Biochemical characterization of yeast mitochondrial Grx5 monothiol glutaredoxin
MetadataShow full item record
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 charac- terized 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 cys- tein 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, in- dicating that Cys-117 plays an essential role in the cat- alytic 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 glutare- doxin 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.
Is part ofThe Journal of Biological Chemistry, 2003, vol. 278, núm 28, p. 25745-25751
European research projects
Showing items related by title, author, creator and subject.
Structure-function analysis of yeast Grx5 monothiol glutaredoxin defines essential amino acids for the function of the protein Bellí i Martínez, Gemma; Polaina, Julio; Tamarit Sumalla, Jordi; Torre Ruiz, M. A. de la; Rodríguez Manzanaque, Maria Teresa; Ros Salvador, Joaquim; Herrero Perpiñán, Enrique (The American Society for Biochemistry and Molecular Biology, 2002)Grx5 defines a family of yeast monothiol glutaredox- ins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in ...
Mitochondrial Hsp60, resistance to oxidative stress, and the labile iron pool are closely connected in Saccharomyces cerevisiae Cabiscol Català, Elisa; Bellí i Martínez, Gemma; Tamarit Sumalla, Jordi; Echave Lozano, Pedro; Herrero Perpiñán, Enrique; Ros Salvador, Joaquim (The American Society for Biochemistry and Molecular Biology, 2002)In the present study, we have analyzed the role of the molecular chaperone Hsp60 in protection of Saccharo- myces cerevisiae against oxidative damage. We con- structed mutant strains in which the levels of Hsp60 protein, ...
Rodríguez-Manzaneque Martínez, María Teresa; Tamarit Sumalla, Jordi; Bellí i Martínez, Gemma; Ros Salvador, Joaquim; Herrero Perpiñán, Enrique (American Society for Cell Biology, 2002)Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the ...