Specific labeling of ironsulfur cluster subsites

Abstract

Desulfovibrio gigas ferredoxin is isolated in different oligomeric forms [1]. These ferredoxins, termed FdI and FdII, are built from one type of monomeric unit of which the sequences of 57 amino acids is known [2, 3]. FdII is a tetramer of molecular weight 24,000. Mössbauer and EPR studies [4] have shown that each monomer of FdII contains one [3Fe-xS] center. The other oligomeric form, the trimeric FdI [5] was shown to contain as a majority species a [4Fe4S] cluster. However the FdI trimer can accomodate a [3FexS] cluster as minority species of variable proportions. Evidence was accumulated that both clusters can be accomodated by the polypeptide chain of this ferredoxin: 1. Controlled reconstitution experiments of the active center of D. gigas FdII (containing only [3FexS] cores) were performed in such a way that a reconstituted protein, Fd R, loaded only with [4Fe4S] cores was obtained [6]. 2. Mössbauer and EPR spectroscopies have been use to extensively study the process of cluster interconversion in D. gigas FdII. The [3FexS] centres could be converted into [4Fe4S] clusters after incubation with Fe 2+ in the presence of dithiothreitol [6]. Based on these observations incubations of FdII were performed using 95% enriched 57Fe in the presence of sulfide and dithiothreitol. Conversion from a [3FexS] into a structure with a [4Fe4S] core occurred; the latter seems to be structurally identical to the cluster of Fd R. The 57Fe Mössbauer ▪ spectra of the newly formed [4Fe4S] cluster [6] correspond to those obtained for the subsites of the Fd R cluster (Fig. 1). The data show that the externally added iron occupies either one subsite or at most two structurally equivalent sites of the [3Fe4S] cluster [6, 7]. The isotopic labelling of specific cluster subsites allows us to study in considerable detail the hyperfine interactions of he [4Fe-4S] core structures. Moreover, this technique will allow us to correlate spectra of subsites in different oxidation states. Application of this technique of isotopic labeling to individual clusters of enzymes with multiple clusters ( e.g., hydrogenase) will greatly simplify the spectral assignments.