Mossbauer spectroscopy aconitase

One large class of non-heme iron-containing biomolecules involves proteins and enzymes containing iron-sulfur clusters. Iron-sulfur clusters are described in Sections 1.7 (Bioorganometallic Chemistry) and 1.8 (Electron Transfer) as well as in Section 3.6 (Mossbauer Spectroscopy). See especially Table 3.2 and the descriptive examples discussed in Section 3.6.4. Iron-sulfur proteins include rubredoxins, ferrodoxins, and the enzymes aconitase and nitrogenase. The nitrogenase enzyme was the subject of Chapter 6 in the hrst edition of this text—see especially Section 6.3 for a discussion of iron-sulfur clusters. In this... 

The rapid progress in the understanding of the active site of aconitase in the 1980 s has primarily originated from the work of H. Beinert and his collaborators. Three essential factors contributed to the success of this work 1) a ready and consistent source of enzyme (gram quantities), 2) a solid chemical and biochemical understanding of aconitase, and 3) close interactions with outstanding collaborators (most notably E. Munck s group for Mossbauer spectroscopy and B. M. 

At the time this suggested that the activation of aconitase required reduction of the Fe-S cluster, rather than the addition of iron. This would relegate iron to the role of an efficient reductant of the Fe-S cluster. Also, while reduction of the cluster by sodium dithionite was immediate, development of full enzyme activity required minutes (41,42). This suggested that perhaps cluster reduction triggered a conformational change which led to activation. Fluorescence studies on the reduction and activation process of aconitase by Ramsay (43,44) supported this idea. Again Mossbauer spectroscopy provided the key to understand the fate of the added iron (39). 

Trinuclear clusters have been detected in over 20 proteins as well as a number of enzymes, among them aconitase, beef heart succinate-ubiquinone oxidoreductase (120), Escherichia coli nitrate reductase (121), E. coli fumarate reductase (122), and succinate dehydrogenase (123). Selected instances of the occurrence of [3Fe-4S] clusters are listed in Table II. Because of the paramagnetic ground states of both oxidation levels, these clusters can be uniquely identified by a number of spectroscopic techniques. Among these, Mossbauer spectroscopy in applied magnetic fields (124, 128, 132, 141-143) and low temperature MCD spectroscopy (127, 138, 144-146) are decisive. While there are small spectroscopic differences among certain [3Fe-4S] centers, the similarities dominate and support the essential structure 3 for all. In a number of the earlier papers on protein... 

The [3Fe-4S]/[4Fe-4S] cluster interconversion of aconitase is clearly subsite-specific because one and the same site is involved in the two reactions. The similar interconversion of Dg Fd II may be equally subsite-specific, but this cannot be proven. In this case, the three subsites b of the [4Fe-4S] cluster cannot actually be equivalent but are indistinguishable by the technique of resolution at hand, Mossbauer spectroscopy. What is... 

Further evidence for direct interaction of the substrate with the cluster was provided by EPR spectroscopy. Although the catalytically most active state of aconitase is the EPR-silent [4Fe S], this state could be reduced to the [4Fe-4S] state for examination by EPR. The [4Fe-4S] state of aconitase appears to retain approximately 30% activity, and exhibits a rhombic EPR signal with g = 2.06, 1.93, 1.86, which is perturbed upon addition of substrates or analogs to a more rhombic signal with g = 2.04, 1.85, and 1.78. " The increased rhombicity upon addition of substrate supported the conclusions from Mossbauer spectroscopy, that addition of substrate causes one iron site to become distinct from the other three sites in the cluster. 

Mossbauer spectroscopy of the [4Fe-4S]+ aconitase in the presence of substrate shows an even more dramatic effect on the unique site than in the case of [4Fe-4S] " aconitase, with the FCa parameters indicating largely ferrous character and a localized valence at this site (Table 1). The Feb sites also differentiate, with two (Fcb2 and Fcbs) essentially unaffected by substrate addition, and the third (Febi), which shows increased ferrous character. That both the Fea and Febi sites show increased ferrous character, while the Fcb2 and Fcbs sites are essentially unchanged upon substrate binding, requires that electron density be withdrawn either from the bound substrate or... 

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. 

The proposal that the sulfonium of AdoMet interacts with a //3-bridging sulfide of the cluster left open the question of the role for the putative unique iron site. A unique iron site was in fact demonstrated in PFE-AE via specific isotopic labeling of the unique iron site with Fe, in the same way described previously for aconitase (see Section 8.27.2.3), which allowed probing of the unique site by Mossbauer spectroscopy. " The Mossbauer parameters for this unique site in the absence of AdoMet were typical for iron in [4Fe-4S] clusters (Table 2). Addition of AdoMet, however, dramatically altered the Mossbauer parameters (Table 2). The new parameters are inconsistent with coordination of a sulfur to the unique site, but are consistent with an increase in coordination number of the unique iron to 5 or 6, and/or coordination of an ionic ligand."" Thus it was proposed that the carboxylate of AdoMet coordinated to the unique iron site of the cluster."" ... 

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