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.

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