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 [Fe₄S₄] cluster, and spinach DHAD, which contains an oxygen-resistant [Fe₂S₂] 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 AtDHAD can accommodate [Fe₂S₂] and [Fe₄S₄] clusters. However, only the [Fe₂S₂] cluster-bound form is catalytically active. We found that the [Fe₂S₂] 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 [Fe₂S₂] cluster-containing NFU2 protein is likely the physiological cluster donor for in vivo maturation of AtDHAD. In summary, AtDHAD binds either one [Fe₄S₄] or one [Fe₂S₂] cluster, with only the latter being catalytically competent and capable of substrate and product binding, and NFU2 appears to be the physiological [Fe₂S₂] cluster donor for DHAD maturation. This work represents the first in vitro characterization of recombinant AtDHAD, providing new insights into the properties, biogenesis, and catalytic role of the active-site Fe-S center in a plant DHAD.