Dihydroxy acid dehydratase from spinach contains a [2Fe-2S] cluster

Area of Science:

• Biochemistry
• Plant Physiology
• Enzymology

Background:

• Dihydroxy acid dehydratase is a key enzyme in the branched-chain amino acid biosynthetic pathway.
• Understanding its structure and function is crucial for plant metabolism research.

Purpose of the Study:

• To purify dihydroxy acid dehydratase from spinach leaves.
• To characterize the enzyme's structure, particularly its iron-sulfur content and redox properties.
• To investigate the role of the iron-sulfur cluster in catalysis.

Main Methods:

• Enzyme purification to homogeneity (5000-fold).
• Sodium dodecyl sulfate and native gel electrophoresis for molecular weight determination.
• Chemical analysis for iron and labile sulfide content.
• Electron Paramagnetic Resonance (EPR) spectroscopy to analyze the iron-sulfur cluster.
• Enzyme activity assays under various redox conditions.

Main Results:

• Purified dihydroxy acid dehydratase is a dimer with monomers of 63,000 Da.
• The enzyme contains a [2Fe-2S] cluster, a novel finding for hydrolyase enzymes.
• The Fe-S cluster has a low redox potential (-470 mV) and its reduction by 6-fold decreases enzyme activity.
• Substrate addition alters the EPR spectrum and visible absorption spectrum, indicating cluster involvement in catalysis.

Conclusions:

• Dihydroxy acid dehydratase from spinach contains a [2Fe-2S] cluster.
• This [2Fe-2S] cluster is directly involved in the catalytic mechanism of the enzyme.
• The findings expand the known roles of iron-sulfur clusters in enzyme catalysis, particularly within the hydrolyase class.