Characterization of iron-sulfur

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. 

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. 

For all known cases of iron-sulfur proteins, J > 0, meaning that the system is antiferromagnetically coupled through the Fe-S-Fe moiety. Equation (4) produces a series of levels, each characterized by a total spin S, with an associated energy, which are populated according to the Boltzmann distribution. Note that for each S level there is in principle an electron relaxation time. For most purposes it is convenient to refer to an effective relaxation time for the whole cluster. 

The characteristic derivative-shaped feature at g 1.94 first observed in mitochondrial membranes has long been considered as the sole EPR fingerprint of iron-sulfur centers. The EPR spectrum exhibited by [4Fe-4S] centers generally reflects a ground state with S = I and is characterized by g values and a spectral shape similar to those displayed by [2Fe-2S] centers (Fig. 6c). Proteins containing [4Fe-4S] centers, which are sometimes called HIPIP, essentially act as electron carriers in the photoinduced cyclic electron transfer of purple bacteria (106), although they have also been discovered in nonphotosynthetic bacteria (107). Their EPR spectrum exhibits an axial shape that varies little from one protein to another with g// 2.11-2.14 and gi 2.03-2.04 (106-108), plus extra features indicative of some heterogeneous characteristics (Pig. 6d). 

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. 

Nuth M,YoonT, Cowan JA. 2002. Iron-sulfur cluster biosynthesis Characterization of iron nucleation sites for the assembly of the [2Fe-2S] cluster core in IscU proteins. J Am Chem Soc 124 8774-5. 

The complexity of the low temperature MCD spectra of the oxidized and reduced trinuclear cluster shows the multiplicity of the predominantly S — Fe charge transfer transitions that contribute to the absorption envelope. While MCD spectroscopy provides a method of resolving the electronic transitions, assignment cannot be attempted without detailed knowledge of the electronic structure. However, the complexity of the low temperature MCD spectra is useful in that it furnishes a discriminating method for determining the type and redox state of protein bound iron-sulfur clusters. Each well characterized type of iron-sulfur cluster, i.e. [2Fe-2S], [3Fe-4S], and [4Fe-4S], has been shown to have a characteristic low temperature MCD spectrum in each paramagnetic redox state (1)... 

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. 

E. M. Maes, "Structural Characterization of Iron and Copper Active Sites by Resonance Raman Spectroscopy Nitrophorin, Nitrite Reductase, and Iron-Sulfur Proteins, PhD. Dissertation, University of Houston, 2000. 

In eukaryotes, the formation of iron/sulfur clusters proceeds inside mitochondria (13). The mitochondrial enzymes are orthologs of the eubacterial isc proteins and are characterized... 

This review is a survey of the research on the direct electron transfer (DET) between biomolecules and electrodes for the development of reagentless biosensors. Both the catalytic reaction of a protein or an enzyme and the coupling with further reaction have been used analytically. For better understanding and a better overview, this chapter begins with a description of electron transfer processes of redox proteins at electrodes. Then the behaviour of the relevant proteins and enzymes at electrodes is briefly characterized and the respective biosensors are described. In the last section sensors for superoxide, nitric oxide and peroxide are presented. These have been developed with several proteins and enzymes. The review is far from complete, for example, the large class of iron-sulfur proteins has hardly been touched. Here the interested reader may consult recent reviews and work cited therein [1,19]. 

Chloroplast ferredoxin containing the [(2Fe-2S)-(S-Cys)4] cluster is one common type of iron-sulfur protein. Another [2Fe-2S]-type protein is the Rieske iron-sulfur protein, present in the Cyt >6/complex as well as the Cyt Ac, complex. The pair of iron atoms in the cluster ofthe Rieske iron-sulfur protein are bound to two cysteine and two histidine residues, in addition to two sulfur atoms. The three-dimensional structures of ferredoxins and that of the Rieske iron-sulfur protein have been determined by X-ray crystallography (see Chapters 34 and 35, respectively, for the structure ofthe chloroplast ferredoxin and the Rieske iron-sulfur protein). The sulfide ions in iron-sulfur proteins urt acid-labile this provides a simple means for detecting the iron-sulfur proteins, as the sulfide is released as H2S upon acidification. The oxidized and reduced states of iron-sulfur clusters differ by just one unit of formal charge, corresponding to and Fe. Iron-sulfurproteins are commonly characterized by optical absorption, circular-dichro-... 

Here we will review the work of Sauer, Mathis, Acker and van Best in their original attempt to characterize the iron-sulfur center FeS-X, an electron carrier with a more negative redox potential and positioned ahead of FeS-A and FeS-B in the electron-transfer chain, and thus an intermediary electron donor to FeS-A and FeS-B. When the oxidized FeS-A/B proteins serve as secondary acceptors, the photochemically reduced forms have been found to re-reduce the photooxidized primary donor P700. In parallel experiments, Sauer etal. characterized FeS-X indirectly by determining the decay kinetics of photooxidized PTOO" in its recombination with reduced FeS-X" by kinetic spectrophotometric measurements. In the process, some insight was also gained indirectly regarding the nature of FeS-A and FeS-B. 

Hg. 6. Laser flash-induced absorbance changes (AA) observed in a core complex at 819 nm (A) and 380 nm (B) at 298 K aA at 819 nm presented on three time scales with parameters derived by computer cun/e fitting AA at 380 nm without and with ferricyanide (C) absorbance difference spectra in the UV/vis region constructed from the 10-//s- and 100-ps decay phases. Figure source Brettel and Golbeck (1995) Spectral and kinetic characterization of electron acceptor A, in a photosystem I core devoid of iron-sulfur centers Fx, Fb and Fa- Photosynthesis Res 45 185,187. 

K Brettel and JH Golbeck (1995) Spectral and kinetic characterization of electron acceptorA in a photosystem I core devoid of iron-sulfur centers Fy, Fg andF/. Photosynthesis Res 45 183-193... 

Most oxidoreductases of these parasites are similar to those in other eukaryotes in using pyridine nucleotides (NAD and NADP) as redox partners. In addition, however, their core metabolism is characterized by an important involvement of iron-sulfur proteins, which is also a characteristic of anaerobic eubacteria. The key step in pyruvate metabolism, its oxidative decarboxylation to acetyl-CoA, is catalyzed by an iron-sulfur enzyme, pyruvateTerredoxin oxidoreductase. The enzyme has been characterized from... 

While most soluble cellular iron is associated with an ill-defined labile iron pool, much of the iron that is used by the cell is found in the form of specific iron cofactors especially as iron-sulfur clusters and hemes. The biosynthesis of iron-sulfur centers has only come of age within the past decade following the characterization of operons encoding cluster assembly genes by Dean and coworkers 4), In eukaryotes, homologous proteins are nuclear transcribed. While predominantly localized within the mitochondrial matrix, one group has reported cytosolic ISU (cluster assembly protein) and NFS (S-donor protein) in human cell lines (5). Alternative cytoplasmic cluster maturation proteins have also been proposed (6). Whether the cytosolic components represent an independent assembly apparatus is unclear, nevertheless, both models support a role for ISU-mediated assembly of [2Fe-2S] building blocks prior to delivery to a target protein (7-P). 

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