The Iron-Sulfur Proteins

The application of ESR has greatly increased knowledge of type, nature, and number of animal mitochondrial electron-transport components. A variety of iron-sulfur centers have been described in their reduced or oxidized forms. The species may be distinguished by utilizing differences in spectroscopic, redox potentiometric, and temperature and power saturation of the ESR spectra. ESR has been used to study the iron-sulfur centers involved in electron transfer pathways in higher plant mitochondria (Rich et al, 1977). 

Many iron-sulfur centers are known to function in the mitochondrial chain. At least five different iron-sulfur centers have been characterized in beef-heart mitochondrial complex I (Albracht et al, 1977). Complex II contains two to three different iron-sulfur centers (Ohnishi et al, 1974a Beinert et al, 1975), whereas complex III contains one iron-sulfur center (Rieske et al, 1964 Orme-Johnson et al, 1974). Complex II exhibits an ESR signal in the oxidized state (Ohnishi et al, 1974b). The ESR spectra and redox properties are similar to those of the signal of the HiPIP from Chromatiwn vinosum. This signal therefore probably occurs for the [4Fe-4S]1-(i- 2-) cluster. The number of iron atoms per center is not known for the other iron-sulfur centers in the respiratory chain. 

The higher-plant mitochondria contain a group of iron-sulfur centers that are paramagnetic and ESR detectable in their oxidized forms, in 

The early isolation and characterization of reconstitutively active succinate dehydrogenase showed that the enzyme molecule contains 8 atoms of nonheme iron and 8 atoms of acid labile sulfide per flavin (Keilin and King, 1958 King, 1963 Davis and Hatefi, 1971 Coles et al, 1972 Zanelti et al, 1972). The iron-sulfur center responsible for the g = 1.94 signal and the free radical signal due to the flavin characterize the enzyme (Beinert and Sands, 1960 King et al, 1961 DerVartanian et al, 1969). 

Recently, with the application of low-temperature ( 77°K) ESR techniques in combination with potentiometric titration, an ESR signal from a second iron-sulfur center, designated as center S2 (Ohnishi et al, 1973), was detected and characterized in particulate preparations such as succinate-ubiquinone reductase or in succinate-cytochrome c reductase, in addition 

---

Wachtershanser has also suggested that early metabolic processes first occurred on the surface of pyrite and other related mineral materials. The iron-sulfur chemistry that prevailed on these mineral surfaces may have influenced the evolution of the iron-sulfur proteins that control and catalyze many reactions in modern pathways (including the succinate dehydrogenase and aconitase reactions of the TCA cycle). 

In 1964, Rieske and co-workers reported the observation of an EPR signal around g = 1.90 in the cytochrome bci complex (1). They succeeded in the isolation of the iron sulfur protein that gave rise to the EPR signal and showed that it contained a [2Fe-2S] cluster. Over the... 

Figure 11-6. Cytochrome P450 hydroxylase cycle in microsomes. The system shown is typical of steroid hydroxylases of the adrenal cortex. Liver microsomal cytochrome P450 hydroxylase does not require the iron-sulfur protein FejSj. Carbon monoxide (CO) inhibits the indicated step.

An additional component is the iron-sulfur protein (FeS nonheme iron) (Figure 12-6). It is associated with the flavoproteins (metallofiavoproteins) and with cytochrome b. The sulfur and iron are thought to take part in the oxidoreduction mechanism between flavin and Q, which involves only a single e change, the iron atom undergoing oxidoreduction between Fe " and Fe k... 

Although heme is absent in Clostridia, it was early recognized that anaerobic bacteria may contain substantial levels of iron (44). To date the best characterized iron compounds from this source are the iron-sulfur proteins termed ferredoxins and rubredoxins. Molecular structures of representatives of both types of protein have been worked out by Jensen and his colleagues by X-ray diffraction analysis (see below). 

The remarkable range of redox potentials in the iron sulfur proteins, already noted, illustrates the principle that nature, having discovered a ligand system, attempts to extract from it the maximum utility. Certainly the outstanding problem awaiting solution in these proteins is an explanation for the relationship between structure and redox potential. Thus... 

There is some evidence that the iron-sulfur protein, FhuF, participates in the mobilization of iron from hydroxamate siderophores in E. coli (Muller et ah, 1998 Hantke, K. unpublished observations). However, a reductase activity of FhuF has not been demonstrated. Many siderophore-iron reductases have been shown to be active in vitro and some have been purified. The characterization of these reductases has revealed them to be flavin reductases which obtain the electrons for flavin reduction from NAD(P)H, and whose main functions are in areas other than reduction of ferric iron (e.g. flavin reductase Fre, sulfite reductase). To date, no specialized siderophore-iron reductases have been identified. It has been suggested that the reduced flavins from flavin oxidoreductases are the electron donors for ferric iron reduction (Fontecave et ah, 1994). Recently it has been shown, after a fruitless search for a reducing enzyme, that reduction of Co3+ in cobalamin is achieved by reduced flavin. Also in this case it was suggested that cobalamins and corrinoids are reduced in vivo by flavins which may be generated by the flavin... 

XAS data comprises both absorption edge structure and extended x-ray absorption fine structure (EXAFS). The application of XAS to systems of chemical interest has been well reviewed (4 5). Briefly, the structure superimposed on the x-ray absorption edge results from the excitation of core-electrons into high-lying vacant orbitals (, ] ) and into continuum states (8 9). The shape and intensity of the edge structure can frequently be used to determine information about the symmetry of the absorbing site. For example, the ls+3d transition in first-row transition metals is dipole forbidden in a centrosymmetric environment. In a non-centrosymmetric environment the admixture of 3d and 4p orbitals can give intensity to this transition. This has been observed, for example, in a study of the iron-sulfur protein rubredoxin, where the iron is tetrahedrally coordinated to four sulfur atoms (6). 

Within organisms, organic sulfur is present predominantly as the amino acids cysteine and methionine, and the algal and bacterial osmolyte, dimethylsulfoniopropionate (DMSP). The latter also serves as an antioxidant and cryoprotectant. Small amounts of organosulfur are also present in some polysaccharides, lipids, vitamins, enzymes, and in the iron-sulfur protein ferrodoxin. Cell lysis and microbial degradation releases... 

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. 

Bovine heart cytochrome bci (PDB 1BE3 and PDB IBGY) as studied by Iwata et al. exists as a dimer in the asymmetric unit cell. Each monomer consists of 11 different polypeptide subunits (SU) with a total of -2165 amino acid residues and a molecular mass of -240 kDa. The protein subunits of the complex occupy three separate regions (1) the intermembrane space (p side) occupied by cytochrome Ci (subunit 4, SU4), the iron-sulfur protein (ISP, SU5) and subunit 8 (2) the transmembrane region occupied by cytochrome b (SU3), the transmembrane helices of cytochrome Ci and the ISP, and subunits 7,10, and 11 and (3) the matrix space (n side) occupied by two large core proteins (subunits 1 and 2) as well as subunits 6 and 9. Subunit 8 is often called the hinge protein and is thought to be essential for proper complex formation between cytochrome c (the exit point for some bci complex electrons) and... 

Figure 1. Schematic models of clusters in the iron-sulfur proteins proposed by...

The iron-sulfur proteins include small proteins that function as remarkably simple electron carriers and large multisubunit complexes with multiple activities. Although many of these enzymes function in electron transfer in bioener-getic or biosynthetic pathways, it has become clear that iron-sulfur proteins catalyze a broad array of reactions not always involving electron transfer. 

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. 

Table I lists isomorphous replacements for various metalloproteins. Consider zinc enzymes, most of which contain the metal ion firmly bound. The diamagnetic, colorless zinc atom contributes very little to those physical properties that can be used to study the enzymes. Thus it has become conventional to replace this metal by a different metal that has the required physical properties (see below) and as far as is possible maintains the same activity. Although this aim may be achieved to a high degree of approximation [e.g., replacement of zinc by cobalt(II)], no such replacement is ever exact. This stresses the extreme degree of biological specificity. The action of an enzyme depends precisely on the exact metal it uses, stressing again the peculiarity of biological action associated with the idiosyncratic nature of active sites. (The entatic state of the metal ion is an essential part of this peculiarity.) Despite this specificity, the replacement method has provided a wealth of information about proteins that could not have been obtained by other methods. Clearly, there will often be a compromise in the choice of replacement. Even isomorphous replacement that should retain structure will not necessarily retain activity at all. However, it has become clear that substitutions can be made for structural studies where the substituted protein is inactive (e.g., in the copper proteins and the iron-sulfur proteins). It is also possible to substitute into metal coenzymes. Many studies have been reported of the...

Massey, V. Iron-sulfur flavoprotein hydroxylases. In The iron-sulfur proteins (Lovenberg, W. ed.) Vol. 1, pp. 301-360, New York, Academic Press 1973... 

FIGURE 19-9 IMADH ubiquinone oxidoreductase (Complex I). Complex I catalyzes the transfer of a hydride ion from NADH to FMN, from which two electrons pass through a series of Fe-S centers to the iron-sulfur protein N-2 in the matrix arm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2, which diffuses into the lipid bilayer. This electron transfer also drives the expulsion from the matrix of four protons per pair of electrons. The detailed mechanism that couples electron and proton transfer in Complex I is not yet known, but probably involves a Q cycle similar to that in Complex III in which QH2 participates twice per electron pair (see Fig. 19-12). Proton flux produces an electrochemical potential across the inner mitochondrial membrane (N side negative, P side positive), which conserves some of the energy released by the electron-transfer reactions. This electrochemical potential drives ATP synthesis. 

Electrons moving through complex III from UQ to cytochrome c follow a circuitous path (fig. 14.11). UQH2 must transfer one of its electrons to the iron-sulfur protein before it can transfer an electron to cytochrome bL. 

Oxidation of UQH2 by the iron-sulfur protein generates the semiquinone UQH , which then serves as the reductant for cytochrome bL. The reduced iron-sulfur protein transfers an electron to cytochrome cx and on to cytochrome c. Meanwhile, the reduced cytochrome bL passes an electron to cytochrome bu, which then contributes the electron for reduction of another molecule of UQ at a second site in the complex. 

---