title: Redox properties and evolution of human glutaredoxins.
abstract: Glutaredoxins (Grxs) are glutathione-dependent oxidoreductases that belong to the thioredoxin superfamily catalyzing thiol-disulfide exchange reactions via active site cysteine residues. Focusing on the human dithiol glutaredoxins having a C-X-Y-C active site sequence motif, the redox potentials of hGrx1 and hGrx2 were determined to be -232 and -221 mV, respectively, using a combination of redox buffers, protein-protein equilibrium and thermodynamic linkage. In addition, a nonactive site disulfide was identified between Cys28 and Cys113 in hGrx2 using redox buffers and chemical digestion. This disulfide confers nearly five kcal mol(-1) additional stability by linking the C-terminal helix to the bulk of the protein. The redox potential of this nonactive site disulfide was determined to be -317 mV and is thus expected to be present in all but the most reducing conditions in vivo. As all human glutaredoxins contain additional nonactive site cysteine residues, a full phylogenetic analysis was performed to help elucidate their structural and functional roles. Three distinct groups were found: Grx1, Grx2, and Grx5, the latter representing a highly conserved group of monothiol glutaredoxins having a C-G-F-S active site sequence, with clear homologs from bacteria to human. Grx1 and Grx2 diverged from a common ancestor before the origin of vertebrates, possibly even earlier in animal evolution. The highly stabilizing nonactive site disulfide observed in hGrx2 is found to be a conserved feature within the deuterostomes and appears to be the only additional conserved intramolecular disulfide within the glutaredoxins.
authors.affiliation: School of Life Sciences, Södertörns Högskola, S-141 89 Huddinge, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Sagemark
authors.forename: Tobias H
authors.initials: TH
authors.lastname: Elgán
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Bürglin
authors.forename: Catrine
authors.initials: C
authors.lastname: Johansson
authors.forename: Arne
authors.initials: A
authors.lastname: Holmgren
authors.forename: Kurt D
authors.initials: KD
authors.lastname: Berndt
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D017124:Conserved Sequence; D004220:Disulfides / Q000032:analysis / Q000737:chemistry; D019143:Evolution, Molecular; D054477:Glutaredoxins; D006801:Humans; D007700:Kinetics; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011489:Protein Denaturation; D013816:Thermodynamics
chemical_list: D004220:Disulfides; C516008:GLRX2 protein, human; D054477:Glutaredoxins; D010088:Oxidoreductases