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Insights into the role of the metal binding site in methionine-R-sulfoxide reductases B

¹ Unité Mixte de Recherche (UMR) Centre National de la Recherche Scientifique (CNRS)-UHP 7567, Maturation des ARN et Enzymologie Molé culaire, Faculté des Sciences, Université Henri Poincaré Nancy I, 54506 Vandoeuvre-lès-Nancy, France
² Laboratoire de Chimie et Toxicologie des métaux, Faculté de Médecine, Université Henri Poincaré Nancy I, 54505 Vandoeuvre-lès-Nancy, France

(RECEIVED July 21, 2005; FINAL REVISION August 26, 2005; ACCEPTED August 26, 2005)

Methionine sulfoxide reductases B (MsrBs) catalyze the reduction of methionine-R-sulfoxide via a three-step chemical mechanism including a reductase step, formation of an intradisulfide bond followed by a thioredoxin recycling process. Fifty percent of the MsrBs, including the Escherichia coli enzyme, possess a metal binding site composed of two CXXC motifs of unknown function. It is located on the opposite side of the active site. The overexpressed E. coli MsrB tightly binds one atom of zinc/iron. Substitution of the cysteines of E. coli MsrB results in complete loss of bound metal and reductase activity, and leads to a low-structured conformation of the protein as shown by CD, fluorescence, and DSC experiments. Introduction of the two CXXC motifs in Neisseria meningitidis MsrB domain leads to a MsrB that tightly binds one atom of zinc/iron, shows a strongly increased thermal stability and displays a reductase activity similar to that of the wild-type but lacking thioredoxin recycling activity. These results demonstrate the stabilizing effect of the metal and the existence of a preformed metal binding site in the nonbound metal MsrB. The data also indicate that metal binding to N. meningitidis MsrB induces subtle structural modifications, which prevent formation of a competent binary complex between oxidized MsrB and reduced thioredoxin but not between reduced MsrB and substrate. The fact that the E. coli and the N. meningitidis MsrBs exhibit a similar thermal stability suggests the existence of other structural factors in the nonbound metal MsrBs that compensate the metal bound stabilizing effect.

Keywords: methionine sulfoxide reductase B; metal binding site; zinc; iron; apoenzyme; thioredoxin

Abbreviations: MetSO, methionine sulfoxide • ROS, reactive oxygen species • Msr, MsrA, and MsrB, methionine sulfoxide reductase, methionine sulfoxide reductase A, methionine sulfoxide reductase B, respectively • eMsrB and nmMsrB, Escherichia coli and Neisseria meningitidis MsrB, respectively • IPTG, isopropyl--D-thiogalactopyranoside • DTT, dithiothreitol • Trx, thioredoxin • TCA, trichloroacetic acid • CD, circular dichroism • ESI, electrospray ionization • DSC, differential scanning calorimetry • ANS, 8-anilino-1-naphtalenesulfonic acid.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051711105.

Reprint requests to: Guy Branlant, UMR CNRS-UHP 7567, Maturation des ARN et Enzymologie Molé culaire, Faculté des Sciences, Université Henri Poincaré Nancy I, Bld. des Aiguillettes, BP 239, 54506 Vandoeuvre-lès-Nancy, France; e-mail: guy.branlant{at}maem.uhp-nancy.fr; fax: +(33) 3-83-68-43-07.

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