Energy generation from the CO oxidation-hydrogen production pathway in Rubrivivax gelatinosus

When incubated in the presence of CO gas, Rubrivivax gelatinosus CBS induces a CO oxidation-H2 production pathway according to the stoichiometry CO + H2O --> CO2 + H2. Once induced, this pathway proceeds equally well in both light and darkness. When light is not present, CO can serve as the sole carbon source, supporting cell growth anaerobically with a cell doubling time of nearly 2 days. This observation suggests that the CO oxidation reaction yields energy. Indeed, new ATP synthesis was detected in darkness following CO additions to the gas phase of the culture, in contrast to the case for a control that received an inert gas such as argon. When the CO-to-H2 activity was determined in the presence of the electron transport uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), the rate of H2 production from CO oxidation was enhanced nearly 40% compared to that of the control. Upon the addition of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), we observed an inhibition of H2 production from CO oxidation which could be reversed upon the addition of CCCP. Collectively, these data strongly suggest that the CO-to-H2 reaction yields ATP driven by a transmembrane proton gradient, but the detailed mechanism of this reaction is not yet known. These findings encourage additional research aimed at long-term H2 production from gas streams containing CO.     

Inhibition of the Conversion of 1-Aminocyclopropane-1-carboxylic Acid to Ethylene by Structural Analogs, Inhibitors of Electron Transfer, Uncouplers of Oxidative Phosphorylation, and Free Radical Scavengers

Cyclopropane carboxylic acid (CCA) at 1 to 5 millimolar, unlike related cyclopropane ring analogs of 1-aminocyclopropane-1-carboxylic acid (ACC) which were virtually ineffective, inhibited C₂H₄ production, and this inhibition was nullified by ACC. Inhibition by CCA is not competitive with ACC since there is a decline, rather than an increase, in native endogenous ACC in the presence of CCA. Similarly, short-chain organic acids from acetic to butyric acid and α-aminoisobutyric acid inhibited C₂H₄ production at 1 to 5 millimolar and lowered endogenous ACC levels. These inhibitions, like that of CCA, were overcome with ACC. Inhibitors of electron transfer and oxidative phosphorylation effectively inhibited ACC conversion to C₂H₄ in pea and apple tissues. The most potent inhibitors were 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) which virtually eliminated ACC-stimulated C₂H₄ production in both tissues. Still other inhibitors of the conversion of ACC to C₂H₄ were putative free radical scavengers which reduced chemiluminescence in the free radical-activated luminol reaction. These inhibitor studies suggest the involvement of a free radical in the reaction sequence which converts ACC to C₂H₄. Additionally, the potent inhibition of this reaction by uncouplers of oxidative phosphorylation (DNP and CCCP) suggest the involvement of ATP or the necessity for an intact membrane for C₂H₄ production from ACC. In the latter case, CCCP may be acting as a proton ionophore to destroy the membrane integrity necessary for C₂H₄ production.     

Enhancing the Production of Hydroxyl Radicals by Pleurotus eryngii via Quinone Redox Cycling for Pollutant Removal

The induction of hydroxyl radical (OH) production via quinone redox cycling in white-rot fungi was investigated to improve pollutant degradation. In particular, we examined the influence of 4-methoxybenzaldehyde (anisaldehyde), Mn²⁺, and oxalate on Pleurotus eryngii OH generation. Our standard quinone redox cycling conditions combined mycelium from laccase-producing cultures with 2,6-dimethoxy-1,4-benzoquinone (DBQ) and Fe³⁺-EDTA. The main reactions involved in OH production under these conditions have been shown to be (i) DBQ reduction to hydroquinone (DBQH₂) by cell-bound dehydrogenase activities; (ii) DBQH₂ oxidation to semiquinone (DBQ⁻) by laccase; (iii) DBQ⁻ autoxidation, catalyzed by Fe³⁺-EDTA, producing superoxide (O₂⁻) and Fe²⁺-EDTA; (iv) O₂⁻ dismutation, generating H₂O₂; and (v) the Fenton reaction. Compared to standard quinone redox cycling conditions, OH production was increased 1.2- and 3.0-fold by the presence of anisaldehyde and Mn²⁺, respectively, and 3.1-fold by substituting Fe³⁺-EDTA with Fe³⁺-oxalate. A 6.3-fold increase was obtained by combining Mn²⁺ and Fe³⁺-oxalate. These increases were due to enhanced production of H₂O₂ via anisaldehyde redox cycling and O₂⁻ reduction by Mn²⁺. They were also caused by the acceleration of the DBQ redox cycle as a consequence of DBQH₂ oxidation by both Fe³⁺-oxalate and the Mn³⁺ generated during O₂⁻ reduction. Finally, induction of OH production through quinone redox cycling enabled P. eryngii to oxidize phenol and the dye reactive black 5, obtaining a high correlation between the rates of OH production and pollutant oxidation.The degradation of lignin and pollutants by white-rot fungi is an oxidative and rather nonspecific process based on the production of substrate free radicals (36). These radicals are produced by ligninolytic enzymes, including laccase and three kinds of peroxidases: lignin peroxidase, manganese peroxidase, and versatile peroxidase (VP) (23). The H₂O₂ required for peroxidase activities is provided by several oxidases, such as glyoxal oxidase and aryl-alcohol oxidase (AAO) (9, 18). This free-radical-based degradative mechanism leads to the production of a broad variety of oxidized compounds. Common lignin depolymerization products are aromatic aldehydes and acids, and quinones (34). In addition to their high extracellular oxidation potential, white-rot fungi show strong ability to reduce these lignin depolymerization products, using different intracellular and membrane-bound systems (4, 25, 39). Since reduced electron acceptors of oxidized compounds are donor substrates for the above-mentioned oxidative enzymes, the simultaneous actions of both systems lead to the establishment of redox cycles (35). Although the function of these redox cycles is not fully understood, they have been hypothesized to be related to further metabolism of lignin depolymerization products that require reduction to be converted in substrates of the ligninolytic enzymes (34). A second function attributed to these redox cycles is the production of reactive oxygen species, i.e., superoxide anion radicals (O₂⁻), H₂O₂, and hydroxyl radicals (OH), where lignin depolymerization products and fungal metabolites act as electron carriers between intracellular reducing equivalents and extracellular oxygen. This function has been studied in Pleurotus eryngii, whose ligninolytic system is composed of laccase (26), VP (24), and AAO (9). Incubation of this fungus with different aromatic aldehydes has been shown to provide extracellular H₂O₂ on a constant basis, due to the establishment of a redox cycle catalyzed by an intracellular aryl-alcohol dehydrogenase (AAD) and the extracellular AAO (7, 10). The process was termed aromatic aldehyde redox cycling, and 4-methoxybenzaldehyde (anisaldehyde) serves as the main Pleurotus metabolite acting as a cycle electron carrier (13). A second cyclic system, involving a cell-bound quinone reductase activity (QR) and laccase, was found to produce O₂⁻ and H₂O₂ during incubation of P. eryngii with different quinones (11). The process was described as the cell-bound divalent reduction of quinones (Q) by QR, followed by extracellular laccase oxidation of hydroquinones (QH₂) into semiquinones (Q⁻), which autoxidized to some extent, producing O₂⁻ (Q⁻ + O₂ ⇆ Q + O₂⁻). H₂O₂ was formed by O₂⁻ dismutation (O₂⁻ + HO₂ + H⁺ → O₂ + H₂O₂). In an accompanying paper, we describe the extension of this O₂⁻ and H₂O₂ generation mechanism to OH radical production by the addition of Fe³⁺-EDTA to incubation mixtures of several white-rot fungi with different quinones (6). Among them, those derived from 4-hydroxyphenyl, guaiacyl, and syringyl lignin units were used: 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), and 2,6-dimethoxy-1,4-benzoquinone (DBQ), respectively. Semiquinone autoxidation under these conditions was catalyzed by Fe³⁺-EDTA instead of being a direct electron transfer to O₂. The intermediate Fe²⁺-EDTA reduced not only O₂, but also H₂O₂, leading to OH radical production by the Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺).Although OH radicals are the strongest oxidants produced by white-rot fungi (2, 14), studies of their involvement in pollutant degradation are quite scarce. In this context, the objectives of this study were to (i) determine possible factors enhancing the production of OH radicals by P. eryngii via quinone redox cycling and (ii) test the validity of this inducible OH production mechanism as a strategy for pollutant degradation. Our selection of possible OH production promoters was guided by two observations (6). First, the redox cycle of benzoquinones working with washed P. eryngii mycelium is rate limited by hydroquinone oxidation, since the amounts of the ligninolytic enzymes that remained bound to the fungus under these conditions were not large. Second, H₂O₂ is the limiting reagent for OH production by the Fenton reaction.With these considerations in mind, anisaldehyde and Mn²⁺ were selected to increase H₂O₂ production. As mentioned above, anisaldehyde induces H₂O₂ production in P. eryngii via aromatic aldehyde redox cycling (7). Mn²⁺ has been shown to enhance H₂O₂ production during the oxidation of QH₂ by P. eryngii laccase by reducing the O₂⁻ produced in the semiquinone autoxidation reaction (Mn²⁺ + O₂⁻ → Mn³⁺ + H₂O₂ + 2 H⁺) (26). Mn²⁺ was also selected to increase the hydroquinone oxidation rate, since this reaction has been shown to be propagated by the Mn³⁺ generated in the latter reaction (QH₂ + Mn³⁺ → Q⁻ + Mn²⁺ + 2 H⁺). The replacement of Fe³⁺-EDTA by Fe³⁺-oxalate was also planned in order to increase the QH₂ oxidation rate above that resulting from the action of laccase. Oxalate is a common extracellular metabolite of wood-rotting fungi to which the function of chelating iron and manganese has been attributed (16, 45). The use of Fe³⁺-oxalate and nonchelated Fe³⁺, both QH₂ oxidants, has been proven to enable quinone redox cycling in fungi that do not produce ligninolytic enzymes, such as the brown-rot fungus Gloeophyllum trabeum (17, 40, 41). Finally, phenol and the azo dye reactive black 5 (RB5) were selected as model pollutants.     

Laccase-Catalyzed Oxidation of Mn2+ in the Presence of Natural Mn3+ Chelators as a Novel Source of Extracellular H2O2 Production and Its Impact on Manganese Peroxidase

A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kDa oxidized Mn²⁺ to Mn³⁺, as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn³⁺ complexes were produced at 0.15, 0.05, and 0.10 μmol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H₂O₂ formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H₂O₂ production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn²⁺ oxidation and abiotic decomposition of these organic chelators by the resulting Mn³⁺, which leads to formation of superoxide and its subsequent reduction to H₂O₂. A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn³⁺ complexes in assays containing 1 mM Mn²⁺ and 100 mM oxalate or malonate, but omitting an additional H₂O₂ source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn²⁺ oxidation in providing H₂O₂ for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.     

Induction of Extracellular Hydroxyl Radical Production by White-Rot Fungi through Quinone Redox Cycling

A simple strategy for the induction of extracellular hydroxyl radical (OH) production by white-rot fungi is presented. It involves the incubation of mycelium with quinones and Fe³⁺-EDTA. Succinctly, it is based on the establishment of a quinone redox cycle catalyzed by cell-bound dehydrogenase activities and the ligninolytic enzymes (laccase and peroxidases). The semiquinone intermediate produced by the ligninolytic enzymes drives OH production by a Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺). H₂O₂ production, Fe³⁺ reduction, and OH generation were initially demonstrated with two Pleurotus eryngii mycelia (one producing laccase and versatile peroxidase and the other producing just laccase) and four quinones, 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and 2-methyl-1,4-naphthoquinone (menadione [MD]). In all cases, OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H₂O₂ levels and Fe³⁺ reduction rates observed with the four quinones. Between the two P. eryngii mycelia used, the best results were obtained with the one producing only laccase, showing higher OH production rates with added purified enzyme. The strategy was then validated in Bjerkandera adusta, Phanerochaete chrysosporium, Phlebia radiata, Pycnoporus cinnabarinus, and Trametes versicolor, also showing good correlation between OH production rates and the kinds and levels of the ligninolytic enzymes expressed by these fungi. We propose this strategy as a useful tool to study the effects of OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.White-rot fungi are unique in their ability to degrade a wide variety of organopollutants (36, 47), mainly due to the secretion of a low-specificity enzyme system whose natural function is the degradation of lignin (11). Components of this system include laccase and/or one or two types of peroxidase, such as lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP) (31). Besides acting directly, the ligninolytic enzymes can bring about lignin and pollutant degradation through the generation of low-molecular-weight extracellular oxidants, including (i) Mn³⁺, (ii) free radicals from some fungal metabolites and lignin depolymerization products (7, 22), and (iii) oxygen free radicals, mainly hydroxyl radicals (OH) and lipid peroxidation radicals (21). Although OH radicals are the strongest oxidants found in cultures of white-rot fungi (1), studies of their involvement in pollutant degradation are scarce. One of the reasons is that the mechanisms proposed for OH production still await in vivo validation.Several potential sources of extracellular OH based on the Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺) have been postulated for white-rot fungi. In one case, an extracellular fungal glycopeptide has been shown to reduce O₂ and Fe³⁺ to H₂O₂ and Fe²⁺ (45). Enzymatic sources include cellobiose dehydrogenase, LiP, and laccase. Among these, only cellobiose dehydrogenase is able to directly catalyze the formation of Fenton''s reagent (33). The ligninolytic enzymes, however, act as an indirect source of OH through the generation of Fe³⁺ and O₂ reductants, such as formate (CO₂⁻) and semiquinone (Q⁻) radicals. The first time evidence was provided that a ligninolytic enzyme was involved in OH production, oxalate was used to generate CO₂⁻ in a LiP reaction mediated by veratryl alcohol (4). The proposed mechanism consisted of the following cascade of reactions: production of veratryl alcohol cation radical (Valc⁺) by LiP, oxidation of oxalate to CO₂⁻ by Valc⁺, reduction of O₂ to O₂⁻ by CO₂⁻, and a superoxide-driven Fenton reaction (Haber-Weiss reaction) in which Fe³⁺ was reduced by O₂⁻. The OH production mechanism assisted by Q⁻ was inferred from the oxidation of 2-methoxy-1,4-benzohydroquinone (MBQH₂) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH₂) by Pleurotus eryngii laccase in the presence of Fe³⁺-EDTA. The ability of Q⁻ radicals to reduce both Fe³⁺ to Fe²⁺ and O₂ to O₂⁻, which dismutated to H₂O₂, was demonstrated (14). In this case, OH radicals were generated by a semiquinone-driven Fenton reaction, as Q⁻ radicals were the main agents accomplishing Fe³⁺ reduction. The first evidence of the likelihood of this OH production mechanism being operative in vivo had been obtained from incubations of P. eryngii with 2-methyl-1,4-naphthoquinone (menadione [MD]) and Fe³⁺-EDTA (15). Extracellular OH radicals were produced on a constant basis through quinone redox cycling, consisting of the reduction of MD by a cell-bound quinone reductase (QR) system, followed by the extracellular oxidation of the resulting hydroquinone (MDH₂) to its semiquinone radical (MD⁻). The production of extracellular O₂⁻ and H₂O₂ by P. eryngii via redox cycling involving laccase was subsequently confirmed using 1,4-benzoquinone (BQ), 2-methyl-1,4-benzoquinone, and 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone), in addition to MD (16). However, the demonstration of OH production based on the redox cycling of quinones other than MD was still required.In the present paper, we describe the induction of extracellular OH production by P. eryngii upon its incubation with BQ, 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and MD in the presence of Fe³⁺-EDTA. The three benzoquinones were selected because they are oxidation products of p-hydroxyphenyl, guaiacyl, and syringyl units of lignin (MD was included as a positive control). Along with laccase, the involvement of P. eryngii VP in the production of O₂⁻ and H₂O₂ from hydroquinone oxidation has also been reported (13). Since hydroquinones are substrates of all known ligninolytic enzymes, quinone redox cycling catalysis could involve any of them. Here, we demonstrate OH production by P. eryngii under two different culture conditions, leading to the production of laccase or laccase and VP. We also show that quinone redox cycling is widespread among white-rot fungi by using a series of well-studied species that produce different combinations of ligninolytic enzymes.     

NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri

It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP⁺ with reduced ferredoxin (Fd_red) and the endergonic reduction of NADP⁺ with NADH in a reversible reaction: Fd_red²⁻ + NADH + 2 NADP⁺ + H⁺ = Fd_ox + NAD⁺ + 2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H₂ (reaction 1) and in deriving a large (30%) portion of its cell carbon from CO₂. Both the energy metabolism and the pathways of biosynthesis have therefore been the subject of many investigations (for relevant literature, see references 12 and 27). (1)During growth of C. kluyveri on ethanol and acetate, approximately five ethanol and four acetate molecules are converted to three butyrate molecules and one caproate molecule (reaction 1a), and one ethanol molecule is oxidized to one acetate⁻, one H⁺, and two H₂ (reaction 1b) molecules (23, 31). How exergonic reaction 1a is coupled with endergonic reaction 1b and with ATP synthesis from ADP and P_i (ΔG^o′ = +32 kJ/mol) has remained unclear for many years. (1a) (1b)We recently showed (12) that, in Clostridium kluyveri, the exergonic reduction of crotonyl-coenzyme A (crotonyl-CoA) (E_o′ = −10 mV) with NADH (E_o′ = −320 mV) involved in reaction 1a is coupled with the endergonic reduction of ferredoxin (Fd_ox) (E_o′ = −420 mV) with NADH (E_o′ = −320 mV) involved in reaction 1b via the recently proposed mechanism of flavin-based electron bifurcation (7). The coupling reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase-EtfAB complex (reaction 2) (12): (2)The reduced ferredoxin (Fd_red²⁻) is assumed to be used for rereduction of NAD⁺ via a membrane-associated, proton-translocating ferredoxin:NAD oxidoreductase (RnfABCDEG) (reaction 3), and the proton motive force thus generated is assumed to drive the phosphorylation of ADP via a membrane-associated F₁F₀ ATP synthetase (reaction 4): (3) (4)The novel coupling mechanism represented by reactions 2 and 3 allowed for the first time the possibility of formulating a metabolic scheme for the ethanol-acetate fermentation that could account for the observed fermentation products and growth yields and thus for the observed ATP gains (27). One issue, however, remained open, namely, why the formation of butyrate from ethanol and acetate in the fermentation involves both an NADP⁺- and an NAD⁺-specific β-hydroxybutyryl-CoA dehydrogenase (16), considering that, in the oxidative part of the fermentation (ethanol oxidation to acetyl-CoA), only NADH is generated (8, 9, 13).The presence of a reduced ferredoxin:NADP⁺ oxidoreductase was proposed based on results of enzymatic studies performed 40 years ago. Cell extracts of Clostridium kluyveri were found to catalyze the formation of H₂ from NADPH in a ferredoxin- and NAD⁺-dependent reaction (34). The results were interpreted to indicate that C. kluyveri contains a ferredoxin-dependent hydrogenase and an NADPH:ferredoxin oxidoreductase with transhydrogenase activity. H₂ formation from NADPH was strictly dependent on the presence of NAD⁺ and was inhibited by NADH, inhibition being competitive with the presence of NAD⁺, indicating that ferredoxin reduction with NADPH is under the allosteric control of the NAD⁺/NADH couple. The cell extracts also catalyzed the NADH-dependent reduction of NADP⁺ with reduced ferredoxin (21, 34). Purification of the enzyme catalyzing these reactions was not achieved, and no function in the energy metabolism of C. kluyveri was assigned.In this communication, we report on the properties of the recombinant enzyme that catalyzes the NAD⁺-dependent reduction of ferredoxin with NADPH and the NADH-dependent reduction of NADP⁺ with reduced ferredoxin and show that the cytoplasmic heterodimeric enzyme couples the exergonic reduction of NADP⁺ with reduced ferredoxin with the endergonic reduction of NADP⁺ with NADH in a fully reversible reaction. The transhydrogenation reaction is endergonic, because in vivo the NADH/NAD⁺ ratio is generally near 0.3 and the NADPH/NADP⁺ ratio is generally above 1 (2, 30). (5)NADP⁺ reduction is most probably the physiological function of the enzyme, which is why we chose the abbreviation NfnAB (for NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase).     

In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP_n + AMP → ADP + polyP_n−1. The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2′-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2′-dAMP were efficiently phosphorylated to ADP and 2′-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl₂, polyP_n=35, and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2′-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

Reinvestigation of the steady-state kinetics and physiological function of the soluble NiFe-hydrogenase I of Pyrococcus furiosus

Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H₂ evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H₂ production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low K_m for H₂ (~20 μM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 μM dissolved CO). Since the K_m for NADP⁺ is ~37 μM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.     

Fluorescence Quenching and Gas Exchange in a Water Stressed C(3) Plant, Digitalis lanata

A leaf cuvette has been adapted for use with a pulse-modulation fluorometer and an open gas exchange system. Leaf water potential (ψ) was decreased by withholding watering from Digitalis lanata EHRH. plants. At different stages of water deficiency the photochemical (q_Q) and nonphotochemical (q_E) fluorescence quenching was determined during the transition between darkness and light-induced steady state photosynthesis of the attached leaves. In addition, the steady state CO₂ and H₂O gas exchange was recorded. Following a decrease of leaf water potential with increasing water deficiency, the transition of photochemical quenching was almost unaffected, whereas nonphotochemical quenching increased. This is indicative of an enhanced thylakoid membrane energization during the transition and is interpreted as a partial inhibition of either the ATP generating or the ATP consuming reaction sequences. Complete reversion of the stress induced changes was achieved within 6 hours after rewatering. In contrast to the variations during transition, the final steady state values of q_Q and q_E remained unchanged over the entire stress range from −0.7 to −2.5 megapascals. From these results we conclude that, once established, electron transport via photosystem II and the transmembrane proton gradient remain unaffected by water stress. These data are indicative of a protective mechanism against photoinhibition during stress, when net CO₂ uptake is limited.     

Motility of Marichromatium gracile in Response to Light, Oxygen, and Sulfide

The motility of the purple sulfur bacterium Marichromatium gracile was investigated under different light regimes in a gradient capillary setup with opposing oxygen and sulfide gradients. The gradients were quantified with microsensors, while the behavior of swimming cells was studied by video microscopy in combination with a computerized cell tracking system. M. gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen and sulfide. The observed migration patterns could be explained solely by the various phobic responses. In the dark, M. gracile formed an ~500-μm-thick band at the oxic-anoxic interface, with a sharp border toward the oxic zone always positioned at ~10 μM O₂. Flux calculations yielded a molar conversion ratio S_tot/O₂ of 2.03:1 (S_tot = [H₂S] + [HS⁻] + [S²⁻]) for the sulfide oxidation within the band, indicating that in darkness the bacteria oxidized sulfide incompletely to sulfur stored in intracellular sulfur globules. In the light, M. gracile spread into the anoxic zone while still avoiding regions with >10 μM O₂. The cells also preferred low sulfide concentrations if the oxygen was replaced by nitrogen. A light-dark transition experiment demonstrated a dynamic interaction between the chemical gradients and the cell's metabolism. In darkness and anoxia, M. gracile lost its motility after ca. 1 h. In contrast, at oxygen concentrations of >100 μM with no sulfide present the cells remained viable and motile for ca. 3 days both in light and darkness. Oxygen was respired also in the light, but respiration rates were lower than in the dark. Observed aggregation patterns are interpreted as effective protection strategies against high oxygen concentrations and might represent first stages of biofilm formation.     

Reversibility of H+-ATPase and H+-Pyrophosphatase in Tonoplast Vesicles from Maize Coleoptiles and Seeds

Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H⁺-ATPase and H⁺-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to ³²Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H⁺ pumps showed that the H⁺-ATPase was more active than H⁺-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H⁺-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: II. H/ATP Stoichiometry of an Anion-Sensitive H-ATPase

The H⁺/ATP stoichiometry was determined for an anion-sensitive H⁺-ATPase in membrane vesicles believed to be derived from tonoplast. Initial rates of proton influx were measured by monitoring the alkalinization of a weakly buffered medium (pH 6.13) following the addition of ATP to a suspension of membrane vesicles of Beta vulgaris L. Initial rates of ATP hydrolysis were measured in an assay where ATP hydrolysis is coupled to NADH oxidation and monitored spectrophotometrically (A₃₄₀) or by monitoring the release of ³²P from [γ-³²P]ATP. Inasmuch as this anion-sensitive H⁺-ATPase is strongly inhibited by NO₃⁻, initial rates of H⁺ influx and ATP hydrolysis were measured in the absence and presence of NO₃⁻ to account for ATPase activity not involved in H⁺ transport. The NO₃⁻-sensitive activities were calculated and used to estimate the ratio of H⁺ transported to ATP hydrolyzed. These measurements resulted in an estimate of the H⁺/ATP stoichiometry of 1.96 ± 0.14 suggesting that the actual stoichiometry is 2 H⁺ transported per ATP hydrolyzed. When compared with the reported values of the electrochemical potential gradient for H⁺ across the tonoplast measured in vivo, our result suggests that the H⁺-ATPase does not operate near equilibrium but is regulated by cellular factors other than energy supply.     

Relationship between Acid Tolerance, Cytoplasmic pH, and ATP and H+-ATPase Levels in Chemostat Cultures of Lactococcus lactis

The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pH_o). A decrease in either the dilution rate or the pH_o led to a decrease in the cytoplasmic pH (pH_i) of the cells, and similar levels of acid tolerance were observed at any specific pH_i irrespective of whether the pH_i resulted from manipulation of the growth rate, manipulation of the pH_o, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pH_o of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pH_o of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pH_i. The rate of ATP formation was not correlated with pH_i, but a good correlation between the cellular level of H⁺-ATPase and pH_i was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pH_i was established. Each pH_i below 6.6 was characterized by unique levels of ATR, H⁺-ATPase, and ATP. High levels of H⁺-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H⁺-ATPase is one of the ATR proteins induced by acid pH_i through growth at an acid pH_o or a slow growth rate.     

Engineered Respiro-Fermentative Metabolism for the Production of Biofuels and Biochemicals from Fatty Acid-Rich Feedstocks

Although lignocellulosic sugars have been proposed as the primary feedstock for the biological production of renewable fuels and chemicals, the availability of fatty acid (FA)-rich feedstocks and recent progress in the development of oil-accumulating organisms make FAs an attractive alternative. In addition to their abundance, the metabolism of FAs is very efficient and could support product yields significantly higher than those obtained from lignocellulosic sugars. However, FAs are metabolized only under respiratory conditions, a metabolic mode that does not support the synthesis of fermentation products. In the work reported here we engineered several native and heterologous fermentative pathways to function in Escherichia coli under aerobic conditions, thus creating a respiro-fermentative metabolic mode that enables the efficient synthesis of fuels and chemicals from FAs. Representative biofuels (ethanol and butanol) and biochemicals (acetate, acetone, isopropanol, succinate, and propionate) were chosen as target products to illustrate the feasibility of the proposed platform. The yields of ethanol, acetate, and acetone in the engineered strains exceeded those reported in the literature for their production from sugars, and in the cases of ethanol and acetate they also surpassed the maximum theoretical values that can be achieved from lignocellulosic sugars. Butanol was produced at yields and titers that were between 2- and 3-fold higher than those reported for its production from sugars in previously engineered microorganisms. Moreover, our work demonstrates production of propionate, a compound previously thought to be synthesized only by propionibacteria, in E. coli. Finally, the synthesis of isopropanol and succinate was also demonstrated. The work reported here represents the first effort toward engineering microorganisms for the conversion of FAs to the aforementioned products.Concerns about climate change and the depletion and cost of petroleum resources have ignited interest in the establishment of a bio-based industry (5, 49, 61), and the conceptual model of a biorefinery has emerged (27, 28, 45). Given its abundance in nature, the carbohydrate portion of edible crops such as sugarcane, sugar beet, maize (corn), and sorghum is currently used as the primary feedstock in the biological production of fuels and chemicals (12, 49, 52). Although the use of nonedible lignocellulosic sugars has been proposed as an efficient and sustainable avenue to the aforementioned processes, the availability of fatty acid (FA)-rich feedstocks and recent progress in the development of oil-accumulating organisms make FAs an attractive alternative. Edible oil-rich crops such as rapeseed, sunflower, soybean, and palm are currently available and widely used as feedstocks for chemical conversion to biodiesel (6), while oleaginous algae and nonedible FA-rich crops along with industrial by-products are receiving greater attention as longer-term alternatives. These nonedible FA-rich feedstocks are presently generated in large amounts and can be exploited for the biological production of fuels and chemicals (14, 22, 51, 56, 57). Unfortunately, microbial platforms to enable this are at present almost absent.FAs not only are abundant but also offer several advantages when used for fuel and chemical production. For example, their metabolism to the key intermediate metabolite acetyl coenzyme A (acetyl-CoA) is very efficient, as it results in 100% carbon recovery (Fig. ​(Fig.1).1). Since many fuels and chemicals can be derived from acetyl-CoA, high yields can be realized if FAs are used as the carbon source. In contrast, sugar metabolism generates one molecule of carbon dioxide (or formate) per molecule of acetyl-CoA, limiting the yield of products derived from acetyl-CoA (Fig. ​(Fig.1).1). The product yield advantage of FAs over sugars is also supported by the more highly reduced nature of their carbon atoms. Table ​Table11 provides a comparison of maximum theoretical yields, on both weight and carbon bases, for the production of biofuels and biochemicals from FAs and lignocellulosic sugars. Maximum theoretical yields have been calculated from stoichiometry based on the pathways shown in Fig. ​Fig.11 for the utilization of FAs and glucose, the synthesis of products, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. The stoichiometric coefficients were obtained by conducting elemental balances on carbon, hydrogen, and oxygen, and an ATP balance was also included in the analysis. As an example, when production of biofuels (e.g., ethanol and butanol) is considered, utilization of FAs (e.g., palmitic acid [C_16:0]) as a substrate returns product yields 2.7-fold (wt/wt) or 1.4-fold (C/C) higher than those for sugars (calculations are provided for glucose but are equally valid for other lignocellulosic sugars). Although the current prices of feedstocks on a weight basis are comparable (lower than 20¢/pound), the data reported in Fig. S1a in the supplemental material show that the price per carbon for glucose derived from corn is remarkably higher. Regardless of the basis used for calculations (i.e., weight or carbon basis), when maximum theoretical yields and costs of FA and sugar feedstocks are accounted for, the advantages of using FAs are remarkable (see Fig. S1b in the supplemental material).Open in a separate windowFIG. 1.Pathways engineered in E. coli for the conversion of fatty acids to fuels (red) and chemicals (green). Also shown is the catabolism of fatty acids via the β-oxidation pathway (orange) and of glucose through the Embden-Meyerhof-Parnas pathway (blue). Relevant reactions are represented by the names of the genes coding for the enzymes (E. coli genes unless otherwise specified in parentheses as follows: C. acetobutylicum, ca; C. beijerinckii, cb): aceA, isocitrate lyase; aceB, malate synthase A; adc, acetoacetate decarboxylase (ca); ackA, acetate kinase; adh, secondary alcohol dehydrogenase (cb); adhE, acetaldehyde/alcohol dehydrogenase; adhE2, secondary alcohol dehydrogenase (ca); atoA and atoD, acetyl-CoA:acetoacetyl-CoA transferase; atoB, acetyl-CoA acetyltransferase; bcd, butyryl-CoA dehydrogenase (ca); crt, crotonase (ca); etfAB, electron transfer flavoprotein (ca); fadA, 3-ketoacyl-CoA thiolase; fadB, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; fadD, acyl-CoA synthetase; fadE, acyl-CoA dehydrogenase; hbd, β-hydroxybutyryl-CoA dehydrogenase (ca); icd, isocitrate dehydrogenase; pta, phosphate acetyltransferase; sdhABCD, succinate dehydrogenase; scpA, methylmalonyl-CoA mutase; scpB, methylmalonyl-CoA decarboxylase; scpC, propionyl-CoA:succinate CoA transferase; sucA, 2-oxoglutarate dehydrogenase; sucB, dihydrolipoyltranssuccinylase; and sucCD, succinyl-CoA synthetase. Abbreviations: 2[H] = NADH = FADH₂ = QH₂ = H₂; P/O, amount of ATP produced per oxygen consumed in the oxidative phosphorylation.

TABLE 1.

Comparison of maximum theoretical yields for the production of biofuels and biochemicals from fatty acids (palmitic acid) and lignocellulosic sugars (glucose)

Adenosine Triphosphate Stimulates Aquifex aeolicus MutL Endonuclease Activity

Background

Human PMS2 (hPMS2) homologues act to nick 5′ and 3′ to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn⁺⁺ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)₂E(X)₄E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.

Methodologies/Principal Findings

We examined the effect ATP had on the Mn⁺⁺ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL) proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6±0.08×10⁻⁵ s⁻¹ and 4.2±0.3×10⁻⁵ s⁻¹ in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X)₂E(X)₄E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.

Conclusions

ATP stimulated the Mn⁺⁺ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X)₂E(X)₄E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn⁺⁺ induced nicking activity.     

The NADP-Dependent Methylene Tetrahydromethanopterin Dehydrogenase in Methylobacterium extorquens AM1

An NADP-dependent methylene tetrahydromethanopterin (H₄MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO₂ in Methylobacterium extorquens AM1. We report here on the purification of this novel enzyme to apparent homogeneity. Via the N-terminal amino acid sequence, it was identified to be the mtdA gene product. The purified enzyme catalyzed the dehydrogenation of methylene H₄MPT with NADP⁺ rather than with NAD⁺, with a specific activity of approximately 400 U/mg of protein. It also catalyzed the dehydrogenation of methylene tetrahydrofolate (methylene H₄F) with NADP⁺. With methylene H₄F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (V_max/K_m) were approximately 20-fold lower than with methylene H₄MPT. Whereas the dehydrogenation of methylene H₄MPT (E₀ = −390 mV) with NADP⁺ (E₀ = −320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H₄F (E₀ = −300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase from M. extorquens AM1 with those of methylene H₄F dehydrogenases from other bacteria and eucarya and with those of methylene H₄MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).     

Oxygen Activation during Oxidation of Methoxyhydroquinones by Laccase from Pleurotus eryngii

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH₂) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH₂) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH₂ more efficiently than it oxidized MBQH₂; both the affinity and maximal velocity of oxidation were higher for DBQH₂ than for MBQH₂. Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q^·− + O₂ ↔ Q + O₂^·−). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O₂^·− on quinone formation rates. Then, the production of H₂O₂ in laccase reactions, as a consequence of O₂^·− dismutation, confirmed that semiquinones autoxidized. The highest H₂O₂ levels were obtained with DBQH₂, indicating that DBQ^·− autoxidized to a greater extent than did MBQ^·−. Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O₂^·− consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O₂^·−, besides undergoing dismutation, reacts with Mn²⁺, Fe³⁺, and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 μM concentrations of MBQ^·− and DBQ^·− from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ^·− autoxidation rose to 13.8% and that of DBQ^·− increased to 39.9%.     

Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO₂ and either ethanol or lactate but not H₂ gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO₂ reduction, with a formate production rate and methanogenic turnover constant of 660 μM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 μM) than digestor contents, and the free flora was the primary site for formate cleavage to CO₂ and H₂ (90 μM formate per h). Inhibition of methanogenesis by CHCl₃ resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H₂ gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H₂ transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.