Iron sulfur clusters reconstitution

Phylloquinone has become a focus of a variety of structure function relationships due to its central role in electron transfer. The PhQ molecules can be extracted from PS I using dry or water-saturated diethyl ether o or using hexane containing 0.3% methanolWater-saturated ether extraction also removes a significant number of antenna chlorophylls, and aU of the carotenoids (and probably Hpids), while leaving the iron-sulfur clusters unaffected. As might be expected, both extraction procedures block room-temperature electron transfer from Aq to the iron-sulfur clusters. - Reconstitution of one PhQ fully restores electron transfer from A to the iron-sulfur clusters. Iwaki and coworkers reported that PhQ could be replaced by a variety of quinones with appropriate redox potentials. Moreover, an assortment of non-native quinones can be inserted into the PhQ binding site in In particular. 

Whereas many cognate apoenzymes can be reconstituted with iron/sulfur clusters by simple and essentially alchemistic procedures using Fe + and sulfide ions under anaerobic conditions, a highly complex enzymatic machinery is used in vivo for the synthesis of iron/sulfur clusters and their transfer to the target enzymes. Sulfide ions required for cluster synthesis are obtained from cysteine (1) via a persulfide of a protein-bound cysteine residue (2) pyridoxal phosphate is required for the formation of the persulfide intermediate (Fig. 1) (12). 

The sulfur insertion occurs with scrambling of stereochemistry at C-1 and with retention at C-4 [135,136]. The protons at C-2 and at C-3 are not lost during the reaction. 1-Mercaptodethiobiotin may be an intermediate [137,138]. In the reconstituted system, sulfur from cysteine or SAM was not incorporated into biotin [132] suggesting that the iron sulfur cluster is the sulfur source. 

The PS-I core protein and its reconstitution with the PsaC subunit will also be discussed in more detail in Chapter 31 in connection with another PS-I iron-sulfur cluster, FeS-X. As we will see, the forward electron transfer from FeS-X to [FeS-A/FeS-B] can be very efficient in the reconstituted complex. It suffices to say that the PS-I core protein is utilized here as a natural reducing vehicle to carry out photochemical reduction of the secondary electron acceptors FeS-A and FeS-B so they may be characterized by EPR spectroscopy. The premise of the mutation approach was that replacement of a given cysteine might cause an alteration of a specific [4Fe 4S] cluster and thus lead to some interference in the functioning of that iron-sulfur cluster. 

As mentioned above, a 1 1 stoichiometric relationship between the photooxidized donor, P700 and the reduced terminal acceptors has not yet been established for the PS-I reaction center. As previously noted, the total amount of the recognized terminal acceptors reduced at 15 K is, on average, approximately 74% of the P700 photooxidized. Even more intriguing, in the reconstituted PS-I complexes from either the Cys-14->Asp or Cys-51->Asp mutant PsaC protein, the extent of photoreduction of each intact iron-sulfur cluster at 15 K remained nearly the same as in the wild-type preparation. The presence of the other cluster that was made photochemically inactive by site-directed mutagenesis apparently had no effect on the behavior of the unaltered cluster. 

Pyruvate formate-lyase activating enzyme is the member of the radical-SAM family whose cluster properties are most similar to those of aconitase. The cluster in pyruvate formate-lyase activating enzyme is quite labile, and in fact until 1997 it was not known that the enzyme contained an iron-sulfur cluster, as all preparations to that time had been done aerobically, under which conditions the cluster falls apart. It was initially reported that PFL-AE contained a mixture of [2Fe-2S] and [4Fe-4S] clusters, and subsequent reconstitution studies of the apo enzyme provided evidence for a [4Fe-4S] cluster. Further studies showed that anaerobic isolation resulted in purification of a form of PFL-AE that contained primarily [3Fe-4S] clusters, which upon reduction converted to [4Fe-4S] clusters.This reductive cluster conversion from [3Fe S] to [4Fe-4S] clusters even in the absence of added iron was remarkably reminiscent of aconitase (see Section 8.27.2.2), and suggested a labile cluster site. Adding to the similarity to aconitase, Mossbauer spectroscopy provided evidence for a linear [3Fe-4S] cluster in PFL-AE isolated under appropriate conditions.Therefore all of the cluster forms previously identified in aconitase were also found in PFL-AE, and like aconitase it appeared to be relatively simple to interconvert between these cluster forms. 

Reconstitution of the Fx Iron-sulfur Cluster in the Photosystem I Core Protein with Kevin Parrett [ref. 12]... 

Our current work with the Photosystem I core protein reconfirms that the Fx iron-sulfur cluster can be oxidatively converted to zero-valence sulfur by treatment with 3 M urea and 5 mM potassium ferricyanide [13,14]. Under these conditions, the P700 flash-induced optical transient has a lifetime of 5 is due to the relaxation of the P700 triplet state, indicating that the P700 Aq primary charge separation and recombination process has remained intact (Fig. 3a,b). We have now found that the Fx iron-sulfur cluster can be reconstituted by addition of ferrous iron, sodium sulfide, and 6-mercaptoethanol to the oxidatively-denatured Photosystem I core protein [see refs. 15 16 for methodology employed]. After incubation for 24 hr, the 5 is optical transient becomes replaced with a 1.2-ms optical transient that is characteristic of the P7(X)+-Fx backreaction (Fig. 3c). 

Reconstitution of the FaIFb Iron-Sulfur Clusters in the 8.9-kDa Polypeptide with Tetemke Mehari and Kevin Parrett [ref. 17]... 

We found that the oxidatively-denatured Fa/Fb iron-sulfur clusters can also be reconstituted by incubating the apoprotein with FeCls and Na2S in the presence of 6-mercaptoethanol. The ESR spectra of the native Fa/Fb protein, the Fa/Fb apoprotein and the reconstituted Fa/Fb protein are shown in Fig. 5. The chemically-reduced spectmm of... 

The reconstitution protocol outlined here allows complete experimental control over iron-sulfur cluster denaturation and reconstitution in the Fx and Fa/Fb polypeptides, and rebinding of the reconstituted Fa/Fb polypeptide to the Photosystem I core protein to yield the intact Photosystem I complex. Studies which include chemical or genetic modification of the Fx and Fa/Fb apoproteins followed by cluster reconstitution can now be performed prior to in vitro reassembly of the Photosystem I complex. 

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. 

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