PqqC/D,which converts a biosynthetic intermediate to pyrroloquinoline quinone

PqqC/D was purified from Escherichia coli transformant. The purified enzyme converted an intermediate that accumulated in a pqqC mutant of Methylobacterium extorquens AM1 to PQQ. The reaction did not show any dependence of NAD(P)H that was observed in the crude extract before purification. PqqC/D reacted with the intermediate stoichiometrically, but not catalytically. When partially purified proteins from the crude extract of E. coli were added to the reaction mixture, the rate of PQQ production increased dependent on the amount of NADPH added and the total amount of PQQ produced increased.     

Properties of a coenzyme, pyrroloquinoline quinone: generation of an active oxygen species during a reduction-oxidation cycle in the presence of NAD(P)H and O2

The oxidation of NAD(P)H by pyrroloquinoline quinone (PQQ) was non-enzymatically carried out at physiological pH in the presence of O2. The PQQ-NAD(P)H system requires about 1 mol of O2 for the oxidation of 1 mol of NAD(P)H. The oxidation of NAD(P)H occurred at a pseudo-first-order rate with respect to NAD(P)H and was of zero order with respect to PQQ concentration in in the presence of O2: k0[PQQ] [NAD(P)H] = k1 [NAD(P)H], where k0[PQQ] = k1, in which [PQQ] represents the initial concentration of PQQ. k0 values for NADH and NADPH were 3.4.10(2) M-1.min-1 and 2.0.10(2) M-1.min-1, respectively, at 25 degrees C and at 258 microM O2 (initial concentration). The system produced O-2, probably by the interaction of PQQ.H and/or NAD(P).with O2, during the oxidation of NAD(P)H. PQQH2 and PQQ.H were easily oxidized to PQQ in the presence of O2, yielding H2O2.     

Characterization of a protein-generated O₂ binding pocket in PqqC, a cofactorless oxidase catalyzing the final step in PQQ production

PQQ is an exogenous, tricyclic, quino-cofactor for a number of bacterial dehydrogenases. The final step of PQQ formation is catalyzed by PqqC, a cofactorless oxidase. This study focuses on the activation of molecular oxygen in an enzyme active site without metal or cofactor and has identified a specific oxygen binding and activating pocket in PqqC. The active site variants H154N, Y175F,S, and R179S were studied with the goal of defining the site of O(2) binding and activation. Using apo-glucose dehydrogenase to assay for PQQ production, none of the mutants in this "O(2) core" are capable of PQQ/PQQH(2) formation. Spectrophotometric assays give insight into the incomplete reactions being catalyzed by these mutants. Active site variants Y175F, H154N, and R179S form a quinoid intermediate (Figure 1) anaerobically. Y175S is capable of proceeding further from quinoid to quinol, whereas Y175F, H154N, and R179S require O(2) to produce the quinol species. None of the mutations precludes substrate/product binding or oxygen binding. Assays for the oxidation of PQQH(2) to PQQ show that these O(2) core mutants are incapable of catalyzing a rate increase over the reaction in buffer, whereas H154N can catalyze the oxidation of PQQH(2) to PQQ in the presence of H(2)O(2) as an electron acceptor. Taken together, these data indicate that none of the targeted mutants can react fully to form quinone even in the presence of bound O(2). The data indicate a successful separation of oxidative chemistry from O(2) binding. The residues H154, Y175, and R179 are proposed to form a core O(2) binding structure that is essential for efficient O(2) activation.     

An integrated NAD+-dependent enzyme-functionalized field-effect transistor (ENFET) system: development of a lactate biosensor

An integrated NAD+-dependent enzyme field-effect transistor (ENFET) device for the biosensing of lactate is described. The aminosiloxane-functionalized gate interface is modified with pyrroloquinoline quinone (PQQ) that acts as a catalyst for the oxidation of NADH. Synthetic amino-derivative of NAD+ is covalently linked to the PQQ monolayer. An affinity complex formed between the NAD+/PQQ-assembly and the NAD+-cofactor-dependent lactate dehydrogenase (LDH) is crosslinked and yields an integrated biosensor ENFET-device for the analysis of lactate. Biocatalyzed oxidation of lactate generates NADH that is oxidized by PQQ in the presence of Ca2+-ions. The reduced catalyst, PQQH2, is oxidized by O2 in a process that constantly regenerates PQQ at the gate interface. The biocatalyzed formation of NADH and the O2-stimulated regeneration of PQQ yield a steady-state pH gradient between the gate interface and the bulk solution. The changes in the pH of the solution near the gate interface and, consequently, the gate potential are controlled by the substrate (lactate) concentration in the solution. The device reveals the detection limit of 1 x 10(-4) M for lactate and the sensitivity of 24+/-2 mV dec(-1). The response time of the device is as low as 15 s.     

Role of calcium in the reaction between pyrroloquinoline quinone and pyridine nucleotides monomers and dimers.

Redox reactions were carried out in aerobiosis and anaerobiosis between NAD(P) dimers or NAD(P)H and pyrroloquinoline quinone (PQQ) in different buffers. The buffer system and pH significantly affected the oxidation rates of nucleotides and the ESR signal intensity of the PQQ(*) radical formed in anaerobiosis by comproportion between the quinone and quinol forms. The relative reactivity of the four nucleotides toward PQQ was affected by pH and buffer nature. PQQ, which behaves as an electron shuttle from nucleotides to oxygen, was first converted to PQQH(2) and then rapidly reoxidized by oxygen, with formation of hydrogen peroxide. Both NAD(P) dimers and NAD(P)H consumed 1 mol of oxygen per mole of reacted molecule of pyridine nucleotide, yielding 1 or 2 mol of NAD(P)(+) from NAD(P)H or from NAD(P) dimers, respectively. Chelating agents such as EDTA and phytate strongly decreased the reaction rate and the PQQ(*) radical signal intensity. Kinetics carried out in the presence of metal ions showed instead an increased reaction rate in the order Ca(2+) > Mg(2+) > Na(+) > K(+). Spectrofluorimetric measurements of PQQ with increasing concentrations of Ca(2+) showed a fluorescence quenching and shift of the maximum emission toward lower wavelengths, while other metal ions showed minor effects, if any. Therefore, it is demonstrated that Ca(2+) binds to PQQ, probably forming a complex which is more reactive with both one-electron (NAD(P) dimers) or two-electron donors (NAD(P)H) in nonenzymic reactions. It is important to recall that Ca(2+) was already found to play active role in PQQ-containing enzymes.     

The vanadate-stimulated oxidation of NAD(P)H by biomembranes is a superoxide-initiated free radical chain reaction

Rat liver microsomes catalyze a vanadate-stimulated oxidation of NAD(P)H, which is augmented by paraquat and suppressed by superoxide dismutase, but not by catalase. NADPH oxidation was a linear function of the concentration of microsomes in the absence of vanadate, but was a saturating function in the presence of vanadate. Microsomes did not catalyze a vanadate-stimulated oxidation of reduced nicotinamide mononucleotide (NMNH), but gained this ability when NADPH was also present. When the concentration of NMNH was much greater than that of NADPH a minimal average chain length could be calculated from 1/2 the ratio of NMNH oxidized per NADPH added. The term chain length, as used here, signifies the number of molecules of NMNH oxidized per initiating event. Chain length could be increased by increasing [vanadate] and [NMNH] and by decreasing pH. Chain lengths in excess of 30 could easily be achieved. The Km for NADPH, arrived at from saturation of its ability to trigger NMNH oxidation by microsomes in the presence of vanadate, was 1.5 microM. Microsomes or the outer mitochondrial membrane was able to catalyze the vanadate-stimulated oxidation of NADH or NADPH but only the oxidation of NADPH was accelerated by paraquat. The inner mitochondrial membrane was able to cause the vanadate-stimulated oxidation of NAD(P)H and in this case paraquat stimulated the oxidation of both pyridine coenzymes. Our results indicate that vanadate stimulation of NAD(P)H oxidation by biomembranes is a consequence of vanadate stimulation of NAD(P)H or NMNH oxidation by O-2, rather than being due to the existence of vanadate-stimulated NAD(P)H oxidases or dehydrogenases.     

Increased phospholipid methylation in the myocardium of alcoholic rats

NAD(P)H is known to be oxidized by singlet molecular oxygen, perhydroxyl radical, and hydroxyl radical. In marked contrast to these reactive oxygen species, NAD(P)H is stable in the presence of micromolar concentrations of H2O2. The experiments herein demonstrate that NADPH is rapidly oxidized by H2O2 in the presence of a heme-peptide. The oxidation product is enzymatically active NADP+. In the absence of NADPH, the heme-peptide undergoes rapid degradation via reaction with H2O2. In the presence of NADPH, the reduced nucleotide is oxidized to NADP and the heme-peptide is partially protected from oxidation. It is suggested that under certain conditions the reduced nucleotides may contribute to the protection of intracellular heme moieties from degradation engendered by endogenous or exogenous H2O2.     

On Dioxygen Permeation through a Dehydrogenase‐Pyrroloquinoline Quinone Complex. A Molecular‐Dynamics Investigation

In this work, an all atom model of the quinoprotein dehydrogenase PqqC in complex with the PQQ (=4,5‐dihydro‐4,5‐dioxo‐1H‐pyrrolo[2,3‐f]quinoline‐2,7,9‐tricarboxylic acid) cofactor and dioxygen (O₂), solvated with TIP3 water in periodic boxes, was subjected to random‐acceleration molecular dynamics (RAMD). It was found that O₂ leaves the active binding pocket, in front of PQQ, to get to the solvent, as easily as with a variety of other O₂‐activating enzymes, O₂ carriers, and gas‐sensing proteins. The shortest pathway, orthogonal to the center of the mean plane of PQQ, was largely preferred by O₂ over pathways slightly deviating from this line. These observations challenge the interpretation of an impermeable active binding pocket of PqqC‐PQQ, as drawn from both X‐ray diffraction data of the crystal at low temperature and physiological experimentation.     

Synthesis of pyrroloquinoline quinone in vivo and in vitro and detection of an intermediate in the biosynthetic pathway.

In Klebsiella pneumoniae, six genes, constituting the pqqABCDEF operon, which are required for the synthesis of the cofactor pyrroloquinoline quinone (PQQ) have been identified. The role of each of these K. pneumoniae Pqq proteins was examined by expression of the cloned pqq genes in Escherichia coli, which cannot synthesize PQQ. All six pqq genes were required for PQQ biosynthesis and excretion into the medium in sufficient amounts to allow growth of E. coli on glucose via the PQQ-dependent glucose dehydrogenase. Mutants lacking the PqqB or PqqF protein synthesized small amounts of PQQ, however. PQQ synthesis was also studied in cell extracts. Extracts made from cells containing all Pqq proteins contained PQQ. Lack of each of the Pqq proteins except PqqB resulted in the absence of PQQ. Extracts lacking PqqB synthesized PQQ slowly. Complementation studies with extracts containing different Pqq proteins showed that an extract lacking PqqC synthesized an intermediate which was also detected in the culture medium of pqqC mutants. It is proposed that PqqC catalyzes the last step in PQQ biosynthesis. Studies with cells lacking PqqB suggest that the same intermediate might be accumulated in these mutants. By using pqq-lacZ protein fusions, it was shown that the expression of the putative precursor of PQQ, the small PqqA polypeptide, was much higher than that of the other Pqq proteins. Synthesis of PQQ most likely requires molecular oxygen, since PQQ was not synthesized under anaerobic conditions, although the pqq genes were expressed.     

The decrease of NAD(P)H has a prominent role in dopamine toxicity

We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 microM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 microM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.     

Structural studies of mutant forms of the PQQ‐forming enzyme PqqC in the presence of product and substrate

Pyrroloquinoline quinone [4,5‐dihydro‐4,5‐dioxo‐1H‐pyrrolo[2,3‐f]quinoline‐2,7,9‐tricarboxylic acid (PQQ)] is a bacterial cofactor in numerous alcohol dehydrogenases including methanol dehydrogenase and glucose dehydrogenase. Its biosynthesis in Klebsiella pneumoniae is facilitated by six genes, pqqABCDEF and proceeds by an unknown pathway. PqqC is one of two metal free oxidases of known structure and catalyzes the last step of PQQ biogenesis which involves a ring closure and an eight‐electron oxidation of the substrate [3a‐(2‐amino‐2‐carboxyethyl)‐4,5‐dioxo‐4,5,6,7,8,9‐hexahydroquinoline‐7,9‐dicarboxylic acid (AHQQ)]. PqqC has 14 conserved active site residues, which have previously been shown to be in close contact with bound PQQ. Herein, we describe the structures of three PqqC active site variants, H154S, Y175F, and the double mutant R179S/Y175S. The H154S crystal structure shows that, even with PQQ bound, the enzyme is still in the “open” conformation with helices α5b and α6 unfolded and the active site solvent accessible. The Y175F PQQ complex crystal structure reveals the closed conformation indicating that Y175 is not required for the conformational change. The R179S/Y175S AHQQ complex crystal structure is the most mechanistically informative, indicating an open conformation with a reaction intermediate trapped in the active site. The intermediate seen in R179S/Y175S is tricyclic but nonplanar, implying that it has not undergone oxidation. These studies implicate a stepwise process in which substrate binding leads to the generation of the closed protein conformation, with the latter playing a critical role in O₂ binding and catalysis. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The enzymatic reaction-induced configuration change of the prosthetic group PQQ of methanol dehydrogenase

Methanol dehydrogenase is a heterotetrameric enzyme containing the prosthetic group pyrroloquinoline quinone (PQQ), which catalyzes the oxidation of methanol to formaldehyde. The crystal structure of methanol dehydrogenase from Methylophilus W3A1, previously determined at high resolution, exhibits a non-planar configuration of the PQQ ring system and lends support for a hydride transfer mechanism of the enzymatic reaction catalyzed by the enzyme. To investigate why PQQ is in the C5-reduced form and to better understand the catalytic mechanism of the enzyme, three structures of this enzyme in a new crystal form have been determined at higher resolution. Two of the three crystals were grown in the presence of 1 and 50 mM methanol, respectively, both structures of which show non-planar configurations of the PQQ ring system, confirming the previous conclusion; the other was crystallized in the presence of 50 mM ethanol, the structure of which displays a planar ring system for PQQ. Comparison of these structures reveals that the configuration change of PQQ is induced by the enzymatic reaction. The reaction takes place and the C5-reduced PQQ intermediate is produced when the enzyme co-crystallizes with methanol, but the enzymatic reaction does not take place and the PQQ ring retains a planar configuration of the oxidized orthoquinone form when ethanol instead of methanol is present in the crystallization solution.     

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation.     

Regulation of poly-β-hydroxybutyrate biosynthesis by nicotinamide nucleotide in Alcaligenes eutrophus

Abstract Poly-β-hydroxybutyrate biosynthesis was studied in Alcaligenes eutrophus under various nutrient-limiting conditions. When the cells were cultivated in nitrogen-limited media, both the levels of NAD(P)H and the ratios of NAD(P)H/NAD(P) were higher than those under nitrogen-sufficient conditions. The specific poly-β-hydroxybutyrate production rate was found to increase with the values of both NADH/NAD and NADPH/NADP, indicating that poly-β-hydroxybutyrate synthesis is directly regulated by the ratios of nicotinamide nucleotides. The effects of nicotinamide nucleotides on poly-β-hydroxybutyrate biosynthesis was investigated with regard to enzyme kinetics. Citrate synthase activity was significantly inhibited by NADH and NADPH, indicating that poly-β-hydroxybutyrate accumulation could be enhanced by facilitating the metabolic flux of acetyl-CoA to poly-β-hydroxybutyrate synthetic pathway. It was also found that cellular NADPH was a limiting substrate for NADPH-linked reductase, controlling the overall biosynthetic activity of poly-/3-hydroxybutyrate in this strain.     

NAD-dependent, PQQ-containing methanol dehydrogenase: a bacterial dehydrogenase in a multienzyme complex

Cell-free extracts of methanol-grown Nocardia sp. 239 only show significant dye-linked methanol-oxidizing activity when NAD+ is added to the assay mixture. This activity resides in a multienzyme complex which could be resolved into 3 components, namely the methanol dehydrogenase, NAD-dependent aldehyde dehydrogenase and NADH dehydrogenase. In its dissociated form, the methanol dehydrogenase no longer shows dye reduction and although rises in the absorbance values around 340 nm are seen on addition of methanol plus NAD+ to the enzyme, this is not due to NADH production. However, dye reduction (NAD dependent) could be restored on incubating methanol dehydrogenase with the corresponding NADH dehydrogenase, obtained from the enzyme complex. It is concluded that this novel methanol dehydrogenase transfers the reducing equivalents, derived from methanol, directly to its associated NADH dehydrogenase via a mechanism in which NAD+ and PQQ are involved.     

Distribution and properties of the genes encoding the biosynthesis of the bacterial cofactor, pyrroloquinoline quinone

Pyrroloquinoline quinone (PQQ) is a small, redox active molecule that serves as a cofactor for several bacterial dehydrogenases, introducing pathways for carbon utilization that confer a growth advantage. Early studies had implicated a ribosomally translated peptide as the substrate for PQQ production. This study presents a sequence- and structure-based analysis of the components of the pqq operon. We find the necessary components for PQQ production are present in 126 prokaryotes, most of which are Gram-negative and a number of which are pathogens. A total of five gene products, PqqA, PqqB, PqqC, PqqD, and PqqE, are identified as being obligatory for PQQ production. Three of the gene products in the pqq operon, PqqB, PqqC, and PqqE, are members of large protein superfamilies. By combining evolutionary conservation patterns with information from three-dimensional structures, we are able to differentiate the gene products involved in PQQ biosynthesis from those with divergent functions. The observed persistence of a conserved gene order within analyzed operons strongly suggests a role for protein-protein interactions in the course of cofactor biosynthesis. These studies propose previously unidentified roles for several of the gene products, as well as identifying possible new targets for antibiotic design and application.     

Superoxide generated by glutathione reductase initiates a vanadate-dependent free radical chain oxidation of NADH.

Vanadate V(V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O2 and the accumulation of H2O2. Superoxide dismutases completely eliminated this effect of V(V), whereas catalase was without effect, as was exogenous H2O2 added to 0.1 mM. These effects could be seen equally well in phosphate- or in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid-buffered solutions. Under anaerobic conditions there was no V(V)-stimulated oxidation of NADPH. Approximately 4% of the electrons flowing from NADPH to O2, through GSSG reductase, resulted in release of O2-. The average length of the free radical chains causing the oxidation of NADPH, initiated by O2- plus V(V), was calculated to be in the range 140-200 NADPH oxidized per O2- introduced. We conclude that GSSG reductase, and by extension other O2(-)-producing flavoprotein dehydrogenases such as lipoyl dehydrogenase and ferredoxin reductase, catalyze V(V)-stimulated oxidation of NAD(P)H because they release O2- and because O2- plus V(V) initiate a free radical chain oxidation of NAD(P)H. There is no reason to suppose that these enzymes can act as NAD(P)H:V(V) oxidoreductases.     

The cofactor preference of glucose-6-phosphate dehydrogenase from Escherichia coli--modeling the physiological production of reduced cofactors

In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. The first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), is generally considered an exclusive NADPH producer, but a rigorous assessment of cofactor preference has yet to be reported. In this work, the specificity constants for NADP and NAD for G6PDH were determined using a pure enzyme preparation. Absence of the phosphate group on the cofactor leads to a 410-fold reduction in the performance of the enzyme. Furthermore, the contribution of the phosphate group to binding of the transition state to the active site was calculated to be 3.6 kcal·mol(-1). In order to estimate the main kinetic parameters for NAD(P) and NAD(P)H, we used the classical initial-rates approach, together with an analysis of reaction time courses. To achieve this, we developed a new analytical solution to the integrated Michaelis-Menten equation by including the effect of competitive product inhibition using the ω-function. With reference to relevant kinetic parameters and intracellular metabolite concentrations reported by others, we modeled the sensitivity of reduced cofactor production by G6PDH as a function of the redox ratios of NAD/NADH (rR(NAD)) and NADP/NADPH (rR(NADP)). Our analysis shows that NADPH production sharply increases within the range of thermodynamically feasible values of rR(NADP), but NADH production remains low within the range feasible for rR(NAD). Nevertheless, we show that certain combinations of rR(NADP) and rR(NAD) sustain greater levels of NADH production over NADPH.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Hepatocyte susceptibility to glyoxal is dependent on cell thiamin content

Glyoxal, a reactive dicarbonyl, is detoxified primarily by the glyoxalase system utilizing glutathione (GSH) and by the aldo-keto reductase enzymes which utilizes NAD[P]H as the co-factor. Thiamin (Vitamin B(1)) is an essential coenzyme for transketolase (TK) that is part of the pentose phosphate pathway which helps maintain cellular NADPH levels. NADPH plays an intracellular role in regenerating glutathione (GSH) from oxidized GSH (GSSG), thereby increasing the antioxidant defenses of the cell. In this study we have focused on the prevention of glyoxal toxicity by supplementation with thiamin (3mM). Thiamin was cytoprotective and restored NADPH levels, glyoxal detoxification and mitochondrial membrane potential. Hepatocyte reactive oxygen species (ROS) formation, lipid peroxidation and GSH oxidation were decreased. Furthermore, hepatocytes were made thiamin deficient with oxythiamin (3mM) as measured by the decreased hepatocyte TK activity. Under thiamin deficient conditions a non-toxic dose of glyoxal (2mM) became cytotoxic and glyoxal metabolism decreased; while ROS formation, lipid peroxidation and GSH oxidation was increased.