Fig. 4. Deficiency in Grx3/4 leads to functional impairment of di-iron enzymes.

(A) Permeabilised wild-type (WT) and Grx4-depleted Gal-GRX4 cells were assayed for specific ribonucleotide reductase activity (see also Fig. S3). (B) WT and Gal-GRX4 cells were grown in SD medium for 40 and 64 h, radiolabeled, and ⁵⁵Fe binding to Rnr2 was analyzed by immunoprecipitation and scintillation counting. Protein levels of Rnr2 and Por1 were determined by immunoblotting (insert). (C) The substrate (demethoxyubiquinol DMQ₆) and product (ubiquinone CoQ₆) of mitochondrial mono-oxygenase Coq7 were analyzed by electrochemical detection coupled to HPLC separation of mitochondrial lipid extracts from WT and Grx4-depleted Gal-GRX4 cells. CoQ₄ is a commercial standard. (D) Ratio of DMQ₆ and CoQ₆ levels in mitochondria isolated from WT, Gal-GRX4 cells (depleted for 40 h or 64 h) containing either vector pCM189 or pCM189-GRX4 (+Grx4). (E) WT and Grx4-depleted Gal-GRX4 cells overproducing Fe-only superoxide dismutase from E. coli (FeSod) were analyzed for superoxide dismutase in-gel activities and FeSod by immunoblotting.