Function and maturation of the Fe-S center in dihydroxyacid dehydratase from Arabidopsis .

Dihydroxyacid dehydratase (DHAD) is the third enzyme required for branched-chain amino acid biosynthesis in bacteria, fungi, and plants. DHAD enzymes contain two distinct types of active-site Fe-S clusters. The best characterized examples are Escherichia coli DHAD, which contains an oxygen-labile [Fe4 S4 ] cluster, and spinach DHAD, which contains an oxygen-resistant [Fe2 S2 ] cluster. Although the Fe-S cluster is crucial for DHAD function, little is known about the cluster-coordination environment or the mechanism of catalysis and cluster biogenesis. Here, using the combination of UV-visible absorption and circular dichroism and resonance Raman and electron paramagnetic resonance, we spectroscopically characterized the Fe-S center in DHAD from Arabidopsis thaliana ( At ). Our results indicated that At DHAD can accommodate [Fe2 S2 ] and [Fe4 S4 ] clusters. However, only the [Fe2 S2 ] cluster-bound form is catalytically active. We found that the [Fe2 S2 ] cluster is coordinated by at least one non-cysteinyl ligand, which can be replaced by the thiol group(s) of dithiothreitol. In vitro cluster transfer and reconstitution reactions revealed that [Fe2 S2 ] cluster-containing NFU2 protein is likely the physiological cluster donor for in vivo maturation of At DHAD. In summary, At DHAD binds either one [Fe4 S4 ] or one [Fe2 S2 ] cluster, with only the latter being catalytically competent and capable of substrate and product binding, and NFU2 appears to be the physiological [Fe2 S2 ] cluster donor for DHAD maturation. This work represents the first in vitro characterization of recombinant At DHAD, providing new insights into the properties, biogenesis, and catalytic role of the active-site Fe-S center in a plant DHAD.