Biochemical characterization of yeast mitochondrial Grx5 monothiol glutaredoxin.
Grx5 is a yeast mitochondrial protein involved in iron-sulfur biogenesis that belongs to a recently described family of monothiolic glutaredoxin-like proteins. No member of this family has been biochemically characterized previously. Grx5 contains a conserved cysteine residue (Cys-60) and a non-conserved one (Cys-117). In this work, we have purified wild type and mutant C60S and C117S proteins and characterized their biochemical properties. A redox potential of -175 mV was calculated for wild type Grx5. The pKa values obtained by titration of mutant proteins with iodoacetamide at different pHs were 5.0 for Cys-60 and 8.2 for Cys-117. When Grx5 was incubated with glutathione disulfide, a transient mixed disulfide was formed between glutathione and the cystein 60 of the protein because of its low pKa. Binding of glutathione to Cys-60 promoted a decrease in the Cys-117 pKa value that triggered the formation of a disulfide bond between both cysteine residues of the protein, indicating that Cys-117 plays an essential role in the catalytic mechanism of Grx5. The disulfide bond in Grx5 could be reduced by GSH but at a rate at least 20 times slower than that observed for the reduction of glutaredoxin 1 from E. coli, a dithiolic glutaredoxin. This slow reduction rate could suggest that GSH may not be the physiologic reducing agent of Grx5. The fact that wild type Grx5 efficiently reduced a glutathiolated protein used as a substrate indicated that Grx5 may act as a thiol reductase inside the mitochondria.
Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, Rovira Roure 44, 25198 Lleida, Spain.
Jordi
J
Tamarit
Gemma
G
Belli
Elisa
E
Cabiscol
Enrique
E
Herrero
Joaquim
J
Ros
D002256:Carbonic Anhydrases / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors
D004220:Disulfides; D004791:Enzyme Inhibitors; D054477:Glutaredoxins; D011506:Proteins; D010088:Oxidoreductases; D002256:Carbonic Anhydrases; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen; D019803:Glutathione Disulfide; D007460:Iodoacetamide