Rubredoxin and Desulforedoxin

An exception to this coordination pattern is observed in Desulfovibrio (D.) gigas desulforedoxin (Dx). The protein is a 8-kDa homodimer where it was found that two of the coordinating cysteines were contiguous in the amino acid sequence (10, 11) (Fig. 2). This fact im- 

While crystal structures of rubredoxins have been known since 1970 (for a full review on rubredoxins in the crystalline state, see Ref. (15)), only recently have both crystal and solution structures of Dx been reported (16, 17) (Fig. 3). The protein can be described as a 2-fold symmetric dimer, firmly hydrogen-bonded and folded as an incomplete /3-barrel with the two iron centers placed on opposite poles of the molecule, 16 A apart. Superimposition of Dx and Rd structures reveal that while some structural features are shared between these two proteins, significant differences in the metal environment and water structure exist. They can account for the spectroscopic differences described earlier. 

Mutants of Dx were constructed, introducing Gly and Pro-Val sequences between Cys 28 and Cys 29 residues, altering the spacing between the adjacent coordinating cysteines. The properties of the mutated proteins are altered and the center became more close to that in rubredoxin (18). 

Different metals are readily incorporated into rubredoxin-type centers after reconstitution of apoprotein with appropriate metal salts (5). Rd and Dx derivatives containing Ni and Co have been analyzed by UV-vis, NMR, EPR, electrochemistry, and MCD (13, 19-22). Fe replacement has been used for Mossbauer studies and an indium de- 

It has always been assumed that these simple proteins act as electron-transfer proteins. This is also a fair conclusion if we take in account that different proteins were isolated in which the Fe(RS)4 center is in association with other non-heme, non-iron-sulfur centers. In these proteins the Fe(RS)4 center may serve as electron donor/ac-ceptor to the catalytic site, as in other iron-sulfur proteins where [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters are proposed to be involved in the intramolecular electron transfer pathway (see the following examples). 

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In the Desulfovibrio species, a large variety of metalloproteins has been isolated and the rubredoxin- and desulforedoxin-like centers can be found in association with other types of mononuclear or dinuclear iron centers as shown in Section 20.5. 

Single-metal replacement data in rubredoxin and desulforedoxin and crystal structures... 

Single-metal replacement data in rubredoxin and desulforedoxin 351 Table 20-2 Comparison of metal coordination geometry in different metal-substituted rubredoxins... 

The classification of iron-sulfur proteins typically uses the number of irons contained in the coordination. Although at least two irons are necessary to accommodate the inorganic sulfur, the simple Fe(S)4 coordination with cysteinyl sulfur also counts to the group of iron-sulfur proteins. This motif is rare and realized only in few proteins, e.g. rubredoxin or desulforedoxin. 

A protein from Desulfovibrio gigas, called desulforedoxin, ° appears to resemble rubredoxins in some respects, but the two Fe atoms in the 7.6-kDa protein appear to be spectroscopically and structurally distinct from the Fe atoms in rubredoxins. A protein from Desulfovibrio vulgaris called ruberythrin has a single rubredoxin site as well as a strongly coupled 2Fe site resembling that of hemerythrin. Its physiological function is unknown. Table 7.1 lists some of the known rubredoxins and their properties. 

No EPR spectra have yet been reported to our knowledge in the case of a protein containing a well-characterized reduced FeS4 center, although a spectrum has been observed in the case of a model complex (24 ). The lack of EPR signals in the case of proteins is apparently directly related to the D and E values, which are equal to D = +7.5 cm E D = 0.28 in the case of C. pasteurianum rubredoxin (15), and D = 6 cm E D = 0.19 in that of D. gigas desulforedoxin (18),... 

A protein named desulforedoxin was isolated from a sulfate reducer organism, Desulfovibrio gigas, by Moura et al.47) and which represents a variation on the basic rubredoxin type structure. Its spectroscopic features will be discussed in relation to those of rubredoxin isolated from D.gigas and Clpasteurianum. 

The comparison of the amino-acid sequence of the two proteins8,12) can give a clue to the nature of these differences. The spacing of the four cysteine residues in sequences of the type Cys(6)-a-b-Cys(9)-Gly and Cys(39)-c-d-Cys(42)-Gly as observed in rubredoxin are replaced by the sequences Cys(9)-x-y-Cys(12)-Gly and Cys(28)-Cys(29)-Gly. This unusual arrangement may impose the stereochemical constraints which are responsible for the spectral differences between desulforedoxin and rubredoxin. The existence of a dimer structure opens also the question of the possibility that the four cysteines that bind the active center do not belong to the same polypeptide chain, as schematically proposed in Fig. 3 73). These seem to be the only two possibilities since the two iron atoms should be equivalent according to the spectroscopic data. 

The visible spectra of Rd and Dx are, in general terms, quite similar, another indication that both proteins contain similar types of iron centers [31]. The typical red color of oxidized Rd and Dx is due to a S — Fe charge-transfer band at 490 and 507 nm, respectively [34]. However, Mossbauer and EPR data for Dx [34, 35] show some differences when compared to Rd, suggesting that the metal ligand geometry and/or coordination environment of these proteins differ. Desulforedoxin and rubredoxin backbones share some similar structural features but show significant differences in terms of metal environment (due to the different cysteine spacing) and water network. 

Figure 20-4 Stereo representation of a superposition of the Ca atoms of D. gigas rubredoxin (dark gray) and one monomer of the desulforedoxin molecule (light gray), for segments 4A-18A ( rubredoxin knuckle ), and 33A-36A (Dx numbering) with ordered water molecules of Dx. Figure prepared with TURBO-FRODO [84].

Figure 20-5 (A) Stereo view of the D. gigas rubredoxin Fe(SCys)4 center viewed along the pseudo two-fold axis, with NH-S hydrogen bonds marked. The orientation is approximately the same as that of (B) for the Dx center. (B) Stereo view of the Fe SCys)4 center from desulforedoxin with NH-S hydrogen bonds marked. Water molecules, W27 and W28 are conserved in all metal-replaced Dx structures and are therefore considered structurally part of the desulforedoxin molecule. The water molecule W3010 is only observed for the Cd- and Hg-substituted Dx structures and makes a hydrogen bond to the Sy of Cys 29, the most exposed cysteine ligand. Figures prepared with TURBO-FRODO [84].

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