Aconitase Iron-sulfur clusters

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. 

FIGURE 20.7 (a) The aconitase reaction converts citrate to cis-aconitate and then to isocitrate. Aconitase is stereospecific and removes the pro-/ hydrogen from the pro-/ arm of citrate, (b) The active site of aconitase. The iron-sulfur cluster (red) is coordinated by cysteines (yellow) and isocitrate (white). 

When induced in macrophages, iNOS produces large amounts of NO which represents a major cytotoxic principle of those cells. Due to its affinity to protein-bound iron, NO can inhibit a number of key enzymes that contain iron in their catalytic centers. These include ribonucleotide reductase (rate-limiting in DNA replication), iron-sulfur cluster-dependent enzymes (complex I and II) involved in mitochondrial electron transport and cis-aconitase in the citric acid cycle. In addition, higher concentrations of NO,... 

To successfully describe the structure and function of nitrogenase, it is important to understand the behavior of the metal-sulfur clusters that are a vital part of this complex enzyme. Metal-sulfur clusters are many, varied, and usually involved in redox processes carried out by the protein in which they constitute prosthetic centers. They may be characterized by the number of iron ions in the prosthetic center that is, rubredoxin (Rd) contains one Fe ion, ferredoxins (Fd) contain two or four Fe ions, and aconitase contains three Fe ions.7 In reference 18, Lippard and Berg present a more detailed description of iron-sulfur clusters only the [Fe4S4] cluster typical of that found in nitrogenase s Fe-protein is discussed in some detail here. The P-cluster and M center of MoFe-protein, which are more complex metal-sulfur complexes, are discussed in Sections 6.5.2. and 6.5.3. 

Iron-sulfur clusters, such as those found in the enzyme aconitase discussed in Section 7.9.2.1, cannot be treated using the 16-e or 18-e rules. Other frameworks exist to treat large metal clusters, and these have some utility in treating [Fe,cSj,]" clusters. One method treats the number of metal atoms and the metal-metal bonds in a cluster according to the following formula ... 

In this text, iron-sulfur clusters are discussed because they appear in proteins and enzymes (1) cytochrome b(6)f, Rieske [2Fe-2S] cluster (Section 7.5 and Figure 7.26) (2) cytochrome bci, Rieske [2Fe-2S] cluster (Section 7.6 and Figure 7.30) and (3) aconitase, [4Fe-4S] cluster (Section 7.9.2.1, and Figure 7.50). The iron-sulfur protein (ISP) component of the cytochrome b(6)f and cytochrome bci complexes, now called the Rieske ISP, was first discovered and isolated by John S. Rieske and co-workers in 1964 (in the cytochrome bci complex). More information about the RISP is found in Section 7.5.1. Section 7.9.2 briefly discusses other proteins with iron-sulfur clusters—rubredoxins, ferrodoxins, and the enzyme nitrogenase. The nitrogenase enzyme was the subject of Chapter 6 in the hrst edition of this text— see especially the first edition s Section 6.3 for a discussion of iron-sulfur clusters. In this second edition, information on iron-sulfur clusters in nitrogenase is found in Section 3.6.4. See Table 3.2 and the descriptive examples discussed in Section 3.6.4. 

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... 

Figure 7 0 Important amino acid residues surrounding the isocitrate substrate (black carbon atoms) and the iron-sulfur cluster of aconitase (PDB 7ACN). Visuahzed using The PyMOL Molecular Graphics System and ChemDraw Ultra, version 10.0. (Printed with permission of Delano Scientific, LLC and CambridgeSoft Corporation.) (See color plate)...

Proteins containing iron-sulfur clusters are ubiquitous in nature, due primarily to their involvement in biological electron transfer reactions. In addition to functioning as simple reagents for electron transfer, protein-bound iron-sulfur clusters also function in catalysis of numerous redox reactions (e.g., H2 oxidation, N2 reduction) and, in some cases, of reactions that involve the addition or elimination of water to or from specific substrates (e.g., aconitase in the tricarboxylic acid cycle) (1). 

Iron-sulfur clusters (7) occur as prosthetic groups in oxidoreductases, but they are also found in lyases—e.g., aconitase (see p. 136) and other enzymes. Iron-sulfur clusters consist of 2-4 iron ions that are coordinated with cysteine residues of the protein (-SR) and with anorganic sulfide ions (S). Structures of this type are only stable in the interior of proteins. Depending on the number of iron and sulfide ions, distinctions are made between [Fe2S2], [Fe3S4], and [Fe4S4] clusters. These structures are particularly numerous in the respiratory chain (see p. 140), and they are found in all complexes except complex IV. 

Fridovich recently summarized important aspects concerning the accurate detection and measurement of superoxide. He indicates that univalent reduction of O2 to superoxide is a facile process, but the instability of superoxide in aqueous solutions hinders its detection and measurement. To measure intracellular superoxide, he favors use of the rapid inactivation of [4Fe-4S]-containing dehydratases (such as aconitase) by oxidation of their iron-sulfur clusters. See Oxygen, Oxides Oxygen Radicals... 

A brief historical note on the structure of the iron-sulfur clusters in ferredoxins is relevant. After the first analytical results revealed the presence of (nearly) equimolar iron and acid-labile sulfur, it was clear that the metal center in ferredoxins did not resemble any previously characterized cofactor type. The early proposals for the Fe S center structure were based on a linear chain of iron atoms coordinated by bridging cysteines and inorganic sulfur (Blomstrom et al., 1964 Rabino-witz, 1971). While the later crystallographic analyses of HiPIP, PaFd, and model compounds (Herskovitz et al., 1972) demonstrated the cubane-type structure of the 4Fe 4S cluster, the original proposals have turned out to be somewhat prophetic. Linear chains of sulfide-linked irons are observed in 2Fe 2S ferredoxins and in the high-pH form of aconitase. Cysteines linked to several metal atoms are present in metallothionein. The chemistry of iron-sulfur clusters is rich and varied, and undoubtedly many other surprises await in the future. 

It is now clear that in addition to their widespread involvement in electron transfer pathways, iron-sulfur clusters function as catalytic centers in a wide variety of enzymes. The first example of such an enzyme is aconitase. It was at first thought that the role of the iron-sulfur group was regulatory, but it is now clear that in this enzyme the iron-sulfur group is part of the catalytic site. One of the iron atoms can coordinate water or hydroxyl and plays a key role in the isomerization catalyzed by the enzyme (Emptage et al., 1983). 

The iron responsive element, a critical factor in the control of proteins involved in iron utilization, has been identified as the cytoplasmic form of the iron-sulfur protein aconitase (Kennedy et al., 1992). Activated macrophages have been shown to activate this element, presumably by attack of the iron-sulfur cluster by NO (Drapier et al., 1993). It has been claimed that this attack is mediated by peroxynitrite (Castro et al., 1994 Hausladen and Fridovich, 1994, but this conclusion is not universally accepted. 

It is noteworthy that except for the Rieske center in Complex III, Complexes I and 11 are home to all the iron-sulfur clusters in the mitochondrial electron transfer chain and consequently most of the iron-containing carriers in the entire sequence. Hibbs subsequently showed that CAM-injured cells lose a substantial portion of their total intracellular iron (Hibbs et al., 1984) [later studies specifically identified loss of mitochondrial iron (Wharton et al., 1988)] and Drapier and Hibbs (1986) showed that the activity of another iron-sulfur-containing enzyme, aconitase, is also lost. In early 1987 Hibbs reported that the cytostatic actions of CAMs requires the presence of only one component in culture medium, L-arginine (Hibbs et al., 1987b). Thus, the stage was set for the discovery of a unique reactive species that targets intracellular iron, produced by CAMs. 

Aconitase contains iron in the form of an Fe4S4 iron-sulfur cluster (Fig. 13-4).81-83 However, the enzyme is usually isolated in a form that does not show its maximum activity until it has been incubated with ferrous iron (Fe2+). The inactive form of the enzyme is thought to contain an Fe3S4 cluster (Chapter 16) which is converted back to the Fe4S4 cluster by the incubation... 

Aconitase exists as both mitochondrial and cytosolic isoenzyme forms of similar structure. However, the cytosolic isoenzyme has a second function. In its apoenzyme form, which lacks the iron-sulfur cluster, it acts as the much-studied iron regulatory factor, or iron-responsive element binding protein (IRE-BP). This protein binds to a specific stem-loop structure in the messenger RNA for proteins involved in iron transport and storage (Chapter 28).86/9°... 

The role of the iron-sulfur clusters in many of the proteins that we have just considered is primarily one of single-electron transfer. The Fe-S cluster is a place for an electron to rest while waiting for a chance to react. There may sometimes be an associated proton pumping action. In a second group of enzymes, exemplified by aconitase (Fig. 13-4), an iron atom of a cluster functions as a Lewis acid in facilitating removal of an -OF group in an a,P dehydration of a carboxylic acid (Chapter 13). A substantial number of other bacterial dehydratases as well as an important plant dihydroxyacid dehydratase also apparently use Fe-S clusters in a catalytic fashion.317 Fumarases A and B from E. coli,317 L-serine dehydratase of a Pepto-streptococcus species,317-319 and the dihydroxyacid... 

Some of the earlier approaches to the characterization of iron-sulfur clusters have proved to be inadequate in the presence of mixtures of clusters and, particularly, three-iron clusters. Thus the technique of core extrusion has worked well for [2Fe-2S] and [4Fe-4S] cores, but led to confusing results for aconitase and Fdl from A vinelandii as [2Fe-2S] cores were extruded. 

Fig. 2.2. Cubane of iron-sulfur cluster (4Fe-4S), prostetic group of cytosolic aconitase...

Aconitase was the first protein to be identified as containing a catalytic iron-sulfur cluster [24-26]. It was also readily established that the redox properties of the [4Fe-4S](2+ 1+) cluster do not play a role of significance in biological functioning the 1 + oxidation state has some 30% of the activity of the 2+ state [25], Since then several other enzymes have been identified or proposed to be nonredox iron-sulfur catalysts. They are listed in Table 2. It appears that all are involved in stereospecific hydration reactions. However, these proteins are considerably less well characterized than aconitase. In particular, no crystal structural information is available yet. Therefore, later we summarize structural and mechanistic information on aconitase, noting that many of the basic principles are expected to be relevant to the other enzymes of Table 2. 

Hail DJ, Rouaoult TA, Tang CK, Chin J, Harford JB, Klausner RD (1992) Reciprocal control of RNA-binding and aconitase activity in the regulation of the iron-responsive element binding protein role of the iron-sulfur cluster. Proc Natl Acad Sci USA 89 7536-7540... 

The stmctural, electronic, biological, and magnetic properties of iron sulfur clusters in metalloproteins has been reviewed focussing particularly on [4Fe-4S ]"+ (n = 1-3) centres, and on synthetic [Fe3S4] and heterometallic [MFe3 S4] clusters. The chemistry and properties of aconitase have been reviewed in the context of its spectroscopic properties, structure, mechanistic function, and its relationship to the iron-regulatory protein. ... 

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