Characterization of the yeast peroxiredoxin Ahp1 in its reduced active and overoxidized inactive forms using NMR.
Peroxiredoxins (Prx's) are a superfamily of thiol-specific antioxidant proteins present in all organisms and involved in the hydroperoxide detoxification of the cell. The catalytic cysteine of Prx's reduces hydroperoxides and is transformed into a transient sulfenic acid (Cys-SOH). At high hydroperoxide concentration, the sulfenic acid can be overoxidized into a sulfinate, or even a sulfonate. We present here the first peroxiredoxin characterization by solution NMR of the Saccharomyces cerevisiae alkylhydroperoxide reductase (Ahp1) in its reduced and in vitro overoxidized forms. NMR (15)N relaxation data and ultracentrifugation experiments indicate that the protein behaves principally as a homodimer (2 x 19 kDa) in solution, regardless of the redox state. In vitro treatment of Ahp1 by a large excess of tBuOOH leads to an inactive form, with the catalytic cysteine overoxidized into sulfonate, as demonstrated by (13)C NMR. Depending on the amino acid sequence of their active site, Prx's are classified into five different families. In this classification, Ahp1 is a member of the scarcely studied D-type Prx's. Ahp1 is unique among the D-type Prx's in its ability to form an intermolecular disulfide. The peptidic sequence of Ahp1 was analyzed and compared to other D-type Prx sequences.
Trivelli
Xavier
X
Laboratoire de RMN Biomoléculaire associé au CNRS, Université Claude Bernard-Lyon 1, Ecole Supérieure de Chimie Physique Electronique de Lyon, 69622 Villeurbanne, France.
Krimm
Isabelle
I
Ebel
Christine
C
Verdoucq
Lionel
L
Prouzet-Mauléon
Valérie
V
Chartier
Yvette
Y
Tsan
Pascale
P
Lauquin
Guy
G
Meyer
Yves
Y
Lancelin
Jean-Marc
JM
D000595:Amino Acid Sequence; D002384:Catalysis; D017124:Conserved Sequence; D019281:Dimerization; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D054464:Peroxiredoxins; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D013816:Thermodynamics; D014461:Ultracentrifugation
C515936:AHP1 protein, S cerevisiae; D009587:Nitrogen Isotopes; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D010544:Peroxidases; D054464:Peroxiredoxins