NAD(P)H:(Quinone-Acceptor) Oxidoreductase of Tobacco Leaves Is a Flavin Mononucleotide-Containing Flavoenzyme

Abstract

The soluble NAD(P)H:(quinone-acceptor) oxidoreductase [NAD(P)H-QR, EC 1.6.99.21 of Nicotiana fabacum L. leaves and roots has been purified. NAD(P)H-QR contains noncovalently bound flavin mononucleotide. Pairs of subunits of 21.4 kD are linked together by disulfide bridges, but the active enzyme is a homotetramer of 94 to 100 kD showing an isoelectric point of 5.1. NAD(P)H-QR is a B-stereospecific dehydrogenase. NADH and NADPH are electron donors of similar efficiency with K,,,:K, ratios (with duroquinone) of 6.2 x 10' and 8.0 x 10' M -~ s-l, respectively. Hydrophilic quinones are good electron acceptors, although ferricyanide and dichlorophenolindophenol are also reduced. The quinones are converted to hydroquinones by an obligatory two-electron transfer. No spectral evidente for a flavin semiquinone was detected following anaerobic photoreduction. Cibacron blue and 7-iodo-acridone-4-carboxylic acid are inhibitory. Tobacco NAD(P)H-QR resembles animal DT-diaphorase in some respects (identical reaction mechanism with a two-electron transfer to quinones, unusually high catalytic capability, and donor and acceptor substrate specificity), but it differs from DT-diaphorase in molecular structure, flavin cofactor, stereospecificity, and sensitivity to inhibitors. As in the case with DT-diaphorase in animals, the main NAD(P)H-QR function in plant cells may be the reduction of quinones to quinols, which prevents the production of semiquinones and oxygen radicals. The enzyme appears to belong to a widespread group of plant and funga1 flavoproteins found in different cell compartments that are able to reduce quinones. ~

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

“…1B), these data are in agreement with the finding that plant NQRs are homotetramers of 21.5-kD subunits (Sparla et al, 1996(Sparla et al, , 1999. Beyond that, the enzyme demonstrates an immunological relationship to AtNQR identified by Sparla et al (1999) rather than to AtFQR1 identified by Laskowski et al (2002).…”

“…Examples of this class are the NAD(P)H:quinone-acceptor oxidoreductases known as DT-diaphorases in animals (Lind et al, 1990;Ross, 1997). Functionally related flavoenzymes have also been found in plants (Spitsberg and Coscia, 1982;Luster and Buckhout, 1989;Valenti et al, 1990;Serrano et al, 1994;Rescigno et al, 1995;Trost et al, 1995Trost et al, , 1997Sparla et al, 1996Sparla et al, , 1998 (Iyanagi and Yamazaki, 1970;Lind et al, 1990;Sparla et al, 1996;Ross, 1997). A purified quinone oxidoreductase investigated by Trost et al (1997) was shown to be insensitive to DPI.…”

A Single-Electron Reducing Quinone Oxidoreductase Is Necessary to Induce Haustorium Development in the Root Parasitic Plant Triphysaria

“…Transcripts for both QR1 and QR2 are rapidly upregulated in Triphysaria roots as a primary response to treatment with DMBQ and other quinones (Matvienko et al, 2001b). Based on sequence homologies, QR1 was classified as a member of the z-crystallin-like quinone oxidoreductases (EC 1.6.5.5) (Thorn et al, 1995;Edwards et al, 1996) and QR2 as a quininereducing flavoprotein (EC 1.6.5.2) (Sparla et al, 1996;Matvienko et al, 2001b). Later purifications of the QR2 enzyme showed that it catalyzes NAD(P)H-dependent quinone reduction with substrate and inhibitor specificity consistent with its placement into the Diaphorase family (Sparla et al, 1999;Wrobel et al, 2002).…”

Redox enzymes in the plant plasma membrane and their possible roles

“…Since the activity of the soluble form was hardly sensitive to DPI, it seems possible that the DPI-binding site and the actual QR site are not on the same polypeptide. Since the soluble enzyme contains FMN (Sparla et al 1996), the PM-bound QR can also be considered to be a flavoprotein. However, also in this case the PM preparation was not stripped properly.…”

“…The QR activities from PM vesicles of zucchini hypocotyls were studied by Trost et al (1997) in order to reveal differences and similarities between the PM-bound and the soluble forms of NAD(P)H-(quinone-acceptor) oxidoreductases. A 24 kDa soluble form had previously been purified and characterized from sugar beet cells (Trost et al 1995) and tobacco leaves (Sparla, Tedeschi & Trost 1996) as soluble QR. In the case of zucchini, the PM-bound form showed three bands at 27, 24, and 18 kDa whereas the soluble form showed one band at 24 kDa.…”