(Keceived 8 Augusi IOYS) -EJB 95 1316/4 NAD(P)H :(quinone-acceptor) oxidoreductase [NAT)(P)H-QR], a plant cytosolic protein, was purified froni cultured sugarbeet cells by a combination of ainnlonium sulfatc fractionation, FPLC Superdex 200 gel filtration, Q-Sepharosc anion-cxchange chromatography, and a final Blue Sepharose CL-6B affinity chromatography with an NADPH gradient. The subunit molecular niass is 24 kDH and thc activc protein (94 kna) is a tetrarner. The isoelectric point is 4.9. The enzyme was churactcrized by ping-pong kinetics and extremely elevated catalytic capacity. It prcfcrs NADPH over NADH as electron donor (k,,, lK,,, ratios of 1.7X108 M-I s-' and 8.3X lo7 M-' s-! for NADPH and NADH, respectively, with benzoquinoiie as clcctron acccptor). Thc acridonc derivativc 7-iodo-acridone-4-carboxylic acid is an efficient inhi bitor M). dicurnarol is weakly inhibitory. The best acceptcx substrates are hydrophilic, shortchain quinoncs such :IS ubiquinonc-0 (Q-O), bcnzoquinonc and menndione, followed by duroquinone and ferricyanide, whereas hydrophobic quinones, cytochronie I: and oxygen are reduced at negligible rates at best. Quinone acceptors are reduced by a two-electron reaction with no apparent release of free semiquinonic intermediates. ?'his and the above properties suggesr some relationship of NAD(P)H-QR to DTdiaphorase, an animal flavoprotein which, however, has distinct structural properties and is strongly inhibited by dicumarol. It is proposed that NAD(P)H-QR by scavenging unreduced quinones and making thein prone to conjugation may act in plant tissues as a functional equivalent of DT-diaphorase. Ke-ywords: NAD(P)H: (quinone-acceptor) oxidoredudase; DT-diaphorase; quinones: NAD(P)H dehydrogenase; Beta vulgaris. = SXThe presence of pyridine-nucleotide-dependent dehydrogcnnscs using a quinone as the principal electron acceptor has bccn documented for plant cells since the pioneering work of Wosilait and Nason (1953) and Wosilai1 et al. (1953), who reported on the characterization of a quinonc oxidoreductase of pea seeds.Scvcral quinone oxidoreductases have recently bccn found in non-photosynthetic mcmbriines. Rotenone insensitive NAD( P)H dehydrogenascs are thought to be peripherally bound to thc inner membrane of plant mitochondria (Mdlcr el al., 1993; Knudten et al., 1994; Luethy et al., 1995) and to effect triinsnienibrane electron transport by donation lo rnernbrane quinones (Douce and Neuburger, 1989;Zottini et al., 1993). Attention has also been paid to quinone oxidorcductases of microsomal tnernbrancs and the plasmalemma, because of the many interactions of this membrane with the apoplast and the external environment (Rubinstein and Imter, 1993; Serr:ino et al., 1994). The presence of quinones in the plant plasma ineinbrane is still unproven, however. Artificial water-soluble quinones have been used in the study of NAD(P)H dehydrogcnases in plants (De Luca ct al., 1984; MQller and Cranc, 1990) and of the DT-diaphorase of animal cells (Lind et al., 1990). Hydrophilic quinones are c...

Fifty-eight binucleate Rhizoctonia isolates were collected over six years from strawberry plants displaying symptoms of black root rot in Italy. Almost all isolates were able to produce necrosis on strawberry roots, most of them also showed this ability on faba bean and, with lower frequency, on a crucifer and a cereal crop used in rotation with strawberry in Italy. The sequence alignment of Internal Transcribed Spacer (ITS) regions of 51 binucleate Rhizoctonia were analyzed and compared with a set of eight sequences representative of Rhizoctonia isolate Anastomosis Groups (AG) already found to be pathogenic on strawberry (

The dorC gene of the dimethyl sulfoxide respiratory (dor) operon of Rhodobacter capsulatus encodes a pentaheme c-type cytochrome that is involved in electron transfer from ubiquinol to periplasmic dimethyl sulfoxide reductase. DorC was expressed as a C-terminal fusion to an 8-amino acid FLAG epitope and was purified from detergent-solubilized membranes by ion exchange chromatography and immunoaffinity chromatography. The DorC protein had a subunit M r ‫؍‬ 46,000, and pyridine hemochrome analysis indicated that it contained 5 mol heme c/mol DorC polypeptide, as predicted from the derived amino acid sequence of the dorC gene. The reduced form of DorC exhibited visible absorption maxima at 551.5 nm (␣-band), 522 nm (␤-band), and 419 nm (Soret band). Redox potentiometry of the heme centers of DorC identified five components (n ‫؍‬ 1) with midpoint potentials of ؊34, ؊128, ؊184, ؊185, and ؊276 mV. Despite the low redox potentials of the heme centers, DorC was reduced by duroquinol and was oxidized by dimethyl sulfoxide reductase.The ability to use Me 2 SO and trimethylamine-N-oxide (TMAO) 1 as electron acceptors is widespread among facultative aerobic bacteria and the organization of the Me 2 SO and TMAO respiratory chains is now well defined (1). In the Me 2 SO respiratory system of purple photosynthetic bacteria such as Rhodobacter capsulatus, electrons are transferred from primary dehydrogenases via the ubiquinone pool to a periplasmic Me 2 SO reductase (2). Recent sequence and mutational analysis of the Me 2 SO respiratory gene cluster from photosynthetic bacteria has identified a pentaheme c-type cytochrome (DorC) as the likely mediator of electron transfer from ubiquinol to Me 2 SO reductase (3).2 The TMAO respiratory (Tor) system of Escherichia coli (5) is very similar to the Dor system of R. capsulatus, and they differ from the Me 2 SO respiratory (Dms) system of E. coli. The Me 2 SO reductase (DmsABC) of E. coli can be purified as a menaquinol-oxidizing Me 2 SO reductase complex that lacks c-type cytochromes (6). Me 2 SO reductase from R. capsulatus has been purified as a monomeric protein containing a pterin molybdenum cofactor (Moco) as its only prosthetic group (7). This property of Me 2 SO reductase has many advantages for spectroscopic characterization of Moco, but a major deficiency has been the lack of a physiologically relevant electron donor. To overcome this problem we describe in this paper the purification and characterization of DorC.The derived amino acid sequence of Rhodobacter DorC indicated that it was related to members of the NirT class of tetraheme c-type cytochromes (3).2 However, DorC is predicted to be a pentaheme cytochrome with a fifth c-type heme binding motif in the C-terminal polypeptide, which is absent from the tetraheme members of the NirT class. Almost all of the members of the NirT class are involved in an anaerobic respiratory pathway, and their role appears to be to catalyze electron transfer from the Q-pool to a periplasmic terminal reductase (8). Although molecular geneti...

Quinone oxidoreductase activities dependent on pyridine nucleotides are associated with the plasma membrane (PM) i n ZUC-chini (Cucurbifa pepo L.) hypocotyls. I n the presence of NADPH, lipophilic ubiquinone homologs with up t o three isoprenoid units were reduced by intact PM vesicles with a K,,, of 2 t o 7 p M. Affinities for both NADPH and N A D H were similar (K, of 62 and 51 p ~ , respectively). Two NAD(P)H:quinone oxidoreductase forms were identified. The first, labeled as peak I i n gel-filtration experiments, behaves as an intrinsic membrane complex of about 300 kD, it slightly prefers N A D H over NADPH, it is markedly sensitive t o the inhibitor diphenylene iodonium, and it is active with lipophilic quinones. The second form (peak II) is an NADPH-preferring oxidoreductase of about 90 kD, weakly bound t o the PM. Peak II is diphenylene iodonium-insensitive and resembles, in many properties, the soluble NAD(P)H:quinone oxidoreductase that is also present i n the same tissue. Following purification of peak I, however, the latter gave rise t o a quinone oxidoreductase of the soluble type (peak II), based o n substrate and inhibitor specificities and chromatographic and electrophoretic evidence. It is proposed that a redox protein of the same class as the soluble NAD(P)H:quinone oxidoreductase (F. Sparla, C. Tedeschi, and P. Trost 11 9961 Plant Physiol. 1 1 2:249-258) is a component of the diphenylene iodonium-sensitive PM complex capable of reducing lipophilic quinones. ~ ~ Plant cells appear to contain severa1 types of NAD(P)H-dependent quinone oxidoreductases in addition to those of energy-conserving reactions of plastids and mitochondria. The purification of some of these plant proteins has been accomplished, but their biochemical characterization is still at an initial stage. Recently, NAD(P)H-QR from plant tissues was purified and characterized (Rescigno et al., 1995; Trost et al., 1995; Sparla et al., 1996). This plant oxidoreduc-tase represents a functional equivalent of animal DT-diaphorase, since it reduces short-chain quinones to quin-01s by two-electron donation without semiquinone intermediates, thereby resulting in enhanced quinone conjugation and low probability of formation of active oxygen species (Trost et al., 1995). However, a number of properties are different from animal-type DT-diaphorase, i.e. the NAD(P)H-QR contains FMN, it has a mass of about 90 kD with subunits of 21.4 kD (by MS), and the hydride transfer

The capability of high potential iron-sulfur proteins (HiPIPs) and soluble cytochromes to shuttle electrons between the bc1 complex and the terminal oxidase in aerobically grown cells of Rhodoferax fermentans and Rhodospirillum salinarum, two facultative phototrophs, was evaluated. In Rs. salinarum, HiPIP and a c-type cytochrome (alpha-band at 550 nm, Em,7=+290 mV) are both involved in the electron transfer step from the bc1 complex to the terminal oxidase. Kinetic studies indicate that cytochrome c550 is more efficient than HiPIP in oxidizing the bc1 complex, and that HiPIP is a more efficient reductant of the terminal oxidase as compared to cytochrome c550. Rs. salinarum cells contain an additional c-type cytochrome (asymmetric alpha-band at 556 nm, Em,7=+180 mV) which is able to reduce the terminal oxidase, but unable to oxidize the bc1 complex. c-type cytochromes could not be isolated from Rf. fermentans, in which HiPIP, the most abundant soluble electron carrier, is reduced by the bc1 complex (zero-order kinetics) and oxidized by the terminal oxidase (first-order kinetics), respectively. These data, taken together, indicate for the first time that HiPIPs play a significant role in bacterial respiratory electron transfer.