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Abstract
Iron-sulfur (Fe-S) clusters are ancient and ubiquitous cofactors utilized by proteins for a wide variety of biological functions. The objectives of this work were to elucidate the properties and functions of Azotobacter vinelandii (Av) IscU and Arabidopsis thaliana Nfu2, which are generally considered to be primary scaffold proteins for the biogenesis of Fe-S clusters in bacteria and plant chloroplasts, respectively. A series of biological and biophysical methods were employed in order to understand the nature, properties and coordination environments of the clusters assembled on these proteins and their interactions with physiological partner proteins. Experimental techniques include but not limited to site-directed mutagenesis, protein recombinant expression and purification, enzyme activity assays, UV-visible absorption and circular dichroism (CD), resonance Raman (RR), electron paramagnetic resonance (EPR) and Mssbauer spectroscopies. Characterization of the Asp39-to-Ala mutant of Av IscU, especially studies of the pH effects, indicated that the mutation results in replacement of the original aspartate ligand of the [2Fe-2S] cluster with a histidine ligand. This finding indicates that the Asp39-to-Ala mutant is not an appropriate model for studying the cluster assembly and transfer functions of WT IscU. The combination of spectroscopic, analytical and gel filtration data also indicate that the [4Fe-4S] cluster bound in IscU is coordinated within one subunit with at least one non-cysteinyl ligand, and that the [4Fe-4S] cluster-bound forms of IscU are predominantly in the form of tetramers in solution. Our studies on At Nfu2 demonstrated that this protein was able to assemble both [2Fe-2S] and [4Fe-4S] clusters in vitro under different reconstitution conditions. In the presence of EDTA or oxygen, the [4Fe-4S] cluster-bound Nfu2 underwent conversion to the [2Fe-2S] cluster-bound form. Moreover, the [4Fe-4S] cluster assembled on Nfu2 can be rapidly and quantitatively transferred to adenosine 5'-phosphosulfate reductase 1, an important sulfur metabolism enzyme in plant chloroplasts. In addition, cluster transfer studies indicate that the [2Fe-2S] cluster on Nfu2 is effective for maturation of dihydroxy-acid dehydratase and the cluster carrier protein, glutaredoxin S16. These results support a role for At Nfu2 as a scaffold protein in chloroplasts that is involved in biogenesis of both [2Fe-2S] and [4Fe-4S] clusters.