Reactions of iron-sulfur clusters in proteins

Thesis

This thesis describes the investigation of reactions of iron-sulfur clusters in proteins using direct electrochemistry. The influence of potential on metal uptake to generate the [M3Fe-4S] cluster from the [3Fe-4S] cluster of Desulfovibrio africanus Fd III is studied. The influence of potential was complex: rapid and reversible interconversions (M = Fe and Zn) occurred only between the states [M3Fe-4S]²⁺ and [3Fe-4S]⁰, with [3Fe-4S]¹⁺ having little affinity for M. The [M3Fe-4S]¹⁺ cubanes and the hyper-reduced [3Fe-4S]^2- were relatively unreactive.

The reactivity of the transformed cluster, [M3Fe-4S] (M = Fe, Zn, Co), from the 7Fe Fd of Desulfovibrio africanus was studied and was found to react with a number of small thiol molecules, indicating that either ligand addition or exchange takes place at the transformed M site of the cluster. No reaction was observed with oxygenic ligands. In all cases, with the exception of imidazole, negative shifts in reduction potentials were observed.

Reactions of the [2Fe-2S] cluster from the ferredoxin of Clostridium pasteurianum and a number of site-directed mutants of this ferredoxin are studied. The cysteine ligands of the cluster were identified and evidence was obtained for serinate ligation of the cluster in a number of mutants. The reduction potentials of these serinateligated clusters were found to have a notable dependence on pH. A mutant ferredoxin containing only three cysteine ligands was investigated, which was found to interact with an exogenous thiolate ligand and, in addition, displayed a second reduction couple, indicating the formation of the [2Fe-2S]⁰ state.

Reactions of the [3Fe-4S] cluster and various [M3Fe-4S] adducts, from the ferredoxin of the hyperthermophile Pyrococcus furiosus, are studied. The [3Fe-4S] cluster exhibited a complex pH dependence over a wide pH range. The formation of the hyper-reduced [3Fe-4S]^2- state was observed, which required 3H⁺ for the overall 3e‧ reduction from [3Fe-4S]¹⁺. Metal uptake reactions for M = Fe, Zn, Cd, were found to be slower than for its mesophilic counterpart, the 7Fe Fd III from Desulfovibrio africanus. Conversely, Tl uptake was found to be rapid, suggesting that co-ordination of Tl does not require reorganisation of the protein structure.

Authors: Fawcett, S

• Peer review status: Peer reviewed
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford