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

Abstract

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 O 2 Á2 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-electrontransferring 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 O 2 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 O 2 . The possible function of this enzyme in the generation of O 2 Á2 and H 2 O 2 at the plasma membrane of plants in vivo is discussed.

Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes

“…The mean and standard deviation of five measurements are shown plant cell walls in great abundance (Mika et al 2004(Mika et al , 2010. Consistent with these conclusions only minor traces of peroxidase activity could be previously detected in purified plasma membranes from the growing part of soybean hypocotyls, supporting the notion that peroxidases are not obligatory components of such membranes (Schopfer et al 2008). In contrast, high peroxidase activity can be found in the cell walls of this tissue (data not shown).…”

“…1 the pellets were resuspended in 100 ml of washing buffer (1.5 M KCl, 20 mM Hepes, pH 7.5), pelleted (45 min at 91,000g, 4°C), resuspended in 20 mM Hepes buffer (pH 7.5) and stored at -70°C. Data documenting the purity of plasma membranes prepared by this procedure have been reported previously (Schopfer et al 2008). …”

Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

“…quinone reductases; Serrano et al, 1994;Trost et al, 1997;Schopfer et al, 2008;and MDA reductase;Bérczi and Møller, 1998), b-type cytochromes Bérczi et al, 2003;Preger et al, 2005), and peroxidases (Mika et al, 2008). Moreover, molecular biology approaches led to the discovery of flavocytochromes of the FRO family that use cytosolic pyridine nucleotides to reduce apoplastic ferrichelates (Robinson et al, 1999).…”

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism

“…Levels of these metabolites in the culture medium, a site of apoplastic H 2 O 2 formation, were not determined. Other possible apoplastic ROS-producing enzymes include plasma membrane-located quinone oxidoreductases (Schopfer et al, 2008;Lüthje et al, 2013;Kärkönen et al, 2014). In the cell culture, two quinone oxidoreductaseencoding genes (FLAVOPROTEIN WrbA) were induced during lignin formation.…”