Iron-sulfur proteins with cuboidal [Fe4S4] clusters exhibit a remarkable functional diversity. Insights on the factors determining the function of the protein can be obtained by modifications of the metal site by incorporation of metals other than iron in the active site of the protein. This approach further has interesting perspectives in the design of new biologically based catalytic systems. This project presents two strategies for incorpora¬tion of non-natural metals in iron-sulfur proteins. These studies are based on the ferredoxin from Pyrococcus furiosus. This ferredoxin can contain either a [Fe3S4] cluster or a [Fe4S4] cluster. One new protein was syn¬the¬sized by incor¬porating cobalt into the [Fe3S4] cluster thus creating a [CoFe3S4] cluster. The other arti¬ficial protein was designed by replacement of the iron-sulfur cluster with a synthetic [Mo4S4] cluster. The synthesis, purification and characteri¬za¬tion of the two new proteins were carried out. The P. furiosus ferredoxin was studied as a reference for the two artificial proteins. The P. furiosus [Fe3S4] and [Fe4S4] ferre¬doxins were studied with cyclic voltam¬¬me¬try as re¬fe¬rence for the work on the artificial proteins. The effects of different buffer systems and additives were tested to find the optimal conditions for electro¬chemi¬cal charac¬te¬ri¬za¬tion. Different buffer systems did not have a significant effect, but the voltammo¬grams were strongly dependent on the NaCl content as NaCl had an attenua¬ting effect on the redox signals of the P. furiosus ferredoxins. The P. furiosus ferredoxin with the heterometallic [CoFe3S4] cluster was synthesized and purified in the oxidized [CoFe3S4]2+ state. The chromatographic, mass spectro¬metric and EPR spec¬troscopic results indicated that the [CoFe3S4]2+ ferredoxin was purified to high purity and that the pro¬tein was stable under the used conditions. These results are in dis¬agree¬ment with pre¬vious reports of readily oxidative degrada¬tion of the [CoFe3S4]2+ ferredoxin to [Fe3S4]+ ferredoxin. Experiments with chemical reduc¬tion and oxi¬dation sugges¬ted a redox active protein and this was confirmed by cyclic voltammetry. One well-defined pair of redox peaks appear¬ed and the pair was assig¬ned to the [CoFe3S4]2+/+ redox couple and had a formal potential of -177 mV versus SHE. Unlike the naturally occurring iron-sulfur cluster the molybdenum-sulfur cluster is not incorporated into the ferredoxin by self-assem¬bly. Instead the molybdenum-sulfur ana¬lo¬gue was synthesized by addition of pre-prepared [Mo4S4(H2O)12]Cl5 to the apo-ferredoxin which was stabilized by sulfonation. The purification of the molyb¬denum-sulfur ana¬lo¬gue revealed two closely related species and that the ratio between the two species depended on the experimental conditions. The two purified species were subjected to EPR moni¬tored redox titration and the obtained EPR spectra were compared to the spectra of [Mo4S4(H2O)12]5+. The results confirmed the incor¬poration of the intact [Mo4S4] cluster and sug¬ges¬ted stabilization of the cluster in three oxidation states; the 4+, 5+ and 6+ states. The formal potentials of the transitions be¬tween the three oxidation states were deter¬mi¬ned to -195 mV and -295 mV versus SHE for the first species and -205 mV and -380 mV versus SHE for the other species. The obtained spectra after oxidative titration suggested oxidative break down of the [Mo4S4] cluster. It is not possible to identify the ligands on the cluster based on these studies, however, the spectra suggest that the difference between the two species in variations in the ligand environment. These studies have given important insights on these new proteins and have increased the understanding of ferredoxins with re-designed active sites. This is an important step for use of these proteins in new catalytic systems.