The glutaredoxin Grx4 is a Fe-S cluster-containing protein.

<p>(A) Cells pellets of <i>E</i>. <i>coli</i> over-expressing GST-HA-Grx4 are brown. Cells transformed with plasmids pGEX-2T-TEV (GST) or p400 (pGEX-2T-TEV-GST-<i>HA-grx4</i>; GST-Grx4) were grown into LB, and protein expression was induced by IPTG. Cells were pelleted on Elisa plates and photographed. (B and C) Reconstitution of the Fe-S cluster of Grx4. UV-visible absorption spectra of reconstituted Fe-HA-Grx4 (Fe-Grx4). The red dashed line indicates the spectrum of the apo-protein, obtained in the absence of added Fe. (D) The reconstituted Fe-S cluster of Grx4 is sensitive to oxygen. UV-visible absorption spectra of reconstituted Fe-HA-Grx4 (Fe-Grx4) protein before (solid line) and after (red dashed line) 15 minutes of oxygen exposure. (E) GSH is required for reconstitution of the Grx4 Fe-S cluster. UV-visible absorption spectra of Grx4 reconstitution reactions performed in the presence (solid line) or absence (red dashed line) of GSH. (F) Cell lacking Gcs1, auxotrophic for GSH, display constitutive activation of the Fe starvation response after 8 h of GSH withdrawal. Strains 972 (WT), NG81 (<i>Δgrx4</i>) and NG77 (<i>Δgcs1</i>) were grown in YE media, and shifted to MM without GSH, when DIP was added. When indicated for strain NG77, growth proceeded for 8 h prior to DIP addition. Total RNA was analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g001" target="_blank">Fig. 1E</a>. (G) Same as in F, with strains 972 (WT), NG81 (<i>Δgrx4</i>) and JE7 (<i>Δgcs1 Δgrx4</i>).