Detoxification of superoxide without production of H2O2: Antioxidant activity of superoxide reductase complexed with ferrocyanide

doi:10.1073/pnas.0510828103

Detoxification of superoxide without production of H₂O₂: Antioxidant activity of superoxide reductase complexed with ferrocyanide

*Département de Réponse et Dynamique Cellulaires/Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, Unité Mixte de Recherche (UMR) 5047, Commissariat à l’Energie Atomique (CEA)/Centre National de la Recherche Scientifique (CNRS)/Université Joseph Fourier, 17 Avenue des Martyrs, 38054 Grenoble Cedex 9, France;
^‡Laboratoire de Chimie Physique, UMR 8000, CNRS/Université Paris-Sud, Bâtiment 350, 91405 Orsay Cedex, France;
^§Département d’Ecophysiologie Végétale et Microbiologie/Laboratoire des Interactions Proteine Metal, UMR 6191, CEA-Cadarache, 13108 Saint Paul-lez-Durance, France;
^¶Institut Jacques Monod, CNRS/Universités Paris 6 et Paris 7, 2 Place Jussieu, 75251 Paris Cedex 5, France;
^‖Institut National de la Santé et de la Recherche Médicale Unité 612 and Institut Curie, Bâtiment 110–112, 91405 Orsay Cedex, France; and
**European Synchrotron Radiation Facility, BP 220, 38043 Grenoble Cedex, France

Edited by Perry A. Frey, University of Wisconsin, Madison, WI, and approved August 14, 2006 (received for review December 15, 2005)

Abstract

The superoxide radical O₂ ^·̅ is a toxic by-product of oxygen metabolism. Two O₂ ^·̅ detoxifying enzymes have been described so far, superoxide dismutase and superoxide reductase (SOR), both forming H₂O₂ as a reaction product. Recently, the SOR active site, a ferrous iron in a [Fe²⁺ (N-His)₄ (S-Cys)] pentacoordination, was shown to have the ability to form a complex with the organometallic compound ferrocyanide. Here, we have investigated in detail the reactivity of the SOR–ferrocyanide complex with O₂ ^·̅ by pulse and γ-ray radiolysis, infrared, and UV-visible spectroscopies. The complex reacts very efficiently with O₂ ^·̅. However, the presence of the ferrocyanide adduct markedly modifies the reaction mechanism of SOR, with the formation of transient intermediates different from those observed for SOR alone. A one-electron redox chemistry appears to be carried out by the ferrocyanide moiety of the complex, whereas the SOR iron site remains in the reduced state. Surprisingly, the toxic H₂O₂ species is no longer the reaction product. Accordingly, in vivoexperiments showed that formation of the SOR–ferrocyanide complex increased the antioxidant capabilities of SOR expressed in an Escherichia coli sodA sodB recA mutant strain. Altogether, these data describe an unprecedented O₂ ^·̅ detoxification activity, catalyzed by the SOR–ferrocyanide complex, which does not conduct to the production of the toxic H₂O₂ species.

Footnotes

^††To whom correspondence should be addressed. E-mail: vniviere{at}cea.fr
Author contributions: F.P.M.-H. and V.N. designed research; F.P.M.-H., C.H.-L., C.B., D.T., E.T., and V.N. performed research; V.F. and V.A. contributed new reagents/analytic tools; F.P.M.-H., C.H.-L., C.B., D.T., and V.N. analyzed data; and V.N. wrote the paper.
↵ ^†Present address: Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla y Consejo Superior de Investigaciones Cientificas, Américo Vespucio 49, 41092 Sevilla, Spain.
The authors declare no conflict of interest.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

SOD,

superoxide dismutase;

SOR,

superoxide reductase.