Iron-sulfur clusters characteristics

Resonance Raman studies of the recombinant proteins showed vibrational bands at the 200-430 cm region characteristic of iron-sulfur clusters (124). Most interestingly, on Fe and O isotope sensitive band was detected at 801 cm which could be attributed to either a Fe(IV)=0 species or a monobridged Fe-O-Fe structure. This observation, together with Mossbauer analysis, which indicated a mixed N, 0, and S ligand environment for cluster 2, suggests a Fe-O-Fe or Fe=0 unit as part of the structure for cluster 2. 

In the hydrogenosomal membranes, EPR spectra showed no trace of the highly characteristic features of the iron-sulfur clusters of complex I (NADH ubiquinone reductase) and the Rieske protein of complex III of the mitochondrial respiratory chain. This is consistent with the absence of... 

Some characteristic properties of iron-sulfur clusters in proteins result from interactions with invariant amino acid fragments around the clusters. In particular, clusters in peptide environments exhibit positive shifts in redox potentials relative to those in nonpeptide environments. Such shifts are observed for a variety of oligopeptide model complexes of IFe, 2Fe-2S, and 4Fe-4S proteins. 

The complexity of the low temperature MCD spectra of the oxidized and reduced trinuclear cluster shows the multiplicity of the predominantly S — Fe charge transfer transitions that contribute to the absorption envelope. While MCD spectroscopy provides a method of resolving the electronic transitions, assignment cannot be attempted without detailed knowledge of the electronic structure. However, the complexity of the low temperature MCD spectra is useful in that it furnishes a discriminating method for determining the type and redox state of protein bound iron-sulfur clusters. Each well characterized type of iron-sulfur cluster, i.e. [2Fe-2S], [3Fe-4S], and [4Fe-4S], has been shown to have a characteristic low temperature MCD spectrum in each paramagnetic redox state (1)... 

The reduction state of the pterin was a point of uncertainty throughout these studies of molybopterin derivatives. The absence of fluorescence in anaerobic molybdopterin samples suggested a reduced pterin. Redox titration of XO and SO both indicated that the pterin could undergo a two-electron oxidation reaction (73, 74). Sulfite oxidase, for example, produced the fluorescence characteristic of an oxidized pterin after addition of 2 equiv of ferricyanide. However, titrating XO was problematic due to interfering redox processes of the iron-sulfur clusters. 

Aconitase was first suggested to have an iron-sulfur cluster by virtue of the presence of acid-labile sulfide, as well as a g = 2.01 EPR signal, in the purified protein. The iron-sulfur cluster giving rise to this g = 2.01 signal was later shown to be a [3Fe-4S]" " cluster, and was characteristic of inactive enzyme. " " Activation by addition of iron and reductant, or by addition of reductant alone, provided the enzymatically active aconitase, although in the latter case only approximately 75% of full activity was obtained. As the active form of aconitase was subsequently shown to be the [4Fe-4S] form, " addition of iron and reductant provided the iron needed to occupy the fourth metal site in the cluster, while addition of reductant alone resulted in rearrangement of the clusters, labilizing iron to allow reassembly of [4Fe-4S] clusters in 3/4 of the cluster sites. 

Scheme 6 Some of the important iron-sulfur cluster units found in metalloenzymes. [2Fe-2S] rhombus cluster is characteristic of [2Fe-2S] feiredoxins and Rieskie proteins, [4Fe-4S] cubane -e.g., in [4Fe-4S] feiredoxins, aconitase [3Fe-4S] clusters are present in the inactive form of aconitase, [3Fe-4S] feiredoxins. The iron vertices, designated as [Fe], have high-spin tetrahedral FeS4 coordination. Reprinted from [140], with the permission of Elsevier, copyright 2000...

Our current work with the Photosystem I core protein reconfirms that the Fx iron-sulfur cluster can be oxidatively converted to zero-valence sulfur by treatment with 3 M urea and 5 mM potassium ferricyanide [13,14]. Under these conditions, the P700 flash-induced optical transient has a lifetime of 5 is due to the relaxation of the P700 triplet state, indicating that the P700 Aq primary charge separation and recombination process has remained intact (Fig. 3a,b). We have now found that the Fx iron-sulfur cluster can be reconstituted by addition of ferrous iron, sodium sulfide, and 6-mercaptoethanol to the oxidatively-denatured Photosystem I core protein [see refs. 15 16 for methodology employed]. After incubation for 24 hr, the 5 is optical transient becomes replaced with a 1.2-ms optical transient that is characteristic of the P7(X)+-Fx backreaction (Fig. 3c). 

The reductase from M.capsulatus (Bath) is composed of a single polypeptide of Mr = 44.6 kDa and 1 mol of FAD and 1 mol of 2Fe2S cluster per mol of protein [24]. The reductase is responsible for an electron transfer from NADH to the hydroxylase [25]. All reductases from the other methanotrophs have the same cofactors, FAD and 2Fe2S cluster, and their molecular weights are about 40 kDa. Electrons from NADH are transferred to FAD to form fully reduced FADH2, which transfers one electron very rapidly to the iron-sulfur cluster to form a flavin semiquinone radical [15, 45]. The semiquinone radical is detected as a free radical signal at g = 2.004 by means of ESR spectroscopy [46]. Upon complete reduction of the reductase with sodium dithionite, the ESR spectrum of the reductase shows the g values at 2.04, 1.96, and 1.87 (gav = 1-96) characteristic of a reduced 2Fe2S cluster [46]. 

---