Human mitochondrial glutaredoxin reduces S-glutathionylated proteins with high affinity accepting electrons from either glutathione or thioredoxin reductase.
Glutaredoxins catalyze glutathione-dependent thiol disulfide oxidoreductions via a GSH-binding site and active cysteines. Recently a second human glutaredoxin (Grx2), which is targeted to either mitochondria or the nucleus, was cloned. Grx2 contains the active site sequence CSYC, which is different from the conserved CPYC motif present in the cytosolic Grx1. Here we have compared the activity of Grx2 and Grx1 using glutathionylated substrates and active site mutants. The kinetic studies showed that Grx2 catalyzes the reduction of glutathionylated substrates with a lower rate but higher affinity compared with Grx1, resulting in almost identical catalytic efficiencies (k(cat)/K(m)). Permutation of the active site motifs of Grx1 and Grx2 revealed that the CSYC sequence of Grx2 is a prerequisite for its high affinity toward glutathionylated proteins, which comes at the price of lower k(cat). Furthermore Grx2 was a substrate for NADPH and thioredoxin reductase, which efficiently reduced both the active site disulfide and the GSH-glutaredoxin intermediate formed in the reduction of glutathionylated substrates. Using this novel electron donor pathway, Grx2 reduced low molecular weight disulfides such as CoA but with particular high efficiency glutathionylated substrates including GSSG. These results suggest an important role for Grx2 in protection and recovery from oxidative stress.
Johansson
Catrine
C
Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden.
Lillig
Christopher Horst
CH
Holmgren
Arne
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D017124:Conserved Sequence; D003600:Cytosol / Q000737:chemistry; D004220:Disulfides / Q000378:metabolism; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010088:Oxidoreductases; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*
D004220:Disulfides; C516008:GLRX2 protein, human; D054477:Glutaredoxins; D011506:Proteins; D011994:Recombinant Proteins; D009249:NADP; D010088:Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione