Ferredoxin reconstitution

In the simplest of metalloproteins, spontaneous association can be observed in vitro. For example, in IFe-containing rubredoxins and small [2Fe-2S] and [4Fe-4S] ferredoxins reconstitution occurs under reducing conditions with high concentrations of Fe and sulfide. However, these examples are exceptional and it is unlikely that such... 

Malkin R, Rabinowitz JC. 1966. The reconstitution of clostridial ferredoxins. Biochem Biophys Res Commun 23 822-7. 

Conversion of four-iron clusters into three-iron clusters by treatment with ferricyanide has been demonstrated for ferredoxin from Clostridium pasteurianum797 and from Bacillus stearother-mophilus.798 The reverse process, the conversion of a three-iron cluster into a [4Fe-4S] cluster, has been achieved by incubation with Fe2+ and S2 in the presence of dithiothreitol for ferredoxin from Desulfovibrio gigas.799 The reconstitution of [4Fe-4S] clusters allows the possibility of incorporating an isotopic label into the cluster. 

Bayer and his associates 16) are of the opinion, however, that the inorganic sulfide is of amino acid origin, being released from cysteine when ferredoxin is denatured. They reported the detection of inorganic sulfide from certain synthetic cysteine-iron derivatives, as well as a reconstitution of ferredoxin after the iron had been completely removed by treatment with a,a -dipyridyl. Since these data are in conflict with those discussed above, the resolution of this discrepancy will be awaited with interest. 

Bayer et al. (7) succeeded in reconstituting clostridial ferredoxin from a, a -dipyridyl-treated apoferredoxin in the presence of ferrous ion without the presence of a labile sulfur source. Based on this observation, they proposed that labile sulfur originates from cysteinyl residues in the presence of iron. Contrary to their finding, Rabinowitz and his coworkers (21, 33) demonstrated that the reconstitution of clostridial ferredoxin from mersaryl-, a, a -dipyridyl-, and trichloroacetic acid-treated apo-ferredoxins absolutely requires the addition of hydrogen sulfide. Consequently, they concluded that cysteine is not the source of labile sulfur . 

In contrast to their previous report, Bayer et al. (5) recently reported the requirement of Na2S in reconstituted spinach ferredoxin from a, a -dipyridyl-treated apoferredoxin in the presence of ferrous ion and 2-mercaptoethanol. In this report, they mentioned that, if 2-mercapto-ethanol is not purified before use, the addition of Na2S is not required. Therefore, one can assume that unpurified 2-mercaptoethanol contains H2S. We checked for the presence of H2S in once distilled 2-mercapto-ethanol by gas chromatography. The presence of H2S in samples of 2-mercaptoethanol could not be detected by this method. Since an excess amount of 2-mercaptoethanol is required for the reconstitution experiment, minute contamination of H2S in the sample may serve for the labile sulfur source, or H2S may be produced from 2-mercaptoethanol under the conditions of the reaction. 

Packer, L. and Cullingford, W. 1977. Stoichiometry of H2 production by an in vitro chloroplast, ferredoxin, hydrogenase reconstituted system. Z. Naturforsch. 33c, 113-115. 

Baginsky and Hatefi (155, 156) showed that loss of reconstitution activity appears to be related to a damage in the iron-sulfur system of the enzyme which is not detectable by assay for iron and labile sulfide content. They obtained a preparation of succinate dehydrogenase from complex II which exhibited no reconstitution activity but had an iron labile sulfide flavin ratio close to 8 8 1. They were then able to reactivate this enzyme for reconstitution by treating it with NajS, ferrous ions, and mercaptoethanol, essentially in the same manner as apoferredoxin had been previously converted to ferredoxin (181, 18Z). The reactivated preparation was able to reconstitute with alkali-treated submitochondrial particles or complex II. Analyses showed that the preparation had acquired additional iron and labile sulfide, but control experiments indicated that reconstitution activity was not a spurious effect. The reactiva-... 

The clusters thus generated have been found to have properties closely matching those of native proteins. Further studies have shown that N-H- -S hydrogen bonding to sulfur atoms within the cluster creates a small positive shift in redox potentials. The sequence Cys-(X)3-Cys-(X)2-Cys-(X)2-Cys, which includes sequence (6) and is often found in ferredoxins, has been shown to support efficient cluster reconstitution. ... 

Perhaps the best characterized example of a subsite differentiated [4Fe-4S] protein is aconitase, which catalyzes the citrate-isocitrate isomerization in the citric acid cycle (257). Aconitase isolated aerobically is inactive and contains a [3Fe-4S] cluster. Activity is restored by incubation with Fe and this also reconstitutes the [4Fe-4S] cluster. Oxidation of the core results in loss of the fourth iron atom, regenerating the [3Fe-4S] form. Mossbauer studies have demonstrated that only one of the four iron sites is exchanged (258). X-ray studies on both [3Fe-4S] and [4Fe-4S] forms of pig heart aconitase 258a) showed that insertion of iron into [3Fe-4S] occurs isomorphously. The positions of the common atoms in the two forms of the core agree to within 0.1 A, supporting the view of the [3Fe-4S] cluster as an iron-voided cubane. A similar result was obtained for the seven iron ferredoxin from Azo-... 

The EPR results for thePsaC Cysl4->Asp andPsaC Cys-51->Asp mutant proteins reconstituted with the PS-I core complex, combined with knowledge available on cysteine coordination patterns in bacterial ferredoxins containing two [4Fe 4S] clusters, as discussed below, permitted Zhao et a/. to conclude that cysteine coordination to the two iron-sulfur centers in PsaC assumes the same pattern as in bacterial ferredoxin. 

Fig. 13. Decay kinetics of flash-induced absorbance changes at 832 nm of intact PS-l complex (first column), of FeS-B-less PS-I complex prepared by HgClj treatment (second column), and FeS-B-reconstituted PS-l complex (third column). All samples contain 50 jxM PMSHj (reduced by dithionite). Samples in the top row contain no exogenous acceptors samples in the bottom row contain exogenously added ferredoxin (16 pM). Four consecutive short flashes spaced at 15-ms intervals were applied to the sample during each excitation cycle. Signals from three flash cycles were individually averaged, with a 1-minute dark interval between each cycle. Figure source Vassiliev, Jung, Yang and Golbeck (1998) PsaC subunit of photosystem I is oriented with iron-sulfur duster Fg as the immediate eiectron donor to ferredoxin and flavodoxin. Biophys J 74 2031.

IR Vassiliev Y-S Jung Pi ang and JH Golbeck (1998) PsaC subunit of photosystem I is oriented with iron-sulfur dusterpB as the immediate electron donor to ferredoxin and flavodoxin. Biophys J 74 2029-2035 Y-S Jung L Yu and JH Golbeck (1995) Reconstitution of iron-sulfur center Fb results in complete restoration of NADP photoreduction in Hg-treated photosystem I complexes from Synechococcus sp PCC 6301. Photosynthesis Res 46 249-255... 

The hexane-extracted particles that still retained one Q per P700 retained 81 % of the NADP -reduction activity ofthe unextracted control. In contrast, particles extracted with hexane-0.3% methanol had all the OQ removed and the NADP -reduction activity was completely lost. The NADP -reduction activity of the hexane/methanol-extracted particles could be reactivated by readdition of exogenous but only when the hexane extract was also added back. Exogenous OQ alone, even at a rather high concentration, could not reactivate NADP photoreduction and the hexane extract alone was also not effective, presumably because of its low Q content. The nature ofthe component in the hexane extract that contributes to reconstitution is as yet unknown. As the hexane extract contains OQ,chlorophylls,carotenoids,lipidsand other nonpolar molecules, some critical component is probably needed to ensure the correct membrane structure for binding ferredoxin and/or Fd-NADP -reductase. This conclusion is supported by the fact that activity of prior terminal acceptors such as the iron-sulfur centers do not require the hexane extract. 

An interesting fact is that when Fdll is converted into the apoprotein by treatment with trichloroacetic acid and the iron-sulfur center is reconstituted by the addition of iron and sulfide171 the protein becomes a [4 Fe-4 S] ferredoxin. Indeed the isotropic type signal of the oxidized form is no longer observed and upon reduction with dithionite a g = 1.94 type signal indicative of a + 1 oxidation state is observed at 4.2 K (see Fig. 8). This reconstituted material exhibits Mossbauer spectra almost identical to the observed for the reduced ferredoxin from Bacillus stearother-mophilus46) a protein for which a [4 Fe-4 S] center is firmly established (Fig. 9). 

Fig. 8. EPR spectrum of reconstituted D. gigas ferredoxin II in the reduced form. The spectrum was recorded at the following instrumental settings microwave power 2 mW frequency 9.223 GHz modulation amplitude 10 G temperature 18 K and gain 1000...

Fig. 9.a Mossbauer spectrum of reduced reconstituted D. gigas ferredoxin II taken at 4.2 K in a field of 600 G applied parallel to the observed 7-radiation b Mossbauer spectra of reduced D. gigas ferredoxin I (subtraction of 25 % of a spectrum of a three-iron center run in the same conditions was made). Spectrum obtained in natural abundance Fe56. Experimental conditions as for spectrum (a). 

The uper trace is the simulated Mossbauer spectra obtained using B. stearothermophilus [4 Fe-4 S] ferredoxin parameters for the reduced form46) and AEq and 6 values determined for reduced reconstituted D. gigas ferredoxin II. (Our unpublished results in collaboration with Drs. B.H. Huynh and E. Miinck)... 

The results obtained with the oligomeric forms of D.gigas ferredoxin and reconstituted material, implicate that the same amino-acid polypeptide chain can accomodate both [3 Fe—xS] and [4 Fe—4 S] centers. Fdll represents a very pure and homogeneous preparation in respect to the type of centers present, being an example of a protein containing only [3 Fe—xS] cores. The oligomeric Fdl is isolated in a form that contains both molecules with one [4 Fe-4 S] center and a small number (up to 25 %) of molecules with one (3 Fe-xS] center. This situation is different from that of the AvFe—Sill protein where in each molecule one center of the novel structure is associated with one [4 Fe-4 S] center of HiPIP type . 

It is possible to reconstitute ferredoxins from the isolated apoprotein by the addition of iron and sulfide (52). There are a variety of methods that can be used for reconstitution, but essentially it involves the conversion of the apoprotein to itr sulfhydryl form and the addition of either ferrous or ferric salts, in the presence of sodium sulfide (53) or elemental sulfur (5i). Normally, because of the diflSculties involved in mixing the sulfide and the iron, it is done in the presence of the reducing agent, such as mercaptoethanol, which seems to complex with the iron and sulfide to yield some soluble form of these elements that react with the apoprotein to form a ferredoxin that is indistinguishable from the native material. 

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