A Rhizobium meliloti ferredoxin (FdxN) purified from Escherichia coli donates electrons to Rhodobacter capsulatus nitrogenase. 764 75

A soluble low-potential cytochrome c549 has been purified in milligram quantities from the cyanobacterium Synechocystis sp. PCC 6803. The protein exhibits an acid isoelectric point of 3.9, a molecular mass of 15.8 kDa, and a midpoint redox potential value of -250 mV at pH 7.0 EPR and 1H NMR studies suggest a low-spin heme iron with bis-histidine coordination at the fifth and sixth positions. EDTA-photoreduced 5-deazariboflavin has been used as the electron-donating system to study, by laser flash absorption spectroscopy, the electron transfer reactions between Synechocystis cytochrome c549 and redox proteins involved in the cyclic electron flow around photosystem I. The second-order rate constants (k2) obtained for ferredoxin (or flavodoxin) oxidation by Synechocystis cytochrome c549 are rather low (ca. 10(5) M-1 s-1), thus suggesting that this low-potential heme-protein does not operate as the primary electron carrier for either transferring electrons to the cytochrome b6f complex in cyclic photophosphorylation or to hydrogenase during anaerobic metabolism. The k2 values for plastocyanin reduction by cytochrome c549 are about 100 times higher (ca. 10(7) M-1 s-1), but it remains to be determined whether or not this reaction actually reflects a physiological process.
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PMID:Purification and physicochemical properties of the low-potential cytochrome C549 from the cyanobacterium Synechocystis sp. PCC 6803. 772 71

EPR spectroscopy was used to investigate the cytochrome P-450-dependent steroid hydroxylase ecdysone 20-mono-oxygenase of the cotton leafworm (Spodoptera littoralis) and the redox centres associated with membranes from the fat-body mitochondrial fraction. Intense features at g = 2.42, 2.25 and 1.92 from oxidized mitochondrial membranes have been assigned to the low-spin haem form of ferricytochrome P-450, probably of ecdysone 20-mono-oxygenase. High-spin cytochrome P-450 (substrate-bound) was tentatively assigned to a signal at g = 8.0, which was detectable from membranes as prepared. An EPR signal characteristic of a [2Fe-2S] cluster detected from the soluble mitochondrial matrix fraction has been shown to be distinct from the signals associated with mitochondrial NADH dehydrogenase and succinate dehydrogenase, and has therefore been attributed to a ferredoxin. We conclude that the S. littoralis fat-body mitochondrial electron-transport system involved in steroid 20-hydroxylation comprises both ferredoxin and cytochrome P-450 components, and thus resembles the enzyme systems of adrenocortical mitochondria. EPR signals characteristic of the respiratory chain were also observed from fat-body mitochondria and assigned to the iron-sulphur clusters associated with Complex I (Centres N1, N2), Complex II (Centres S1, S3), Complex III (the Rieske centre), and the copper centre of Complex IV, demonstrating similarities to mammalian mitochondria. The reduced membrane fraction also yielded a major resonance at g = 2.09 and 1.88 characteristic of the [4Fe-4S] cluster of electron-transferring flavoprotein: ubiquinone oxidoreductase. As the fat-body is the major metabolic organ of insects, this protein is presumably required for the beta-oxidation of fatty acids in mitochondria. High-spin haem signals in the low-field region of spectra also demonstrated that the mitochondrial fraction contains relatively high concentrations of catalase.
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PMID:EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm). 774 2

Detailed redox titrations monitored by EPR and UV-visible spectroscopies have been carried out on the dimeric ferredoxins I and II from Desulfovibrio vulgaris Miyazaki. Ferredoxin II contains a unique [4Fe-4S] cluster per subunit characterized by a midpoint potential of -417 mV at 24 degrees C. The enthalpic and entropic contributions to the redox free energy variation of this cluster have been determined from the temperature dependence of the midpoint potential and compared to the data reported for other iron-sulfur proteins. The molecular arrangement of the two subunits is such that two [4Fe-4S]i+ clusters are magnetically coupled in the fully reduced state of the protein. Ferredoxin I contains one [3Fe-4S] and one [4Fe-4S] cluster per subunit, whose spectral and redox properties are very similar to those of the same clusters in ferredoxin III from Desulfovibrio africanus. The strong heterogeneity in the redox properties of the [3Fe-4S] center supports a bridging position between the N-terminal and C-terminal parts of the protein.
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PMID:EPR and redox characterization of ferredoxins I and II from Desulfovibrio vulgaris Miyazaki. 777 85

A novel iron-sulfur protein from the photosynthetic purple bacterium Rhodopseudomonas palustris was purified to homogeneity and identified as a ferredoxin on the basis of its physicochemical properties. Based on the uv/vis spectrum, iron quantitation, cyclic voltammetry, EPR, and 1H NMR data, the ferredoxin is found to contain two iron-sulfur clusters, one [3Fe-4S] and one [4Fe-4S], which places this protein in the class of 7Fe ferredoxins. The voltammetric peak potentials of the two clusters are -0.260 and -0.560 V at pH 8.0. The molecular mass around 19 kDa makes this protein the heaviest known in this class. This paper further demonstrates the diagnostic power of magnetic resonance spectroscopies in recognition of the two types of clusters in iron-sulfur proteins.
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PMID:Magnetic resonance of Fe-S clusters: isolation and characterization of a 7Fe ferredoxin from Rhodopseudomonas palustris. 779 75


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