Effects of multiple ligand binding on kinetic isotope effects in PQQ-dependent methanol dehydrogenase

The reaction of PQQ-dependent methanol dehydrogenase (MDH) from Methylophilus methylotrophus has been studied by steady-state and stopped-flow kinetic methods, with particular reference to multiple ligand binding and the kinetic isotope effect (KIE) for PQQ reduction. Phenazine ethosulfate (PES; an artificial electron acceptor) and cyanide (a suppressant of endogenous activity), but not ammonium (an activator of MDH), compete for binding at the catalytic methanol-binding site. Cyanide does not activate turnover in M. methylotrophus MDH, as reported previously for the Paracoccus denitrificans enzyme. Activity is dependent on activation by ammonium but is inhibited at high ammonium concentrations. PES and methanol also influence the stimulatory and inhibitory effects of ammonium through competitive binding. Reaction profiles as a function of ammonium and PES concentration differ between methanol and deuterated methanol, owing to force constant effects on the binding of methanol to the stimulatory and inhibitory ammonium binding sites. Differential binding gives rise to unusual KIEs for PQQ reduction as a function of ammonium and PES concentration. The observed KIEs at different ligand concentrations are independent of temperature, consistent with their origin in differential binding affinities of protiated and deuterated substrate at the ammonium binding sites. Stopped-flow studies indicate that enzyme oxidation is not rate-limiting at low ammonium concentrations (<4 mM) during steady-state turnover. At higher ammonium concentrations (>20 mM), the low effective concentration of PES in the active site owing to the competitive binding of ammonium lowers the second-order rate constant for enzyme oxidation, and the oxidative half-reaction becomes more rate limiting. A sequential stopped-flow method is reported that has enabled, for the first time, a detailed study of the reductive half-reaction of MDH and comparison with steady-state data. The limiting rate of PQQ reduction (0.48 s(-1)) is less than the steady-state turnover number, and the observed KIE in stopped-flow studies is unity. Although catalytically active, we propose reduction of the oxidized enzyme generated in stopped-flow analyses is gated by conformational change or ligand exchange. Slow recovery from this trapped state on mixing with methanol accounts for the slow reduction of PQQ and a KIE of 1. This study emphasizes the need for caution in using inflated KIEs, and the temperature dependence of KIEs, as a probe for hydrogen tunneling.     

Superoxide-mediated release of iron from ferritin by some flavoenzymes

NADH-lipoamide dehydrogenase mobilized iron from ferritin under aerobic conditions. Superoxide dismutase strongly inhibited this mobilization, indicating that the superoxide radical is generated by the enzymatic reaction and release iron from ferritin. Addition of lipoamide as an electron acceptor to NADH-lipoamide dehydrogenase increased the release of iron from ferritin and this release was partially inhibited by superoxide dismutase. Similarly, addition of menadione (2-methyl-1, 4-naphthoquinone) as an electron acceptor to xanthine-xanthine oxidase promoted the release of iron from ferritin and this release was strongly inhibited by superoxide dismutase. These results suggest that dihydrolipoamide and semiquinone of menadione can react with oxygen to form the superoxide radical that mediates release of iron from ferritin.     

Photoinhibition of hydroxylamine-extracted photosystem II membranes: studies of the mechanism.

The effects of photosystem II (PSII) exogenous electron donors and acceptors on the kinetics of weak light photoinhibition of NH2OH/EDTA-extracted spinach PSII membranes were examined. Under aerobic conditions, Mn2+ (approximately 1 Mn/reaction center; Km approximately 400 nM) inhibited photoinactivation and approximately 1 Mn/reaction center plus 100 microM NH2NH2 gave almost complete protection. In the absence of electron donors, strict anaerobiosis greatly inhibited photoinactivation even in the presence of an electron acceptor. Under aerobic conditions, the addition of electron acceptors (FeCN, DCIP), oxyradical scavengers, or superoxide dismutase strongly suppressed rates of photodamages. Increase in the concentrations of superoxide above those produced by illuminated NH2OH/EDTA-photosystem II membranes increased the rates of damage in the light but gave no damage in the dark. Scavengers of hydroxyl radicals and singlet oxygen did not suppress the rates of aerobic photoinhibition. These findings, along with others, lead us to conclude that photodamage of the secondary donors of the PSII reaction center occurs by two mechanisms: (1) a rapid superoxide and tyrosine YZ+ dependent process and (2) a slower process in which P680+/Chl+ catalyze the damages.     

Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway.

Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ). PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate. Aerobically, E. coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E. coli in a defined glucose-salts medium. Thus, for E. coli ZSC113 the Enter-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway. In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor. Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism. Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed. We suggest that E. coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment.     

Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway.

Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ). PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate. Aerobically, E. coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E. coli in a defined glucose-salts medium. Thus, for E. coli ZSC113 the Enter-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway. In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor. Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism. Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed. We suggest that E. coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment.     

A likely molecular basis of the susceptibility of Giardia lamblia towards oxygen

Giardia lamblia is an amitochondrial protozoan susceptible to oxygen, but the molecular basis for it remains unclear. A Giardia NAD(P)H:menadione oxidoreductase (DT-diaphorase) is known to catalyse a single electron transfer reaction with quinones as the likely two-electron acceptor when oxygen is absent. Here we overexpressed this enzyme in Giardia trophozoites and observed a significantly enhanced susceptibility of the cells towards oxygen. A knock-down of this enzyme resulted, however, in more oxygen-tolerant Giardia cells growing equally well under anaerobic and aerobic conditions. The function of DT-diaphorase could be thus a major, if not the only, cause for the oxygen susceptibility of Giardia. Overexpressed DT-diaphorase is accompanied by increased intracellular hydrogen peroxide. An overexpression of Fe-superoxide dismutase in Giardia led also to a similarly heightened sensitivity to oxygen. Thus, generation of H2O2 from superoxide anion likely produced from DT-diaphorase catalysed reaction using oxygen as electron acceptor may constitute the molecular basis for Giardia susceptibility to oxygen. A functional homologue of DT-diaphorase in Giardia, NADH oxidase, uses oxygen as the preferred electron acceptor and reduces it to water. Overexpression of this enzyme in Giardia resulted in significantly enhanced growth under aerobic conditions. Giardia NADH oxidase could be thus an instrumental enzyme for the organism to adapt to and to tolerate an aerobic living environment.     

Properties of the Membrane-Bound Quinoprotein Aldehyde Dehydrogenase from Acetobacter rancens

The n-alkane metabolizing strain Acetobacter rancens CCM 1774 possesses a dye-linked membrane-bound aldehyde dehydrogenase. The application of a sequential solubilization procedure at defined protein-detergent ratios allowed fast and effective purification without loss in enzyme activity. Both forms of aldehyde dehydrogenase—the membrane-bound and the solubilized enzyme— exhibited different properties, such as stability and electron transfer to cyto-chromes. By means of spectrophotometric investigations the presence of heme, FAD and other known groups in the purified enzyme could be excluded. Preliminary investigations with regard to the natural electron acceptor indicated the participation of PQQ in electron transfer. The fluorescence spectrum recorded for methanol extracts of the pure enzyme are comparable with those of adducts of PQQ. Inactivated aldehyde dehydrogenase could be reactivated by addition of these extracts, following saturation kinetics. Both enzyme forms catalyzed the oxidation of straight chain aldehydes, initial activities decreasing with increasing carbon chain length. Kinetic and inhibition experiments excluded a ping-pong mechanism as reported for other PQQ-enzymes. The membrane-bound enzyme should follow a compulsory-order mechanism in which the aldehyde substrate binds first whereas the purified enzyme follows a random-order mechanism.     

Microbial metabolism of C1-nitrogen compounds other than cyanide

Abstract The following topics are discussed in this review: the structure of methylamine dehydrogenase and the binding of its pyrrolo-quinoline quinone (PQQ) prosthetic group, the role of the copper protein amicyanin as electron acceptor of the enzyme and the nature of the electron carriers between amicyanin and oxygen in the electron transport chain. Also covered are recent developments in the metabolism of trimethylamine and its N -oxide and N -methylformamides.     

Ca2+-assisted, direct hydride transfer, and rate-determining tautomerization of C5-reduced PQQ to PQQH2, in the oxidation of beta-D-glucose by soluble, quinoprotein glucose dehydrogenase

Spectral and kinetic studies were performed on enzyme forms of soluble glucose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH) in which the PQQ-activating Ca(2+) was absent (Holo X) or was replaced with Ba(2+) (Ba-E) or in which PQQ was replaced with an analogue or a derivative called "nitroPQQ" (E-NPQ). Although exhibiting diminished rates, just like sGDH, all enzyme forms were able to oxidize a broad spectrum of aldose sugars, and their reduced forms could be oxidized with the usual artificial electron acceptor. On inspection of the plots for the reductive half-reaction, it appeared that the enzyme forms exhibited a negative cooperativity effect similar to that of sGDH itself under turnover conditions, supporting the view that simultaneous binding of substrate to the two subunits of sGDH causes the effect. Stopped-flow spectroscopy of the reductive half-reaction of Ba-E with glucose showed a fluorescing transient previously observed in the reaction of sGDH with glucose-1-d, whereas no intermediate was detected at all in the reactions of E-NPQ and Holo X. Using hydrazine as a probe, the fluorescing C5 adduct of PQQ and hydrazine was formed in sGDH, Ba-E, and Holo X, but E-NPQ did not react with hydrazine. When this is combined with other properties of E-NPQ and the behavior of enzyme forms containing a PQQ analogue, we concluded that the catalytic potential of the cofactor in the enzyme is not determined by its adduct-forming ability but by whether it is or can be activated with Ca(2+), activation being reflected by the large red shift of the absorption maximum induced by this metal ion when binding to the reduced cofactor in the enzyme. This conclusion, together with the observed deuterium kinetic isotope effect of 7.8 on transient formation in Ba-E, and that already known on transient decay, indicate that the sequential steps in the mechanism of sGDH must be (1) reversible substrate binding, (2) direct transfer of a hydride ion (reversible or irreversible) from the C1 position of the beta-anomer of glucose to the C5 of PQQ, (3) irreversible, rate-determining tautomerization of the fluorescing, C5-reduced PQQ to PQQH(2) and release (or earlier) of the product, D-glucono-delta-lactone, and (4) oxidation of PQQH(2) by an electron acceptor. The PQQ-activating Ca(2+) greatly facilitates the reactions occurring in step 2. His144 may also play a role in this by acting as a general base catalyst, initiating hydride transfer by abstracting a proton from the anomeric OH group of glucose. The validity of the proposed mechanism is discussed for other PQQ-containing dehydrogenases.     

The role of PQQ and quinoproteins in methylotrophic bacteria

Abstract Quinoprotein dehydrogenases play a non-exclusive role in the dissimilation of C₁ compounds. Methanol and methylamine oxidation occur by covalent catalysis while the reduction equivalents are transferred to the respiratory chain in one-electron steps. Cytochrome c _L is an excellent electron acceptor for methanol dehydrogenase at pH 7.0 and a bad one at pH 9.0. Efficient methanol oxidation (with NH₃ as activator) occurs at pH 9.0, but (due to the failure of NH₃) not at pH 7.0. Since stimulation occurred at the latter condition with a compound prepared from Hyphomicrobium X, most probably methanol oxidation in vivo requires the presence of a natural activator. The finding of pro-PQQ in methylamine dehydrogenase implicates that certain quinoproteins may have a modified tyrosine as cofactor. This type of quinoprotein is involved in assimilation routes which also occur in methylotrophs. l -Tyrosine and l -glutamate are the precursors of PQQ biosynthesis. Free intermediates in the route of biosynthesis have not been found. Most probably the whole process occurs on a protein matrix. In view of the significant amounts found in their culture fluid, methylotrophic bacteria seem particularly well suited for the fermentative production of PQQ.     

Flavin-linked Erv-family sulfhydryl oxidases release superoxide anion during catalytic turnover

Typically, simple flavoprotein oxidases couple the oxidation of their substrates with the formation of hydrogen peroxide without release of significant levels of the superoxide ion. However, two evolutionarily related single-domain sulfhydryl oxidases (Erv2p; a yeast endoplasmic reticulum resident protein and augmenter of liver regeneration, ALR, an enzyme predominantly found in the mitochondrial intermembrane) release up to ~30% of the oxygen they reduce as the superoxide ion. Both enzymes oxidize dithiol substrates via a redox-active disulfide adjacent to the flavin cofactor within the helix-rich Erv domain. Subsequent reduction of the flavin is followed by transfer of reducing equivalents to molecular oxygen. Superoxide release was initially detected using tris(3-hydroxypropyl)phosphine (THP) as an alternative reducing substrate to dithiothreitol (DTT). THP, and other phosphines, showed anomalously high turnover numbers with Erv2p and ALR in the oxygen electrode, but oxygen consumption was drastically suppressed upon the addition of superoxide dismutase. The superoxide ion initiates a radical chain reaction promoting the aerobic oxidation of phosphines with the formation of hydrogen peroxide. Use of a known flux of superoxide generated by the xanthine/xanthine oxidase system showed that one superoxide ion stimulates the reduction of 27 and 4.5 molecules of oxygen using THP and tris(2-carboxyethyl)phosphine (TCEP), respectively. This superoxide-dependent amplification of oxygen consumption by phosphines provides a new kinetic method for the detection of superoxide. Superoxide release was also observed by a standard chemiluminescence method using a luciferin analogue (MCLA) when 2 mM DTT was employed as a substrate of Erv2p and ALR. The percentage of superoxide released from Erv2p increased to ~65% when monomeric mutants of the normally homodimeric enzyme were used. In contrast, monomeric multidomain quiescin sulfhydryl oxidase enzymes that also contain an Erv FAD-binding fold release only 1-5% of their total reduced oxygen species as the superoxide ion. Aspects of the mechanism and possible physiological significance of superoxide release from these Erv-domain flavoproteins are discussed.     

Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).     

Quinoprotein ethanol dehydrogenase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: the unusual disulfide ring formed by adjacent cysteine residues is essential for efficient electron transfer to cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>550</Subscript>

All pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases contain an unusual disulfide ring formed between adjacent cysteine residues. A mutant enzyme that is lacking this structure was generated by replacing Cys105 and Cys106 with Ala in quinoprotein ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa ATCC17933. Heterologously expressed quinoprotein ethanol dehydrogenase in which Cys-105 and Cys-106 have been replaced by Ala (Cys105Ala/Cys106Ala apo-QEDH) was successfully converted to enzymatic active holo-enzyme by incorporation of its cofactor PQQ in the presence of Ca²⁺. The enzymatic activity of the mutant enzyme in the artificial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 did not depend on an activating amine which is essential for wild type activity under these conditions. The mutant enzyme showed increased Michaelis constants for primary alcohols, while the affinity for the secondary alcohol 2-propanol was unaltered. Surprisingly, for all substrates tested the specific activity of the mutant enzyme in the artificial dye test was higher than that found for wild type QEDH. On the contrary, in the ferricyanide test with the natural electron acceptor cytochrome c ₅₅₀ the activity of mutant Cys105Ala/Cys106Ala was 15-fold lower than that of wild type QEDH. We demonstrate for the first time unambiguously that the unusual disulfide ring is essential for efficient electron transfer at pH 7 from QEDH to its natural electron acceptor cytochrome c ₅₅₀.     

Does the intestinal microflora synthesize pyrroloquinoline quinone?

Pyrroloquinoline quinone (PQQ) functions as a cofactor for prokaryotic oxidoreductases, such as methanol dehydrogenase and glucose dehydrogenase. When chemically-defined diets without PQQ are fed to animals, lathyritic changes are observed. In previous studies, it was assumed that PQQ was produced by the intestinal microflora; consequently, antibiotics were routinely added to diets. In the present study this assumption is tested further in mice by: (i) examining the effects of dietary antibiotics on fecal PQQ excretion, (ii) isolating the intestinal flora to identify bacteria known to synthesize PQQ and (iii) determining in vitro if the intestinal microflora synthesizes PQQ from radio-chemically labeled precursors. The results of these experiments indicate that little if any PQQ is synthesized by the intestinal microflora. Rather, when PQQ is present in the intestine, the diet is a more obvious source.     

Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni.

Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name 'quinohaemoprotein alcohol dehydrogenase'. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.     

Aerobic and anaerobic growth of Paracoccus denitrificans on methanol

1. The dye-linked methanol dehydrogenase from Paracoccus denitrificans grown aerobically on methanol has been purified and its properties compared with similar enzymes from other bacteria. It was shown to be specific and to have high affinity for primary alcohols and formaldehyde as substrate, ammonia was the best activator and the enzyme could be linked to reduction of phenazine methosulphate.
2. Paracoccus denitrificans could be grown anaerobically on methanol, using nitrate or nitrite as electron acceptor. The methanol dehydrogenase synthesized under these conditions could not be differentiated from the aerobically-synthesized enzyme.
3. Activities of methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, nitrate reductase and nitrite reductase were measured under aerobic and anaerobic growth conditions.
4. Difference spectra of reduced and oxidized cytochromes in membrane and supernatant fractions of methanol-grown P. denitrificans were measured.
5. From the results of the spectral and enzymatic analyses it has been suggested that anaerobic growth on methanol/nitrate is made possible by reduction of nitrate to nitrite using electrons derived from the pyridine nucleotide-linked dehydrogenations of formaldehyde and formate, the nitrite so produced then functioning as electron acceptor for methanol dehydrogenase via cytochrome c and nitrite reductase.

     

Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans

A new type of sulfite oxidase which utilizes ferric ion (Fe3+) as an electron acceptor was found in iron-grown Thiobacillus ferrooxidans. It was localized in the plasma membrane of the bacterium and had a pH optimum at 6.0. Under aerobic conditions, 1 mol of sulfite was oxidized by the enzyme to produce 1 mol of sulfate. Under anaerobic conditions in the presence of Fe3+, sulfite was oxidized by the enzyme as rapidly as it was under aerobic conditions. In the presence of o-phenanthroline or a chelator for Fe2+, the production of Fe2+ was observed during sulfite oxidation by this enzyme under not only anaerobic conditions but also aerobic conditions. No Fe2+ production was observed in the absence of o-phenanthroline, suggesting that the Fe2+ produced was rapidly reoxidized by molecular oxygen. Neither cytochrome c nor ferricyanide, both of which are electron acceptors for other sulfite oxidases, served as an electron acceptor for the sulfite oxidase of T. ferrooxidans. The enzyme was strongly inhibited by chelating agents for Fe3+. The physiological role of sulfite oxidase in sulfur oxidation of T. ferrooxidans is discussed.     

Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase

Metabolic engineering studies have generally focused on manipulating enzyme levels through either the amplification, addition, or deletion of a particular pathway. However, with cofactor-dependent production systems, once the enzyme levels are no longer limiting, cofactor availability and the ratio of the reduced to oxidized form of the cofactor can become limiting. Under these situations, cofactor manipulation may become crucial in order to further increase system productivity. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. However, cofactor manipulations can potentially become a powerful tool for metabolic engineering. Nicotinamide adenine dinucleotide (NAD) functions as a cofactor in over 300 oxidation-reduction reactions and regulates various enzymes and genetic processes. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. This paper investigates a genetic means of manipulating the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase. More specifically, it explores the effect on the metabolic patterns in Escherichia coli under anaerobic and aerobic conditions of substituting the native cofactor-independent formate dehydrogenase (FDH) by and NAD(+)-dependent FDH from Candida boidinii. The over-expression of the NAD(+)-dependent FDH doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed, increased the final cell density, and provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically. Under anaerobic conditions, the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol-to-acetate ratio. Even more interesting is the observation that during aerobic growth, the increased availability of NADH induced a shift to fermentation even in the presence of oxygen by stimulating pathways that are normally inactive under these conditions.     

Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans.

A new type of sulfite oxidase which utilizes ferric ion (Fe3+) as an electron acceptor was found in iron-grown Thiobacillus ferrooxidans. It was localized in the plasma membrane of the bacterium and had a pH optimum at 6.0. Under aerobic conditions, 1 mol of sulfite was oxidized by the enzyme to produce 1 mol of sulfate. Under anaerobic conditions in the presence of Fe3+, sulfite was oxidized by the enzyme as rapidly as it was under aerobic conditions. In the presence of o-phenanthroline or a chelator for Fe2+, the production of Fe2+ was observed during sulfite oxidation by this enzyme under not only anaerobic conditions but also aerobic conditions. No Fe2+ production was observed in the absence of o-phenanthroline, suggesting that the Fe2+ produced was rapidly reoxidized by molecular oxygen. Neither cytochrome c nor ferricyanide, both of which are electron acceptors for other sulfite oxidases, served as an electron acceptor for the sulfite oxidase of T. ferrooxidans. The enzyme was strongly inhibited by chelating agents for Fe3+. The physiological role of sulfite oxidase in sulfur oxidation of T. ferrooxidans is discussed.