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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Naphthoquinone-Dependent Generation of Superoxide Radicals by Quinone Reductase Isolated from the Plasma Membrane of Soybean^[W]

Universität Freiburg, Institut für Biologie II, D–79104 Freiburg, Germany (P.S., F.D.); and Commissariat à l'Energie Atomique, Institut de Biologie et Technologies de Saclay, CNRS Unité de Recherche Associée 2096, Service de Bioénergétique Biologie Structurale et Mécanisme, F–91191 Gif-sur-Yvette cedex, France (E.H., A.K.-L.)

Using a tetrazolium-based assay, a NAD(P)H oxidoreductase was purified from plasma membranes prepared from soybean (Glycine max) hypocotyls. The enzyme, a tetramer of 85 kD, produces O₂^·– by a reaction that depended on menadione or several other 1,4-naphthoquinones, in apparent agreement with a classification as a one-electron-transferring flavoenzyme producing semiquinone radicals. However, the enzyme displayed catalytic and molecular properties of obligatory two-electron-transferring quinone reductases of the DT-diaphorase type, including insensitivity to inhibition by diphenyleneiodonium. This apparent discrepancy was clarified by investigating the pH-dependent reactivity of menadionehydroquinone toward O₂ and identifying the protein by mass spectrometry and immunological techniques. The enzyme turned out to be a classical NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.5.2, formerly 1.6.99.2) that reduces menadione to menadionehydroquinone and subsequently undergoes autoxidation at pH 6.5. Autoxidation involves the production of the semiquinone as an intermediate, creating the conditions for one-electron reduction of O₂. The possible function of this enzyme in the generation of O₂^·– and H₂O₂ at the plasma membrane of plants in vivo is discussed.

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.118745

^* Corresponding author; e-mail anja.krieger-liszkay{at}cea.fr.

Received March 5, 2008; accepted April 8, 2008; published April 11, 2008.

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