Yeast [FeFe]-hydrogenase-like protein Nar1 binds a [2Fe-2S] cluster

Area of Science:

• Biochemistry
• Molecular Biology
• Bioinorganic Chemistry

Background:

• Nar1 is an essential eukaryotic protein in the cytosolic iron-sulfur protein assembly (CIA) machinery.
• Its precise function as an iron-sulfur (Fe/S) cluster trafficking factor has been hindered by purification challenges.
• Nar1, a [FeFe]-hydrogenase-like protein, possesses binding sites for [4Fe-4S] clusters but lacks native hydrogenase activity.

Purpose of the Study:

• To develop a new preparation procedure for Nar1 to enable detailed biochemical and biophysical characterization.
• To elucidate the Fe/S cluster binding properties and stoichiometry of Nar1.
• To investigate the oxygen sensitivity of Nar1 and its implications for the CIA pathway.

Main Methods:

• Recombinant protein production in E. coli.
• UV-vis, EPR, and Mössbauer spectroscopies.
• Native mass spectrometry and Fe/S reconstitution assays.

Main Results:

• A novel Nar1 preparation yielded protein with bound [4Fe-4S] and unexpected [2Fe-2S] clusters.
• Fe/S reconstitution successfully installed a second [4Fe-4S] cluster, resulting in up to three Fe/S cofactors.
• Nar1 demonstrated significant oxygen sensitivity, with rapid destruction of two Fe/S clusters.

Conclusions:

• The findings validate a direct link between cellular oxygen levels and CIA pathway function.
• The [2Fe-2S] cluster likely occupies a unique cavity in Nar1.
• This work paves the way for in vitro Fe/S cluster transfer studies to understand Nar1's role in Fe/S trafficking.