Rubredoxin iron coordination

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. 

Proteins with dinuclear iron centres comprise some well studied representatives like ribonucleotide reductase (RNR), purple acid phosphatase (PAP), methane monooxygenase hydroxylase (MMOH), ruberythrin and hemerythrin. The latter is an oxygen carrier in some sea worms it has been first characterized within this group and has thus laid the foundation to this class of iron coordination motif. Ruberythrin is found in anaerobic sulfate-reducing bacteria. Its name implies that, in addition to a hemerythrin-related diiron site another iron is coordinated in a mononuclear fashion relating to rubredoxin which is an iron-... 

The simplest NHIP is rubredoxin, in which the single iron atom is coordinated (Fig. 25.9a) to 4 S atoms belonging to cysteine residues in the protein chain. It differs from the other Fe-S proteins in having no labile sulfur (i.e. inorganic sulfur which can be liberated as H2S by treatment with mineral acid sulfur atoms of this type are not part of the protein, but form bridges between Fe atoms.)... 

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).

In this review we will deal with iron-sulfur proteins where the iron atoms are coordinated only by cysteine ligands and bridging sulfurs, as well as rubredoxin (Rd hereafter), which is the initial building block in all subsequent discussions. 

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 ).

Rubredoxins do not have acid-labile sulfur as do ferredoxins, with the iron characterized by a mercaptide coordination. They are small proteins composed of some 50 amino acids. Both oxidized and reduced forms are high spin. 

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). 

Rbo is a homodimeric protein, each subunit of which contains two distinct mononuclear nonheme iron centers in separate domains (Fig. 10.4) (Coehlo et al. 1997). Center I contains a distorted rubredoxin-type [Fe(SCys)4] coordination sphere. [Fe(SCys)4] sites in proteins are known to catalyze exclusively electron transfer, which is, therefore, the putative function for center I. Center II contains a unique [Fe(NHis)4(SCys)] site that is rapidly oxidized by 0, and is, therefore, the likely site of superoxide reduction (Lombard et al. 2000). A blue nonheme iron protein, neelaredoxin (Nlr) from Desulfovibrio gigas (Silva et al. 1999), contains an iron center closely resembling that of Rbo center II (Table 10.1). The blue color is due to the oxidized (i.e., Fe(III)) form [Fe(NHis)4(SCys)] site, which, in both Nlr and Rbo, has a prominent absorption feature at -650 nm. Reduction of center II to its Fe(II) form fully bleaches its visible absorption. These absorption features have been used to probe the reactivity of Rbo with superoxidie. 

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. 

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedrally coordinated by a combination of thiolate and sulfide donors. Of the 10 or more structurally characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein rubredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane structures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane structure (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH3 at a MoFe S8 homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

In oxidized rubredoxin there is a pseudotetrahedral iron(III) coordinated to four cysteine sulfurs. The cysteine protons are broad beyond detection. The PC2H2 signals of 2H labeled cysteines are located far downfield (300-900 ppm) (Figs. 5.14 and 5.15) [30], which correspond to hyperfine coupling constants of about 1-3 MHz for protons. Two of the four 2Ha protons appear downfield (180 and 150 ppm), while the other two appear upfield (—10 ppm, overlapped). Either... 

The active site of rubredoxin (Rd), (I) (Figure 1), consists of one Fe ion tetrahedraUy coordinated by four cysteine thiolate ligands. The iron core serves as the electron-transport site of rubredoxin proteins. 

Negative-ion gas-phase photoelectron spectroscopy has also recently been nsed to evalnate the reorganization energies of tetrahedrally coordinated Fe+ anions that serve as models for the active sites of mononuclear iron sulfur proteins such as rubredoxin that cycles between high spin Fe+ and high spin... 

---