Reduction of 1-Cys peroxiredoxins by ascorbate changes the thiol-specific antioxidant paradigm, revealing another function of vitamin C

*Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, CEP 05508-900, São Paulo, Brazil;
^†Laboratório de Espectrometria de Massa, Centro de Toxinologia Aplicada (CAT/CEPID), Instituto Butantan, CEP 05503-900, São Paulo, Brazil; and
^‡Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CEP 05508-900, São Paulo, Brazil

Communicated by E. R. Stadtman, National Institutes of Health, Bethesda, MD, January 20, 2007 (received for review August 17, 2006)

Abstract

Peroxiredoxins (Prx) are widely distributed peroxidases that can be divided into 1-Cys and 2-Cys Prx groups based on the number of conserved cysteine residues that participate in their catalytical cycle. Prx have been described to be strictly dependent on thiols, but here, we show that ascorbate (vitamin C) also reduces 1-Cys Prx, but not 2-Cys Prx, from several taxonomic groups. Reduction by ascorbate is partly related to the fact that the oxidized form of 1-Cys Prx is a stable sulfenic acid (Cys-SOH) instead of a disulfide. In addition, a histidine residue in the active site is required. In fact, we engineered a 2-Cys Prx with these two features, and it displayed ascorbate peroxidase activity. These data represent a breakthrough in the thiol-specific antioxidant paradigm. Ascorbate may be the long-sought-after biological reductant of 1-Cys Prx. Because ascorbate is present in high amounts in cells, the ascorbate/protein sulfenic acid pair represents an aspect of redox biochemistry that has yet to be explored in vivo.

Footnotes

^§To whom correspondence should be addressed. E-mail: nettoles{at}ib.usp.br
Author contributions: G.M., O.A., and L.E.S.N. designed research; G.M., B.B.H., and D.C.P. performed research; G.M., B.B.H., D.C.P., O.A., and L.E.S.N. analyzed data; and G.M., O.A., and L.E.S.N. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0700481104/DC1.
Abbreviations:

Prx,

peroxiredoxin(s);

RSH,

thiol;

ROS,

reactive oxygen species;

RNS,

reactive nitrogen species;

SOH,

sulfenic acid;

RS−,

thiolate group;

Cysp,

peroxidatic cysteine;

Cysr,

resolving cysteine;

GS,

glutamine synthetase;

DTT,

dithiothreitol;

t-BOOH,

tert-butyl hydroperoxide;

NEM,

N-ethylmaleimide.