Bacterial ferredoxins

Figure 8.39 Fourier transformed Fe extended X-ray absorption fine structure (EXAFS) and retransformation, after applying a 0.9-3.5 A filter window, of (a) a rubredoxin, (b) a plant ferredoxin and (c) a bacterial ferredoxin, whose structures are also shown. (Reproduced, with permission, Ifom Teo, B. K. and Joy, D. C. (Eds), EXAFS Spectroscopy, p. 15, Plenum, New York, 1981)...

A second example is that of an Ala-to-Cys mutation, which causes the fonnation of a rare SH S hydrogen bond between the cysteine and a redox site sulfur and a 50 mV decrease in redox potential (and vice versa) in the bacterial ferredoxins [73]. Here, the side chain contribution of the cysteine is significant however, a backbone shift can also contribute depending on whether the nearby residues allow it to happen. Site-specific mutants have confirmed the redox potential shift [76,77] and the side chain conformation of cysteine but not the backbone shift in the case with crystal structures of both the native and mutant species [78] the latter can be attributed to the specific sequence of the ferre-doxin studied [73]. 

Figure 25.9 Some non-haem iron proteins (a) rubredoxin in which the single Fe is coordinated, almost tetra-hedrally, to 4 cysteine-sulfurs, (b) plant ferredoxin, [Fe2S2(S-Cys)4], (c) [Fe4S4(S-Cys)4] cube of bacterial ferredoxins. (This is in fact distorted, the Fe4 and S4 making up the two interpenetrating tetrahedra, of which the latter is larger than the former).

Fig. 1. Iron-sulfur clusters basic building blocks. In most cases the iron is tetrahe-drally coordinated by sulfur from cysteinyl residues (and labile sulfur). Variability on coordination is allowed (see text). A, Rubredoxin type FeS4 (simplest cluster, no labile sulfur) B, plant-type ferredoxin [2Fe-2S] C, bacterial ferredoxin [3Fe-4S] D, bacterial ferredoxin and HiPIP [4Fe-4S] E, novel cluster [4Fe-2S, 20] ( hybrid cluster ).

At this point it may be valuable to digress a moment and discuss the state-of-the-knowledge in the field of Fe-S proteins by the ntid-1970 s. At this time there were three known structures found in nature, IFe as represented by rubredoxin, the [2Fe-2S] cluster as represented by plant ferredoxins, and the [4Fe-4S] cluster as found in many bacterial ferredoxins (24). The schematic structures and selected properties are listed in Table I. 

Other Iron Compounds of Biological Interest.—The valency of iron in a range of ferredoxin extracts has been determined by ESCA. The structure of the Fe—S complex in a bacterial ferredoxin has been determined. The iron and sulphur atoms occupy alternate corners of a cube and four more sulphur atoms project from the iron atoms.Admission of oxygen to a neutral solution of Fe and excess penicillamine gives a red bis-complex, which is relatively stable in aqueous solution at room temperature. Quantitative... 

Bacterial ferredoxins. Bacterial ferredoxin was first described in 1962 by Mortenson et al. (p who found a low-molecular iron protein involved in electron transfer of pyruvate hydrogenase and nitrogenase in C. pasteurianum. Subsequently, a number of ferredoxins have been found lii widely different types of bacteria such as photosynthetic bacteria and N2-fixing bacteria. These bacterial type ferredoxins have molecular... 

Bacterial ferredoxins function primarily as electron carriers in ferredoxin-mediated oxidation reduction reactions. Some examples are reduction of NAD, NADP, FMN, FAD, sulfite and protons in anaerobic bacteria, CO -fixation cycles in photosynthetic bacteria, nitrogen fixation in anaerobic nitrogen fixing bacteria, and reductive carboxylation of substrates in fermentative bacteria. The roles of bacterial ferredoxins in these reactions have been summarized by Orme-Johnson (2), Buchanan and Arnon (3), and Mortenson and Nakos (31). 

The cluster found in certain bacterial ferredoxins involved in anaerobic metabolism. It consists of a cubane-like cluster of four iron atoms, four labile sulphur atoms, thus Fe4S4, and four cysteine ligands... 

Many bacterial ferredoxins have two such clusters, each of which can be reduced to a paramagnetic state. In other proteins, 4Fe4S clusters can be oxidized to a paramagnetic state. The tetranuclear clusters in these two types of proteins are similar in structure, but they functionally shuttle between different reduction states. In both cases, each iron atom is additionally coordinated by four cysteinyl sulfur ligands. 

Perutz MF, Raidt H. Stereochemical basis of heat stability in bacterial ferredoxins and in haemoglobin A2. Nature 1975 255 256-259. 

The bacterial ferredoxins from Peptococcus, Clostridium (Fig. 16-16B),267/268 Desulfovibrio, and other anaerobes each contain two Fe4S4 clusters with essentially the same structure as that of the Chromatium HIPIP.267 269 Each cluster can accept one electron. 

The soluble electron carriers released from the reaction centers into the cytoplasm of bacteria or into the stroma of chloroplasts are reduced single-electron carriers. Bacterial ferredoxin with two Fe4S4 clusters is formed by bacteria if enough iron is present. In its absence flavodoxin (Chapter 15), which may carry either one or two electrons, is used. In chloroplasts the carrier is the soluble chloroplast ferredoxin (Fig. 16-16,C), which contains one Fe2S2 center. Reduced ferredoxin transfers electrons to NADP+ (Eq. 15-28) via ferredoxin NADP oxidoreductase, a flavoprotein of known three-dimensional structure.367 369... 

Not only are two molecules of ATP hydrolyzed to pump each electron, but the Fe-protein must receive electrons from a powerful (low E°) reductant such as reduced ferredoxin, reduced flavodoxin, or dithionite. Klebsiella pneumoniae contains a pyruvate flavodoxin oxidoreductase (Eq. 15-35) that reduces either flavodoxin or ferredoxin to provide the low potential electron donor.29 30 In some bacteria, e.g., the strictly aerobic Azotobacter, NADPH is the electron donor for reduction of N2. The Fe-protein is thought to accept electrons from a chain that includes at least the ordinary bacterial ferredoxin (Fd) and a special one-electron-accepting azotoflavin, a flavoprotein that is somewhat larger than the flavodoxins (Chapter 15) and appears to play a specific role in N2 fixation.31 In Clostridium and Rhizobium reduced ferredoxins generated by cleavage of pyruvate reduce nitrogenase directly.32... 

Otaka, E and Ooi, T. (1987) Examination of Protein Sequence Homologies IV Twenty-Seven Bacterial Ferredoxins, Journal of Molecular Evolution, 26, 257-268. 

Crystallization of Clostridium pasteurianum ferredoxin demonstration of functional interchangeability of spinach photosynthetic pyridine nucleotide reductase and bacterial ferredoxin suggestion that both be classified as ferredoxins... 

Losada and Arnon (63)). A similar initial treatment with acetone has also been used successfully for the preparation of bacterial ferredoxin (Mor-tenson (73a). 

It was pointed out previously that both bacterial and plant fer-redoxins are colored proteins in the oxidized state. Fig. 3 shows the visible and ultraviolet absorption spectra of a bacterial (C. pasteurianum) and plant (spinach) ferredoxin. Bacterial ferredoxin shows a single peak in the visible region at 390 m(r and a peak in the ultraviolet region at about 280 mp. with a shoulder at 300 mp. The relative height of the peak at 280 mp to the shoulder at 300 mp varies among preparations from different bacteria generally the peak at 280 mp predominates (Loven-berg, Buchanan, and Rabinowitz (65) Bachofen and Arnon (12)). Plant... 

Both plant and bacterial ferredoxins are small molecules. A summary of the determinations of their molecular weight is presented in Table 4. 

Plant ferredoxin, therefore, appears to be twice the size of bacterial ferredoxin with a molecular weight of 13,000, although a higher value would seem possible from the available data. 

One of the characteristics of ferredoxin, as inferred from its name, is the presence of iron. Mortenson, Valentine, and Carnahan (75) reported non-heme iron in bacterial ferredoxin and several investigators independently reported non-heme iron in plant ferredoxin (Tagawa and Arnon (99) Fry and San Pietro (46) Horio and Yamashita (58)). Iron was also found in other clostridial ferredoxins (Lovenberg, Buchanan, and Rabinowitz (65)) and in ferredoxin of photosynthetic bacteria (Evans and Buchanan (41) Bachofen and Arnon (12)). Based on a molecular weight of 13,000, the plant ferredoxins which have been investigated contain 2 atoms of iron per molecule, but the iron content of bacterial ferredoxins differs. Clostridial ferredoxins contain 7 atoms of iron (Loven-... 

In an elegant experiment coupling the oxidation of reduced spinach ferredoxin to the reduction of TPN (a two electron carrier) by a crystalline enzyme, Whatley, Tagawa, and Arnon (114) showed that, like the cytochromes, the reduction of spinach ferredoxin involves the transfer of a single electron. These results were confirmed by Fry et al- (45) for spinach ferredoxin. Sobel and Lovenbarg (96) applied this same technique, as well as hydrogen-hydrogenase, to C. pasteurianum ferredoxin and found that its oxidation and reduction involved transfer of two, rather than one, electron. Reduction of this bacterial ferredoxin was accompanied by the reduction of two ferric iron atoms to the ferrous state. 

The crystallization of bacterial ferredoxin made possible determination of the amino acid composition of ferredoxin from six anaerobic bacteria... 

The bacterial ferredoxins, in general, are similar to each other in amino acid content, but differ in detail with each ferredoxin having a characteristic composition. Each contains about fifty total amino acid residues with an abundance of acidic and a paucity of basic amino acids. The abundance of acidic residues accounts for the affinity of ferredoxin for DEAE-cellulose and its low isoelectric point (Lovenberg, Buchanan, and Rabinowitz (65)). Each of the bacterial ferredoxins lacks histidine, methionine, tryptophan, and at least one additional amino acid, which is characteristic of a particular ferredoxin. The possible significance of these differences in either the conformation or function of these proteins has not been established, although minor differences in enzymic activity do exist (Lovenberg, Buchanan, and Rabinowitz (65)). 

Table 6 shows that spinach ferredoxin contains all the amino acids in bacterial ferredoxin plus one residue each of methionine, histidine, and tryptophan. Like bacterial ferredoxin, spinach ferredoxin has an abundance of acidic and a paucity of basic amino acids. Hill and San Pietro (53) found that parsley ferredoxin is similar to the one from spinach in composition and spectra, but differs in lacking tryptophan. Little is known of the structure of plant ferredoxin. It is known that spinach ferredoxin is a single peptide chain with alanine at both ends (Tsugita et al. (103)). 

The incubation of spinach ferredoxin (Fry and San Pietro (46)) or bacterial ferredoxin (Lovenberg, Buchanan, and Rabinowitz (65)) with the iron chelating agent, o-phenanthroline, results in removal of the iron from the protein and in the formation of a ferrous triphenanthrolate complex. Under these conditions, all of the iron appears to be in the ferrous state, but this does not constitute proof that iron of the native protein is also in the ferrous state. Reduction of bound ferric iron could occur... 

The data indicate that the 2 iron atoms of oxidized spinach ferredoxin are both in the ferric state. Reduction of spinach ferredoxin involves transfer of a single electron (Whatley, Tagawa, and Arnon (114)) and one of the ferric iron atoms is reduced to the ferrous form (Fry, Lazzarini, and San Pietro (45)). At least 2 of the iron atoms of bacterial ferredoxin are in the ferric state, but the valency of the remaining 5 is uncertain, even though Blomstrom et al. (23) detected them as ferrous iron. Sobel and Lovenberg (96) showed that reduction of bacterial ferredoxin is accompanied by an increase of 2 iron atoms detected as ferrous iron. The data, therefore, imply that ferric iron is the active catalytic factor of ferredoxin and is reduced to the ferrous form when ferredoxin is reduced—the number of iron atoms reduced being one for plant ferredoxin and two for bacterial ferredoxin. 

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