Iron-sulfur cluster biosynthesis: Iron-sulfur cluster transfer from holo ISU and ISA to apo ferredoxin

The processes of iron-sulfur cluster transfer from holo ISU and ISA to apo Fd were studied. Direct cluster transfer from holo ISU to apo Fd was demonstrated by M6ssbauer spectroscopy. Experimental data shows that an intact [2Fe-2S] core is transferred to target proteins. Substitution of a key aspartate residue (Asp 37) of ISU is found to decrease the rate of cluster transfer by at least an order of magnitude. The rate constants of cluster transfer from native ISA to Fd are shown to be similar to those of cluster transfer from Cys derivatives of ISA. The factors that contribute to cluster transfer efficiency also appear to be similar, including the importance of cluster lability and solvent accessibility.; Both ISA- and ISU-mediated Fe-S cluster transfer to apo Fd have been shown to be pH-dependent. The pKa of ISA is 7.8, slightly higher than the value of 6.9 found for ISU. This is consistent with deprotonation of a solvent accessible cysteine which would thereby provide a more effective nucleophilic center for facilitating cluster transfer to apo Fd. Viscosity studies show that diffusion is not the rate-limiting step, which is most likely the process of Fe-S cluster transfer between the intermediate complexes, ISU-Fd and ISA-Fd.; The influences of the binding of DnaK to IscU were studied. DnaK can maintain a stable structure of ferric ion-bound IscU and avoids aggregation triggered by ferric ions. Also, DnaK-bound IscU has more stable iron sulfur clusters. Kinetic studies of Fe-S cluster transfer from holo IscU to apo Fd in the presence of DnaK reveal that the binding of DnaK to IscU slows down the rate of Fe-S cluster transfer from IscU, especially formation of IscU-Fd complexes. Binding of DnaK decreases the rates of forming IscU-Fd complexes (greater than 8-fold) and also causes a minor change on the rates of iron sulfur cluster transfer in IscU-Fd complexes. In other words, binding of DnaK to IscU does not facilitate the process of Fe-S cluster transfer from IscU.