Geranylgeranyl reductase involved in the biosynthesis of archaeal membrane lipids in the hyperthermophilic archaeon Archaeoglobus fulgidus

Complete saturation of the geranylgeranyl groups of biosynthetic intermediates of archaeal membrane lipids is an important reaction that confers chemical stability on the lipids of archaea, which generally inhabit extreme conditions. An enzyme encoded by the AF0464 gene of a hyperthermophilic archaeon, Archaeoglobus fulgidus, which is a distant homologue of plant geranylgeranyl reductases and an A. fulgidus menaquinone-specific prenyl reductase [Hemmi H, Yoshihiro T, Shibuya K, Nakayama T, & Nishino T (2005) J Bacteriol187, 1937-1944], was recombinantly expressed and purified, and its geranylgeranyl reductase activity was examined. The radio HPLC analysis indicated that the flavoenzyme, which binds FAD noncovalently, showed activity towards lipid-biosynthetic intermediates containing one or two geranylgeranyl groups under anaerobic conditions. It showed a preference for 2,3-di-O-geranylgeranylglyceryl phosphate over 3-O-geranylgeranylglyceryl phosphate and geranylgeranyl diphosphate in vitro, and did not reduce the prenyl group of respiratory quinones in Escherichia coli cells. The substrate specificity strongly suggests that the enzyme is involved in the biosynthesis of archaeal membrane lipids. GC-MS analysis of the reaction product from 2,3-di-O-geranylgeranylglyceryl phosphate proved that the substrate was converted to archaetidic acid (2,3-di-O-phytanylglyceryl phosphate). The archaeal enzyme required sodium dithionite as the electron donor for activity in vitro, similarly to the menaquinone-specific prenyl reductase from the same anaerobic archaeon. On the other hand, in the presence of NADPH (the preferred electron donor for plant homologues), the enzyme reaction did not proceed.     

Participation of chlorobiumquinone in the transplasma membrane electron transport system of Leishmania donovani promastigote: effect of near-ultraviolet light on the redox reaction of plasma membrane

The respiratory quinone composition of the parasitic protozoa Leishmania donovani promastigote was investigated. 1'-oxomenaquinone-7, a chlorobiumquinone was found to be the major isoprenoid quinone. Substantial level of ubiquinone-9 was also present. Isolation and identification of the quinone from the purified plasma membrane yielded mainly 1'-oxomenaquinone-7 and ubiquinone-9; menaquinone was not detected. Membrane bound 1'-oxomenaquinone-7 could be destroyed by near-ultraviolet irradiation, with a concomitant loss or stimulation of plasma membrane electron transport activities. The abilities of different quinones to restore alpha-lipoic acid and ferricyanide reductase activity in near UV-irradiated cell preparations were compared. The order was; conjugate of chlorobiumquinone and sphingosine base approximately conjugate of 2-methyl-3-(1'-oxooctadecyl)-1,4-napthoquinone and octadecylamine > chlorobiumquinone approximately 2-methyl-3-(1'-oxooctadecyl)-1,4-napthoquinone > menaquinone-4 approximately ubiquinone-10. After irradiation with near-UV light, transmembrane alpha-lipoic acid reduction was inhibited, while transmembrane ferricyanide reduction was stimulated. The result obtained indicates that chlorobiumquinone mediates the plasma membrane electron transport between cytosolic reductant and oxygen as well as alpha-lipoic acid. UV-inactivation of chlorobiumquinone shuts down the plasma membrane oxygen uptake and diverts the electron flux towards ferricyanide reduction via ubiquinone-9. Chlorobiumquinone is the only example of a polyisoprenoid quinone containing a side chain carbonyl group from photosynthetic green-sulphur bacteria. Recent work has revealed numerous genes of trypanosomatid sharing common ancestry with plants and/or bacteria. These observations pose some fascinating questions about the evolutionary biology of this important group of parasitic protozoa.     

Restoration of respiratory electron-transport reactions in quinone-depleted particle preparations from Anacystis nidulans.

Electron transport from H2, NADPH, NADH and succinate to O2 or ferricytochrome c in respiratory particles isolated from Anacystis nidulans in which hydrogenase had been induced was abolished after extraction of the membranes with n-pentane; oxidation of ascorbate plus NNN'N'-tetramethyl-p-phenylenediamine remained unaffected. Incorporation of authentic ubiquinone-10, plastoquinone-9, menaquinone-7 and phylloquinone (in order of increasing efficiency) restored the electron-transport reactions. ATP-dependent reversed electron flow from NNN'N'-tetramethyl-p-phenylenediamine to NADP+ or, via the membrane-bound hydrogenase, to H+ was likewise abolished by pentane extraction and restored by incorporation of phylloquinone. Participation of the incorporated quinones in the respiratory electron-transport reactions of reconstituted particles was confirmed by measuring the degree of steady-state reduction of the quinones. Isolation and identification of the quinones present in native Anacystis membranes yielded mainly plastoquinone-9 and phylloquinone; neither menaquinone nor alpha-tocopherolquinone could be detected. Together with the results from reconstitution experiments this suggests that phylloquinone might function as the main respiratory quinone in Anacystis nidulans.     

Participation of chlorobiumquinone in the transplasma membrane electron transport system of Leishmania donovani promastigote: Effect of near-ultraviolet light on the redox reaction of plasma membrane

The respiratory quinone composition of the parasitic protozoa Leishmania donovani promastigote was investigated. 1′-oxomenaquinone-7, a chlorobiumquinone was found to be the major isoprenoid quinone. Substantial level of ubiquinone-9 was also present. Isolation and identification of the quinone from the purified plasma membrane yielded mainly 1′-oxomenaquinone-7 and ubiquinone-9; menaquinone was not detected. Membrane bound 1′-oxomenaquinone-7 could be destroyed by near-ultraviolet irradiation, with a concomitant loss or stimulation of plasma membrane electron transport activities. The abilities of different quinones to restore α-lipoic acid and ferricyanide reductase activity in near UV-irradiated cell preparations were compared. The order was; conjugate of chlorobiumquinone and sphingosine base ? conjugate of 2-methyl-3-(1′-oxooctadecyl)-1,4-napthoquinone and octadecylamine >> chlorobiumquinone ? 2-methyl-3-(1′-oxooctadecyl)-1,4-napthoquinone > menaquinone-4 ? ubiquinone-10. After irradiation with near-UV light, transmembrane α-lipoic acid reduction was inhibited, while transmembrane ferricyanide reduction was stimulated. The result obtained indicates that chlorobiumquinone mediates the plasma membrane electron transport between cytosolic reductant and oxygen as well as α-lipoic acid. UV-inactivation of chlorobiumquinone shuts down the plasma membrane oxygen uptake and diverts the electron flux towards ferricyanide reduction via ubiquinone-9. Chlorobiumquinone is the only example of a polyisoprenoid quinone containing a side chain carbonyl group from photosynthetic green-sulphur bacteria. Recent work has revealed numerous genes of trypanosomatid sharing common ancestry with plants and/or bacteria. These observations pose some fascinating questions about the evolutionary biology of this important group of parasitic protozoa.     

Function of Ubiquinone in Electron Transport from Reduced Nicotinamide Adenine Dinucleotide to Nitrate and Oxygen in Aerobacter aerogenes

The possible role of quinones in the electron transport system of Aerobacter aerogenes was investigated. The only quinone found in measurable amounts in bacteria grown in minimal media under both aerobic and anaerobic conditions was ubiquinone-8. Membrane-bound ubiquinone-8 could be removed by extraction with pentane, or destroyed by ultraviolet irradiation, with a concomitant loss of both reduced nicotinamide adenine dinucleotide (NADH) oxidase and NADH-linked respiratory nitrate reductase activity. In the extracted membrane preparations, these enzymatic activities could be restored, both to the same degree, by incorporation of ubiquinone-6, -8, or -10, but not by incorporation of menaquinones. The NADH oxidation and the nitrate reduction were sensitive to the respiratory inhibitors dicoumarol, lapachol, and cyanide. The results obtained indicate that ubiquinone-8 mediates the electron transport between NADH and oxygen as well as between NADH and nitrate. Branching of the electron transport chain to oxygen and nitrate occurs after an initial common pathway.     

(S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase from the thermoacidophilic archaeon Sulfolobus solfataricus. Molecular cloning and characterization of a membrane-intrinsic prenyltransferase involved in the biosynthesis of archaeal ether-linked membrane lipids

The core structure of membrane lipids of archaea have some unique properties that permit archaea to be distinguished from the others, i.e. bacteria and eukaryotes. (S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase, which catalyzes the transfer of a geranylgeranyl group from geranylgeranyl diphosphate to (S)-3-O-geranylgeranylglyceryl phosphate, is involved in the biosynthesis of archaeal membrane lipids. Enzymes of the UbiA prenyltransferase family are known to catalyze the transfer of a prenyl group to various acceptors with hydrophobic ring structures in the biosynthesis of respiratory quinones, hemes, chlorophylls, vitamin E, and shikonin. The thermoacidophilic archaeon Sulfolobus solfataricus was found to encode three homologues of UbiA prenyltransferase in its genome. One of the homologues encoded by SSO0583 was expressed in Escherichia coli, purified, and characterized. Radio-assay and mass spectrometry analysis data indicated that the enzyme specifically catalyzes the biosynthesis of (S)-2,3-di-O-geranylgeranylglyceryl phosphate. The fact that the orthologues of the enzyme are encoded in almost all archaeal genomes clearly indicates the importance of their functions. A phylogenetic tree constructed using the amino acid sequences of some typical members of the UbiA prenyltransferase family and their homologues from S. solfataricus suggests that the two other S. solfataricus homologues, excluding the (S)-2,3-di-O-geranylgeranylglyceryl phosphate synthase, are involved in the production of respiratory quinone and heme, respectively. We propose here that archaeal prenyltransferases involved in membrane lipid biosynthesis might be prototypes of the protein family and that archaea might have played an important role in the molecular evolution of prenyltransferases.     

Menaquinone is the sole quinone in the facultatively aerobic green photosynthetic bacterium Chloroflexus aurantiacus

The role of quinones was investigated in Chloroflexus aurantiacus, a thermophilic green bacterium capable of photosynthetic or respiratory growth. Thin-layer chromatography, ultraviolet difference spectroscopy and high-pressure liquid chromatography showed that menaquinone is the only quinone present in both photosynthetic and respiratory Chloroflexus cultures. Menaquinone-10 and menaquinone-8 are the predominant homologues in both cultures. For comparative purposes the quinone compositions in photoheterotrophic cultures of Chromatium vinosum and Chlorobium limicola were also analyzed. Chloroflexus is the only facultatively aerobic photosynthetic bacterium that does not possess ubiquinone. Menaquinone appears to be the only quinone involved in the photosynthetic and oxidative electron transport in this organism.     

Structures of mammalian cytosolic quinone reductases

The metabolism of quinone compounds presents one source of oxidative stress in mammals, as many pathways proceed by mechanisms that generate reactive oxygen species as by-products. One defense against quinone toxicity is the enzyme NAD(P)H:quinone oxidoreductase type 1 (QR1), which metabolizes quinones by a two-electron reduction mechanism, thus averting production of radicals. QR1 is expressed in the cytoplasm of many tissues, and is highly inducible. A closely related homologue, quinone reductase type 2 (QR2), has been identified in several mammalian species. QR2 is also capable of reducing quinones to hydroquinones, but unlike QR1, cannot use NAD(P)H. X-ray crystallographic studies of QR1 and QR2 illustrate that despite their different biochemical properties, these enzymes have very similar three-dimensional structures. In particular, conserved features of the active sites point to the close relationship between these two enzymes.     

The energy-linked transhydrogenase reaction in respiratory mutants of Escherichia coli K 12

1. Energy-linked and non-energy-linked transhydrogenase activities were assayed in membrane preparations from normal Escherichia coli K 12 and from various mutant strains. 2. The energy-linked transhydrogenase, which uses ATP as energy source, was dependent for activity on the presence of a functional Mg(2+)+Ca(2+)-stimulated adenosine triphosphatase. 3. Neither of the quinones formed by E. coli, namely ubiquinone-8 and menaquinone-8, was required for normal ATP-dependent energy-linked transhydrogenase activity. 4. The energy-linked transhydrogenase was inhibited by piericidin A at a site unrelated to the sites of inhibition of the electron-transport chain by piericidin A.     

An NADH:quinone oxidoreductase active during biodegradation by the brown-rot basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k(cat)/K(m) for the quinones is greater than 10(8) M(-1) s(-1). The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

Heterologous expression and biochemical characterization of an NAD(P)H:quinone oxidoreductase from the hemiparasitic plant Triphysaria versicolor

Quinones are widespread secondary metabolites that function as signal molecules between organisms in the rhizosphere. Quinones are particularly important in the exchange of chemical signals between plant roots, a phenomenon classically termed allelopathy. The bioactivity of quinones is due in large part to radical intermediates formed during redox cycling between quinone and hydroquinone states. In order to investigate the role of quinone oxidoreductases in processing quinone signals exchanged between plant roots, we characterized an NAD(P)H-dependent quinone reductase expressed in roots of the parasitic plant Triphysaria versicolor (TvQR2). The predicted amino acid sequence encoded by TvQR2 shares homology with quinone reductases from Archaea, Eubacteria and Eukaryota organisms. The complete TvQR2 cDNA was cloned into the fungus Pichia pastoris and the heterologous protein purified. The recombinant protein reduced a variety of quinones and napthoquinones, including several of allelopathic significance, using either NADH or NADPH as electron donors. The protein had an absorption spectrum consistent with it being a flavoprotein and was inhibited by the quinone reductase inhibitor dicumarol. We propose that the TvQR2 protein functions as a quinone reductase in plant roots to mitigate the toxicity of exogenous quinones in the rhizosphere.     

NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae

The sodium ion-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na⁺-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc₁ and b₆f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na⁺-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na⁺-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na⁺-NQR.     

An NADH:Quinone Oxidoreductase Active during Biodegradation by the Brown-Rot Basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k_cat/K_m for the quinones is greater than 10⁸ M⁻¹ s⁻¹. The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

Quinone reductase 2 is a catechol quinone reductase

The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.     

Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens

A newly isolated gene from Agrobacterium tumefaciens (A. tumefaciens), which encoded a decaprenyl diphosphate synthase, was cloned in Escherichia coli (E. coli), and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1077 bp capable of encoding a 358-amino-acid protein with a calculated isoelectric point of pH 5.16 and a molecular mass of 38 960 Da. The primary structure of the enzyme shared significant homology with prenyl diphosphate synthases from various sources. The deduced amino acid sequence included oligopeptide DDxxD aspartate-rich domains conserved in the majority of prenyl diphosphate synthases. High levels of the active enzyme were expressed in the soluble fraction and were readily purified to homogeneity by Ni-NTA chromatography. E. coli JM109 harboring the dps gene produced ubiquinone-10 in addition to endogenous ubiquinone-8, while E. coli JM109 harboring the dps gene mutated on the DDxxD domain lost the ability to produce ubiquinone-10, which suggests that the A. tumefaciens dps gene is functionally expressed in E. coli and that it encodes a decaprenyl diphosphate synthase.     

FAD semiquinone stability regulates single- and two-electron reduction of quinones by Anabaena PCC7119 ferredoxin:NADP+ reductase and its Glu301Ala mutant

Flavoenzymes may reduce quinones in a single-electron, mixed single- and two-electron, and two-electron way. The mechanisms of two-electron reduction of quinones are insufficiently understood. To get an insight into the role of flavin semiquinone stability in the regulation of single- vs. two-electron reduction of quinones, we studied the reactions of wild type Anabaena ferredoxin:NADP(+)reductase (FNR) with 48% FAD semiquinone (FADH*) stabilized at the equilibrium (pH 7.0), and its Glu301Ala mutant (8% FADH* at the equilibrium). We found that Glu301Ala substitution does not change the quinone substrate specificity of FNR. However, it confers the mixed single- and two-electron mechanism of quinone reduction (50% single-electron flux), whereas the wild type FNR reduces quinones in a single-electron way. During the oxidation of fully reduced wild type FNR by tetramethyl-1,4-benzoquinone, the first electron transfer (formation of FADH*) is about 40 times faster than the second one (oxidation of FADH*). In contrast, the first and second electron transfer proceeded at similar rates in Glu301Ala FNR. Thus, the change in the quinone reduction mechanism may be explained by the relative increase in the rate of second electron transfer. This enabled us to propose the unified scheme of single-, two- and mixed single- and two-electron reduction of quinones by flavoenzymes with the central role of the stability of flavin/quinone ion-radical pair.     

Identification of the quinone species in cyanide-sensitive and cyanide-insensitive mitochondria of Moniliella tomentosa.

Moniliella tomentosa was investigated for the presence of different quinones that might be involved in the cyanide-sensitive and/or cyanide-insensitive electron-transport pathways. The naturally occurring quinone in Moniliella tomentosa was found to be ubiquinone-45. Other quinone species could not be detected. The concentration of ubiquinone-45 in mitochondria is not related to the presence or absence of the alternative oxidase activity.     

Exploring the primary electron acceptor (QA)-site of the bacterial reaction center from Rhodobacter sphaeroides. Binding mode of vitamin K derivatives.

The functional replacement of the primary ubiquinone (QA) in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides with synthetic vitamin K derivatives has provided a powerful tool to investigate the electron transfer mechanism. To investigate the binding mode of these quinones to the QA binding site we have determined the binding free energy and charge recombination rate from QA(-) to D+ (kAD) of 29 different 1,4-naphthoquinone derivatives with systematically altered structures. The most striking result was that none of the eight tested compounds carrying methyl groups in both positions 5 and 8 of the aromatic ring exhibited functional binding. To understand the binding properties of these quinones on a molecular level, the structures of the reaction center-naphthoquinone complexes were predicted with ligand docking calculations. All protein--ligand structures show hydrogen bonds between the carbonyl oxygens of the quinone and AlaM260 and HisM219 as found for the native ubiquinone-10 in the X-ray structure. The center-to-center distance between the naphthoquinones at QA and the native ubiquinone-10 at QB (the secondary electron acceptor) is essentially the same, compared to the native structure. A detailed analysis of the docking calculations reveals that 5,8-disubstitution prohibits binding due to steric clashes of the 5-methyl group with the backbone atoms of AlaM260 and AlaM249. The experimentally determined binding free energies were reproduced with an rmsd of approximately 4 kJ x mol(-1) in most cases providing a valuable tool for the design of new artificial electron acceptors and inhibitors.     

Vectorial redox reactions of physiological quinones. I. Requirement of a minimum length of the isoprenoid side chain.

Physiological quinones carrying isoprenoid side chains have been compared with homologues lacking the side chain, for their ability to carry electrons and protons from dithionite to ferricyanide, trapped in liposomes. Six differential observations were made: (1) Plastoquinone and ubiquinones, with a side chain of more than two isoprene units, are by far better mediators than their short-chain homologues. Also other benzoquinones lacking a long side chain are poor catalysts, except dimethyl-methylenedioxy-p-benzoquinone, a highly autooxidizable compound. Tocopherol is a good catalyst. (2) Vitamin K-1 and K-2 are poor mediators compared to vitamin K-3. (3) The reaction catalyzed by quinones carrying long isoprenoid side chains has an about three-fold higher activation energy, irrespective of the catalytic efficiency. (4) The reaction catalyzed by quinones lacking a long side chain follows pseudo first-order kinetics, while the reaction with quinones carrying a long side chain is of apparently higher order. (5) The rate with ubiquinone-1 is increasing pH, while with ubiquinone-9 it is decreasing. (6) The reaction mediated by short-chain quinones seems to be satuarated at lower dithionite concentration. We conclude that isoprenoid quinones are able to translocate electrons and protons in lipid membranes, and that the side chain has a strong impact on the mechanism. This and the relevance of the model reaction for electron and proton transport in photosynthesis and respiration is discussed.     

Characterization of the Na-dependent respiratory chain NADH:quinone oxidoreductase of the marine bacterium, Vibrio alginolyticus, in relation to the primary Na pump

The Na⁺-dependent respiratory chain NADH: quinone oxidoreductase of the marine bacterium, Vibrio alginolyticus, was extracted from membrane by a detergent, Liponox DCH, and was purified by chromatography on QAE-Sephadex and Bio-Gel HTP. The activity of NADH oxidation was separated into two fractions. The one fraction could react with several artificial electron acceptors including Q-1, but could not reduce ubiquinone and menaquinone such as Q-5 and menaquinone-4, which was called NADH dehydrogenase. The other fraction could reduce Q-5 and menaquinone-4 in addition to the NADH dehydrogenase activity, which was called quinone reductase. The purified NADH dehydrogenase consumed NADH in excess of the amount of Q-1 and the reduced Q-1 (quinol) was not produced at all due to an oxidation-reduction cycle of semiquinone radicals. The quinone reductase, however, consumed NADH with the quantitative formation of quinol on account of a dismutation reaction of semiquinone radicals. Identical to the membrane-bound NADH: quinone oxidoreductase, the quinone reductase specifically required Na⁺ for the activity and was inhibited by 2-heptyl-4-hydroxyquinoline N-oxide. The electron transfer in the quinone reductase was formulated in a form of quinone cycle and the dismutation reaction of semiquinone radicals was assigned to be coupled to the Na⁺ pump in the respiratory chain of this organism.