Biochemical characterization of yeast mitochondrial Grx5 monothiol glutaredoxin
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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
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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 ...
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