Protein cubane sites

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

These studies of protein-bound heterometallic cubanes have amply demonstrated that the heterometal site is redox active and able to bind small molecules. Although they have yet to be identified as intrinsic components of any protein or enzyme (except as part of the nitrogenase FeMo cofactor cluster (254)), they are clearly attractive candidates for the active sites of redox enzymes. 

Fig. 6. A schematic view of the [3Fe-4S] Emd [4Fe-4S] cores, as versatile structures. The absence of one site leads to the formation of a [3Fe-4S] core. The cubane structure can incorporate different metals (in proteins, M = Fe, Co, Zn, Cd, Ni, Tl, Cs), and S, N, O may be coordinating atoms from hgands (Li). The versatihty csm be extended to higher coordination number at the iron site and a water molecule can even be a ligand, exchangeable with substrate (as in the case of aconitase (,87)). The most characteristic binding motifs are schematically indicated, for different situations proteins accommodating [3Fe-4S], [4Fe-4S], [3Fe-4S] + [4Fe-4S], and [4Fe-4S] -I- [4Fe-4S] clusters. A disulfide bridge may replace a cluster site (see text).

MSssbauer studies of Fd n have established that the protein contains a 3-Fe cluster, and that the three iron sites reside in a predominantly tetrahedral environment of sulfur ligands. The cluster has a Fe3S4 stoichiometry (see Beinert and Thomson (17)), and EXAFS data (14) suggest a compact core with an Fe-Fe distance of 2.7 A, just as observed for Fe4 cubanes. The structure is perhaps close to that shown on the left of Figure 1. Thus, we picture the Fe3S4 cluster as a cubane Fc4S4 cluster from which one Fe has been removed. This view is supported by the observation... 

Many microbes contain a hydrogenase that lacks nickel. The best studied of these proteins is the hydrogenase from C. pasteurianum (Adams, 1990). The active site of this protein has been called the H cluster. Two structures of this protein recently appeared, a 1.6 structure of the het-erodimeric Desulfovibrio desuljuricans enzyme (Nicolet et al., 1999) and the 1.8 structure of the Cpl enzyme from Clostridium pasteurianum (Peters et al., 1998). The H cluster at the active site contains 6Fe as proposed earlier (Adams et al., 1989). There are two subcomponents a typical [4Fe64S] cubane bridged by a cysteine residue to an active site binuclear Fe center (Figure 4b). The unusual nature of the iron site in the NiFe hydrogenase is... 

Stmctural data of the cuboidal model compound and of protein sites indicate that the cuboidal cluster is stmcturally closely related to the cubane cluster and shares many of the same stmctural properties. 

Enzyme activity has been recognized in protein-bound 3 1 site-differentiated clusters, in which the cubane [Fe4S4]... 

Electron transport between cubane [Fe4S4] clusters, cytochrome c, or Cu + centers in blue copper proteins " and the periphery of the proteins has been examined by complexing ruthenium species to surface histidines. In the case of the iron sulfur cubane in Chromatium vinosum, four surface histidines served as points of ruthenium attachment. The rates of electron transport from the Fe4S4 core to ruthenium varied over two orders of magnitude and were used to diagnose the preferred channel for electron transport. Cysteine and lysine residues have also been used as binding sites in studies of cytochrome c and cytochrome P450 cam proteins. 

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-... 

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