Conformation changes in 2-Cys peroxiredoxin during catalytic cycle do not involve decamer disruption.

MANTA, B; TRUJILLO, M; GONZALEZ-FLECHA, LF; SANTOS, J; PRITSCH, O; DENICOLA, A

Montevideo, Uruguay,

Congreso; 6th International Conference on Biological Physics and the 5th Southern Cone Biophysics Congress; 2007

Conformation changes in 2-Cys peroxiredoxin during catalytic cycle do not involve decamer disruption Manta, B1; Trujillo, M2; Gonzalez-Flecha, LF3; Santos, J3; Pritsch, O4; Denicola, A5.   1 - Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Igua 4225, 11400, Montevideo, Uruguay / Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800, Montevideo, Uruguay / Unidad de Biofísica de Proteínas, Instituto Pasteur de Montevideo, Mataojo 2020, 11400, Montevideo, Uruguay.   2 - Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Gral. Flores2125, 11800, Montevideo, Uruguay / Center for Free Radical and Bioemdical Research, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800, Montevideo, Uruguay.   3- Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires - CONICET, Argentina   4. Departamento de Inmunobiologia, Facultad de Medicina, Universidad de la República, Gral. Flores2125, 11800, Montevideo, Uruguay / Center for Free Radical and Bioemdical Research, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800, Montevideo, Uruguay / Unidad de Biofísica de Proteínas, Instituto Pasteur de Montevideo, Mataojo 2020, 11400, Montevideo, Uruguay.   5 - Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Igua 4225, 11400, Montevideo, Uruguay / Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800, Montevideo, Uruguay.   2-Cys Peroxiredoxins (PRDX) are a large family of peroxidases which reduces hydroperoxides at expense of thioredoxin or other disulfide reductase proteins. The typical 2-Cys PRDX are obligate homodimers, utilizing a unique intermolecular redox-active disulfide center for the reduction of peroxides, and are known to form, at least, two oligomeric states: head-to-tail homodimers (α2) or doughnut-shaped decamers ((α2)5). Based mostly on crystallographic evidence, it is thought that the oligomeric state of typical 2-Cys PRDX is redox-linked, with oxidization favoring the dimer. These conformational transitions have been included as part of the catalytic cycle: oxidation of the reactive cysteine to a sulfenic acid by the hydroperoxide results in a local conformational change at the active site that approaches the resolving cysteine from the other monomer of the dimer, forming a disulfide. The disulfide formation weakens the oligomerization interface, resulting in decamer disruption. In this work we present experimental evidence supporting the idea that typical 2-Cys PRDX is a stable decamer that suffers a local conformational change along the catalytic cycle but without disrupting the decameric assemble. The oxidation produces a local conformational change, assessed by far UV-circular dichroism (CD), intrinsic fluorescence and Förster resonance energy transfer (FRET). However, no quaternary changes were observed by light scattering (LS), size-exclusion chromatography (SEC), mass-spectrometry or near-UV CD, supporting the idea that this enzyme is a stable decamer even when oxidized. In spite of this, the oxidized and reduced decamers are not identical species, as have been shown by spectroscopic methods and chemical (guanidine chloride) or thermal (differential scanning calorimetry) denaturation. Moreover, the decamer is a stable but not static structure, as we show here by FRET between fluorophore-labeled subunits interchange and mass-spectrometry analysis of recombination of homologues but not identical 2-Cys PRDX. Our results let us to conclude that these enzymes are dynamic decamer-dimer structures, being the decamer the most abundant species at physiological pH and ionic strength. We hypothesize that the decameric form, that is not essential for peroxidatic activity, is central to produce the correct topology for heterologous protein-protein interactions. We will use BIAcore, FRET, SEC and LS to test this hypothesis.