Plant-Type Iron-Sulfur Proteins

Iron-sulfur proteins can be classified on the basis of iron and sulfide content into plant-type iron-sulfur proteins, and bacterial-type iron-sulfur proteins. Plant-type iron-sulfur proteins contain just two Fe and two inorganic S atoms per protein molecule the name refers to the material first isolated from chloroplasts. The bacterial-type iron-sulfur proteins always contain more than two Fe (and S—) atoms per protein molecule in general there are eight Fe and eight S— atoms per protein molecule. 

All iron-sulfur proteins, whether of the plant-type or the bacterial-type have three characteristics in common all contain the acid-labile sulfide in equimolar ratio to iron all show reduction potentials in the range from —240 to —420 mV (E0,pli = 7.0) and when these proteins are chemically-reduced (typically with dithionite), they display an uncommon EPR signal, known as the g = 1.94 signal. The oxidized forms of the proteins are not paramagnetic (159). 

The g = 1.94 EPR signal exhibited in the reduced state of the ferre-doxins was the basis for models of the active site of these proteins. The identification of this EPR signal with an iron complex has been described in a review by Beinert and Palmer (142). The complexity of the iron ligand field which is necessary to produce a g= 1.94 signal was demonstrated by Beinert et al. (143), who proposed a model compound for this signal. This model compound was pentacyanonitrosylferrate (I). The properties of this model compound were later related and expanded by Van Voorst and Hemmerich (144). 

Meanwhile, Blumberg and Peisach (145) showed that the interaction between a low-spin ferrous atom and an adjacent free radical can give rise to a g= 1.94 EPR signal. Brintzinger, Palmer, and Sands (146) proposed the first two-iron model for the active center of a plant-type ferredoxin. Their model, which consisted of two spin-coupled, low-spin ferric atoms in the oxidized protein and one low-spin ferric and one low-spin ferrous atom in the reduced protein, explained much of the chemical data on the proteins. Later, they (Brintzinger, Palmer, and Sands, (147)) presented EPR data for a compound, bis-hexamethylbenzene, Fe(I), which demonstrated all the properties fo the g= 1.94 signal observed in the ferredoxins. 

Several Mossbauer spectroscopic papers have dealt with members of the plant-type ferredoxins. In these papers, the Mossbauer spectra for a particular protein were interpreted to yield information such as the oxidation state and spin state of the iron atoms in the protein, and in some cases this information was extended to validate a proposed model for the active site. However, problems with denatured protein material or incorrect interpretation of the Mossbauer data have prevented any of these models from being accepted as valid. 

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Table 1 lists some of the properties of the plant-type iron sulfur-proteins for which extensive study by EPR and Mossbauer spectroscopy has been reported. The physical properties summarized show that the plant-type iron sulfur proteins have molecular weights in the range from 12,000 to 24,000 and have EPR g-values (gx, gy, gz) all of the g = 1.94" type shown in Fig. 6 but with minor variations reflecting axial or nonaxial symmetry of the paramagnetic center. The amino-acid sequences of four plant-type iron-sulfur-proteins are known alfalfa (136), L. glauca (137), Scenedesmus (138), and spinach (139). Each protein has about 97 residues, all in a single peptide chain these are shown in Table 2. 

Fig. 6. Electron paramagnetic resonance signal showing the g= 1.94 characteristic of the dithionite-reduced spinach ferredoxin, a plant-type iron-sulfur protein. Spectrum taken at 20 °K...

Fig. 7. Mossbauer spectra of oxidized plant-type iron-sulfur proteins in zero applied magnetic field. Abbreviations AZI = A zotobacter Fe-S protein I, 4.6°K AZII = Azoiobacter Fe-S protein II, 4.2 °K Put. = Putidaredoxin, 4.2 °K Ad.— Pig Ad-renodoxin, 4.2 °K Clos. = Clostridial paramagnetic protein, 4.2 °K PPNR = Spinach ferredoxin, 4.5 °K Parsley = Parsley Ferredoxin, 4.2 °K. Velocity scale is relative to iron in platinum...

Fig. 8. Mossbauer spectra of oxidized plant-type iron-sulfur proteins in high applied magnetic field. Abbreviations Ad. = Pig Adrenodoxin, 4.2 °K, 46 kG PPNR = Spinach Ferredoxin, 4.5 °K, 50 kG Clos. = Clostridial Paramagnetic Protein, 4.2 °K, 46 kG AZI = Azotobacter Fe-S Protein I, 4.6°K, 46 kG AZII = Azotobacter Fe-S Protein II, 4.2 °K, 46 kG. Applied magnetic field is parallel to gamma-ray direction...

The bacterial-type iron-sulfur proteins all contain larger amounts of iron and labile sulfide than the plant-type iron-sulfur proteins best estimates for the iron and labile sulfide content being 8 Fe and 8 S per protein molecule (172, 173) for these ferredoxins from Clostridium and from Chromatium. Although these proteins have large amounts of Fe and S, the molecular weights are less than the molecular weights of the... 

Although the redox potential of Rieske-type clusters is approximately 400 mV lower than that of Rieske clusters, it is 300 mV more positive than the redox potential of plant-type ferredoxins (approximately -400 mV). Multiple factors have been considered to be essential for the redox potential of iron sulfur proteins ... 

During the 1960s, research on proteins containing iron—sulfur clusters was closely related to the field of photosynthesis. Whereas the first ferredoxin, a 2[4Fe-4S] protein, was obtained in 1962 from the nonphotosynthetic bacterium Clostridium pasteurianum (1), in the same year, a plant-type [2Fe-2S] ferredoxin was isolated from spinach chloroplasts (2). Despite the fact that members of this latter class of protein have been reported for eubacteria and even archaebacteria (for a review, see Ref. (3)), the name plant-type ferredoxin is often used to denote this family of iron—sulfur proteins. The two decades... 

Putidaredoxin. Cushman et al. (36) isolated a low molecular iron-sulfur protein from camphor-grown Pseudomonas putida. This protein, putidaredoxin, is similar to the plant type ferredoxins with two irons attached to two acid-labile sulfur atoms (37). It has a molecular weight of 12,000 and shows absorption maxima at 327, 425 and 455 nm. Putidaredoxin functions as an electron transfer component of a methylene hydroxylase system involved in camphor hydroxylation by P. putida. This enzyme system consists of putidaredoxin, flavoprotein and cytochrome P.cQ (38). The electron transport from flavoprotein to cytochrome P.cq is Smilar to that of the mammalian mixed-function oxidase, but requires NADH as a primary electron donor as shown in Fig. 4. In this bacterial mixed-function oxidase system, reduced putidaredoxin donates an electron to substrate-bound cytochrome P. g, and the reduced cytochrome P. g binds to molecular oxygen. One oxygen atom is then used for substrate oxidation, and the other one is reduced to water (39, 40). 

In addition to NAD and flavoproteins, three other types of electron-carrying molecules function in the respiratory chain a hydrophobic quinone (ubiquinone) and two different types of iron-containing proteins (cytochromes and iron-sulfur proteins). Ubiquinone (also called coenzyme Q, or simply Q) is a lipid-soluble ben-zoquinone with a long isoprenoid side chain (Fig. 19-2). The closely related compounds plastoquinone (of plant chloroplasts) and menaquinone (of bacteria) play roles analogous to that of ubiquinone, carrying electrons in membrane-associated electron-transfer chains. Ubiquinone can accept one electron to become the semi-quinone radical ( QH) or two electrons to form ubiquinol (QH2) (Fig. 19-2) and, like flavoprotein carriers, it can act at the junction between a two-electron donor and a one-electron acceptor. Because ubiquinone is both small and hydrophobic, it is freely diffusible within the lipid bilayer of the inner mitochondrial membrane and can shuttle reducing equivalents between other, less mobile electron carriers in the membrane. And because it carries both electrons and protons, it plays a central role in coupling electron flow to proton movement. 

These spectra, taken at variable temperatures and a small polarizing applied magnetic field, show a temperature-dependent transition for spinach ferredoxin. As the temperature is lowered, the effects of an internal magnetic field on the Mossbauer spectra become more distinct until they result at around 30 °K, in a spectrum which is characteristic of the low temperature data of the plant-type ferredoxins (Fig. 11). We attribute this transition in the spectra to spin-lattice relaxation effects. This conclusion is preferred over a spin-spin mechanism as the transition was identical for both the lyophilized and 10 mM aqueous solution samples. Thus, the variable temperature data for reduced spinach ferredoxin indicate that the electron-spin relaxation time is around 10-7 seconds at 50 °K. The temperature at which this transition in the Mossbauer spectra is half-complete is estimated to be the following spinach ferredoxin, 50 K parsley ferredoxin, 60 °K adrenodoxin, putidaredoxin, Clostridium. and Axotobacter iron-sulfur proteins, 100 °K. 

Figure 5 The most commonly encountered FeS centers (a) the monoiron center of rubredoxin, (b) the FeySy cluster of plant-type ferredoxins, (c) the FeySy cluster of Rieske proteins, (d) the Fe3S4 cluster of ferredoxins, and (e) the Fe4 4 cluster of ferredoxins and high potential iron-sulfur proteins (FliPiPs).

We have seen the Z-scheme for the two photosystems in green-plant photosynthesis and the electron carriers in these photosystems. We have also described how the photosystems of green plants and photosynthetic bacteria all appear to function with basically the same sort ofmechanisms of energy transfer, primary charge separation, electron transfer, charge stabilization, etc., yet the molecular constituents of the two reaction centers in green plants, in particular, are quite different from each other. Photosystem I contains iron-sulfur proteins as electron acceptors and may thus be called the iron-sulfur (FeS) type reaction center, while photosystem 11 contains pheophytin as the primary electron acceptor and quinones as the secondary acceptors and may thus be called the pheophytin-quinone (0 Q) type. These two types of reaction centers have also been called RCI and RCII types, respectively. 

The FcjS -type ferredoxins can be arranged into three distantly related classes based on amino acid sequence homologies bacterial-, plant-, and vertebrate-type . Extensive information on the function and mechanisms of this system has been gained through work on the bacterial P450cam system in Pseudomonas putida, which catalyzes the conversion of rf-camphor to 5-exo-hydroxy-camphor. In P putida, the iron-sulfur protein is putidaredoxin (Pdx), a 106 amino acid residue ferredoxin. For catalysis, two reducing equivalents are sequentially transferred from NADH... 

The absorption spectra of the main types of the iron-sulfur proteins is shown in Figure 1. There are very significant differences that have been overlooked with regard to the nomenclature of these proteins. All the proteins examined— the chloroplast ferredoxin, the clostridial ferredoxin, and rubredoxin—show absorption at 280 nm in the oxidized form shown here, but the other absorption peaks differ. The clostridial ferredoxin has an absorption peak at 390 nm but that peak is missing in the plant type ferredoxin. Rubredoxin shows an absorption maximum at 390 nm, but it shows other absorption peaks at 500 and 580 nm which are absent from the clostridial type protein. 

Iron and sulfur can be extracted from F. the resulting apoferredoxin is reactivated by iron(II) salts and sulfides. The synthesis of the iron-free protein has been achieved by the Merrifield technique. On account of their properties as redox systems (Fe +e" Fe ") the F. effect electron transport between enzyme systems but do not exhibit any enzymatic activity. They transport electrons in the respiratory chain, in photosynthesis, and in nitrogen fixation. The iron-sulfur protein P439 of the Fc4S4-type (Mr 11600) plays a role in photosynthesis. Conclusions can be drawn about the evolutionary histories of plants from the similarities and differences in the amino acid sequences. For the evolutionary history of F. in photosynthesis, see Lit.K F. with FejSj- and FejSg-clusters also occur in bacteria Lit. TrendsBiochem.Sci. 13,30-33(1988). FEMSMicrobiol. Rev. 54,155-176 (1988) Trends Biochem. Sci. 13,369 f. (1988). 

One of the two important parts of enzyme is a flavo-iron-sulfur protein with NADH-dependent oxidoreductase activity. The reductase is a monomeric 34 kDa (in case of phthalate dioxygenase reductase) flavo-iron-sulfur protein containing flavin mononucleotide (FMN) and a plant-ferredoxin-type [2Fe-2S] center in a 1 1 ratio [372]. Structure of this part of the enzyme has been studied recently by X-ray crystallographic analysis [375], low-temperature EPR [383], and kinetically [378]. 

Another type of frequently studied metalloproteins is the iron-sulfur (Fe-S) protein. It widely exists in all sorts of bacteria, plants, and animals, and plays a part in a wide range of biochemical processes. The best known function of iron sulfur protein is its role in the redox process of electron transfer. The active center is an iron sulfur cluster, through which several proteins link together with the coordination between the iron atoms in the cluster motif and sulfur atoms of cysteines in protein motifs. There are several primary types of... 

Ferredoxins. Ferredoxins are proteins which contain two or four iron atoms bound to cysteine and inorganic sulfur atoms as shown in Fig. IB. There are two types of ferredoxins plant type ferredoxins (top) which consist of two iron and two labile sulfur atoms coordinated to four cysteine residues, and bacterial type ferredoxins (bottom) consisting of four iron and four labile sulfur atoms coordinated to four cysteine residues. 

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