Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe-S Cluster...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Iron-sulfur (Fe-S) clusters are vital prosthetic groups in proteins.
• Their biosynthesis involves scaffold proteins like ISCU and conserved machinery.
• The initial iron insertion mechanism into ISCU remains unclear.

Purpose of the Study:

• To elucidate the mechanism of initial ferrous iron insertion into the ISCU scaffold protein.
• To determine the binding site and coordination of iron during Fe-S cluster biosynthesis.
• To investigate the role of conserved amino acids in ISCU iron binding and sulfur acquisition.

Main Methods:

• Size exclusion chromatography (SEC)
• Circular dichroism (CD) spectroscopy
• Mössbauer spectroscopy
• Site-directed mutagenesis
• Nuclear Magnetic Resonance (NMR) spectroscopy
• X-ray absorption spectroscopy
• Electron paramagnetic resonance (EPR) spectroscopy

Main Results:

• Iron binds to the ISCU assembly site via one or two cysteines (1-Cys and 2-Cys forms).
• The 2-Cys form predominates at pH 8.0 and correlates with Fe-S cluster assembly activity.
• Iron is coordinated by Cys35, Asp37, Cys61, and His103 in a tetrahedral geometry in the 2-Cys form.
• Cys104 is positioned for sulfur transfer, particularly when His103 is absent.

Conclusions:

• The initiation of Fe-S cluster biosynthesis involves ferrous iron insertion into the ISCU assembly site.
• A conserved mechanism of iron binding and coordination by ISCU is established.
• These findings provide structural insights into the Fe-S cluster assembly process.