Rubredoxin structure

Figure 20-1 Ribbon diagram of the D. gigas rubredoxin structure. Figure prepared with MOLSCRIPT [82] and RASTER-3D [83],...

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)...

One example of a sequence determinant of redox potentials that has been identified in this manner is an Ala-to-Val mutation at residue 44, which causes a 50 mV decrease in redox potential (and vice versa) in the rubredoxins [68]. The mutation was identified because the sum of the backbone contributions to ( ) of residues 43 and 44 change by 40 mV due to an —0.5 A backbone shift away from the redox site. This example points out the importance of examining the backbone contributions. The corresponding site-specific mutants have confirmed both the redox potential shift [75] and the structural shift [75]. 

Fig. 5. Structure-based alignment of the sequences of the water-soluble Rieske fragment from bovine heart bci complex (ISF), the water-soluble Rieske fragment from spinach b f complex (RFS), and of the Rieske domain of naphthalene dioxygenase (NDO) and of the metal binding loops of rubredoxin (RXN) and transcriptional factor TFIIS (TFI). The numbering of the j3 strands is the same for the ISF and RFS. The metal binding ligands are highlighted the asterisks indicate those residues that are fully conserved between the three Rieske proteins.

The general topology of rubredoxins is also observed in the general zinc-ribbon motif in RNA polymerases or in transcription factors (59). The first published zinc-ribbon structure was that of the nucleic-acid binding domain of human transcriptional elongation factor TFIIS (PDB file ITFI) 40). These zinc binding domains and rubredoxins... 

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. 

In 1996, the 3D-structure of D. vulgaris Rr was published by de-Mare and collaborators 48), and all the studies earlier published were proved to be correct. The protein is described as a tetramer of two-domain subunits (Fig. 4). Each subunit contains a domain characterized by a four-helix bundle surrounding a diiron-oxo site and a C-terminal rubredoxin-like Fe(RS)4 domain (see Fig. 2). In this last do-... 

Kok M, R Oldenius, MPG van der Linden, CHC Meulenberg, J Kingma, B Witholt (1989a) The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase. J Biol Chem 264 5442-5451. 

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 crystallographic structure of rubredoxin from Clostridium pasteurianum at 2.5 A, a resolution sufficient to reveal the sequence of several of the bulky amino acid side chains, shows the iron coordinated to two pairs of cysteine residues located rather near the termini of the polypeptide chain (Fig. 1). A related rubredoxin, with a three times larger molecular weight, from Pseudomonas oleovorans is believed to bind iron in a similar fashion. This conclusion is based on physical probes, especially electron paramagnetic resonance spectroscopy, all of which indicate that the iron is in each case situated in a highly similar environment however, the proteins display some specificity in catalytic function. 

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). 

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. 

Jensen [3.11] as well as Teeter [3.12] studied by X-ray diffraction the structure of water molecules in the vicinity, at the surface and inside of protein crystals. Jensen used rubredoxin (CEB) crystals to deduce the structure of water from the density distribution of electrons, calculated from diffraction pictures. Jensen found that water molecules which are placed within approx. 60 nm of the protein surface form a net, which is most dense in the distance of a hydrogen bond at the donor- or acceptor- molecules of a protein. In distances larger than 60 nm, the structure of water becomes increasingly blurred, ending in a structureless phase. Water molecules are also in the inside of proteins, but are more strongly bound than... 

Fe-S proteins contain four basic core structures, which have been characterized crystal-lographically both in model compounds (Rao and Holm, 2004) and in iron-sulfur proteins. These are (Figure 3.6), respectively, (A) rubredoxins found only in bacteria, in which the [Fe-S] cluster consists of a single Fe atom liganded to four Cys residues—the iron atom... 

The simplest of these proteins are rubredoxins, which are bacterial proteins having a characteristic red colour (from which their name is derived) containing an FeS4 assembly, consisting of an Fe(III) ion coordinated to four cysteine groups. The typical tetrahedral structure of this group is illustrated in Figure 17 for the rubredoxin isolated from Clostridium pasteurianum (FW 6100).35... 

Figure 17 X-Ray structure of the active site of the rubredoxin from Clostridium pasteurianum...

One simple case of disordered structure involves many of the long charged side chains exposed to solvent, particularly lysines. For example, 16 of the 19 lysines in myoglobin are listed as uncertain past C8 and 5 of them for all atoms past C/J (Watson, 1969) for ribonuclease S Wyckoff et al. (1970) report 6 of the 10 lysine side chains in zero electron density in trypsin the ends of 9 of the 13 lysines refined to the maximum allowed temperature factor of 40 (R. Stroud and J. Chambers, personal communication) and in rubredoxin refined at 1.2 A resolution the average temperature factor for the last 4 atoms in the side chain is 9.2 for one of the four lysines versus 43.6, 74.4, and 79.3 for the others. Figure 57 shows the refined electron density for the well-ordered lysine and for the best of the disordered ones in ru-... 

Fig. 58. Stereo drawing of the rubredoxin backbone with the iron (filled circle) and its cysteine sulfur ligands and all the water molecules (open circles) identified during refinement of the structure at 1.2 A resolution. Adapted from Watenpaugh et al. (1979), Fig. 11, with permission.

Most of the metal-rich proteins form approximately cylindrical two-layer structures with either an up and down (rubredoxin, cytochrome c) or a Greek key (ferredoxin) topology, but in which the elements forming the cylinder are a mixture of helices, /3 strands, and more or less extended portions of the backbone. Cytochrome c3 is perhaps the ultimate example of an S-M protein, with four hemes in just over a hundred residues, and essentially no secondary structure at all except for one helix. 

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