Purification and properties of N5, N10-methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg)

The reduction of N5,N10-methylenetrahydromethanopterin (CH2 = H4MPT) to N5-methyltetrahydromethanopterin (CH3-H4MPT) is an intermediate step in methanogenesis from CO2 and H2. The reaction is catalyzed by CH2 = H4MPT reductase. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) was found to be specific for reduced coenzyme F420 as electron donor; neither NADH or NADPH nor reduced viologen dyes could substitute for the reduced 5-deazaflavin. The reductase was purified over 100-fold to apparent homogeneity. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band at the 36-kDa position. The apparent molecular mass of the native enzyme was determined by gel filtration to be in the order of 150 kDa. The purified enzyme was colourless. It did not contain flavin or iron. The ultraviolet visible spectrum was almost identical to that of albumin, suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentration at different constant concentrations of the second substrate yielded straight lines intersecting at one point on the abscissa to the left of the vertical axis. This intersecting pattern is characteristic of a ternary complex catalytic mechanism. The Km for CH2 = H4MPT and for the reduced coenzyme F420 were determined to be 0.3 mM and 3 microM, respectively. Vmax was 6000 mumol.min-1.mg protein-1 (kcat = 3600 s-1). The CH2 = H4MPT reductase was stable in the presence of air; at 4 C less than 10% activity was lost within 24 h.     

Biochemical characterization of a dihydromethanopterin reductase involved in tetrahydromethanopterin biosynthesis in Methylobacterium extorquens AM1

During growth on one-carbon (C1) compounds, the aerobic alpha-proteobacterium Methylobacterium extorquens AM1 synthesizes the tetrahydromethanopterin (H4MPT) derivative dephospho-H4MPT as a C1 carrier in addition to tetrahydrofolate. The enzymes involved in dephospho-H4MPT biosynthesis have not been identified in bacteria. In archaea, the final step in the proposed pathway of H4MPT biosynthesis is the reduction of dihydromethanopterin (H2MPT) to H4MPT, a reaction analogous to the reaction of the bacterial dihydrofolate reductase. A gene encoding a dihydrofolate reductase homolog has previously been reported for M. extorquens and assigned as the putative H2MPT reductase gene (dmrA). In the present work, we describe the biochemical characterization of H2MPT reductase (DmrA), which is encoded by dmrA. The gene was expressed with a six-histidine tag in Escherichia coli, and the recombinant protein was purified by nickel affinity chromatography and gel filtration. Purified DmrA catalyzed the NAD(P)H-dependent reduction of H2MPT with a specific activity of 2.8 micromol of NADPH oxidized per min per mg of protein at 30 degrees C and pH 5.3. Dihydrofolate was not a substrate for DmrA at the physiological pH of 6.8. While the existence of an H2MPT reductase has been proposed previously, this is the first biochemical evidence for such an enzyme in any organism, including archaea. Curiously, no DmrA homologs have been identified in the genomes of known methanogenic archaea, suggesting that bacteria and archaea produce two evolutionarily distinct forms of dihydromethanopterin reductase. This may be a consequence of different electron donors, NAD(P)H versus reduced F420, used, respectively, in bacteria and methanogenic archaea.     

H2-forming methylenetetrahydromethanopterin dehydrogenase, a novel type of hydrogenase without iron-sulfur clusters in methanogenic archaea.

A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2 = H4MPT) to methenyltetrahydromethanopterin (CH identical to H4MPT+) and H2 and was therefore named H2-forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenase, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either H2 or CH2 = H4MPT. We report here that the purified enzyme from Methanobacterium thermoautotrophicum exhibits the following other unique properties: (a) the colorless protein with a specific activity of 2000 U/mg (Vmax) did not contain iron-sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene, nitrite, cyanide, or azide; (c) the enzyme did not catalyze an isotopic exchange between 3H2 and 1H+; (d) the enzyme catalyzed the reduction of CH identical to H4MPT+ with 3H2 generating [methylene-3H]CH2 = H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA-deduced amino acid sequence of the proteins from M. thermoautotrophicum and Methanopyrus kandleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxin-type iron-sulfur protein. Properties of the H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium wolfei are also described indicating that the enzyme from this methanogenic archaeon is very similar to the enzyme from M. thermoautotrophicum with respect both to molecular and catalytic properties.     

The role of tetrahydromethanopterin and cytoplasmic cofactor in methane synthesis.

A fraction previously isolated from acid-treated supernatant fraction of Methanobacterium thermoautotrophicum by DEAE-Sephadex chromatography [Sauer, Mahadevan & Erfle (1984) Biochem. J. 221, 61-97] which was absolutely required for methane synthesis, has been separated into two compounds, tetrahydromethanopterin (H4MPT) and an as-yet-unidentified cofactor we call 'cytoplasmic cofactor'. H4MPT was identified by its u.v. spectrum and by 13C- and 1H-n.m.r. spectroscopy. The reduction of 2-(methylthio)ethanesulphonic acid (CH3-S-CoM) to methane by the membrane fraction from M. thermoautotrophicum was completely dependent on the addition of cytoplasmic cofactor. Methane synthesis from CO2, however, was only partially dependent on cofactor addition, and 57% of the original activity was retained in its absence. The kinetics of 14C labelling were consistent with the scheme methyl-H4MPT----CH3-S-CoM----methane, as has been proposed. This is the first time that direct experimental evidence has been presented to show that the proposed methyl transfer from H4MPT to coenzyme M (HS-CoM) actually occurs.     

Betaine: New Oxidant in the Stickland Reaction and Methanogenesis from Betaine and l-Alanine by a Clostridium sporogenes-Methanosarcina barkeri Coculture

Growing and nongrowing cells of Clostridium sporogenes fermented betaine with l-alanine, l-valine, l-leucine, and l-isoleucine as electron donors in a coupled oxidation-reduction reaction (Stickland reaction). For the substrate combinations betaine and l-alanine and betaine and l-valine balance studies were performed; the results were in agreement with the following fermentation equation: 1 R- CH(NH(2))-COOH + 2 betaine + 2 H(2)O --> 1 R-COOH + 1 CO(2) + 1 NH(3) + 2 trimethylamine + 2 acetate. Growth and production of trimethylamine were strictly dependent on the presence of selenite in the medium. With cell suspensions it was shown that C. sporogenes was unable to catabolize betaine as a single substrate. Betaine, however, was reduced to trimethylamine and acetate under an atmosphere of molecular hydrogen. For the reduction of betaine by cell extracts of C. sporogenes, dimercaptans such as 1,4-dithiothreitol could serve as electron donors. No betaine reductase activity was detected in cells grown in a complex medium without betaine. The pH optimum of betaine reductase was at pH 7.3. When C. sporogenes was cocultured with Methanosarcina barkeri strain Fusaro on betaine together with l-alanine, an almost complete conversion of the two substrates to CH(4), NH(3), and presumably CO(2) was observed.     

Dephosphorylation of the Rieske iron-sulfur protein after induction of the mitochondrial permeability transition

In the mitochondrial permeability transition (MPT), MPT pores open to cause the mitochondrial inner membrane to become non-selectively permeable to molecules of mass up to 1500 Da. In this study, we used proteomics to investigate protein changes after MPT induction. Isolated rat liver mitochondria were incubated with various MPT inducers, including CaCl2, tert-butylhydroperoxide, and phenylarsine oxide, in the presence and absence of the MPT inhibitor, cyclosporin A. MPT induction was confirmed by an absorbance swelling assay. Mitochondrial membrane proteins prepared from control and treated mitochondria were separated by two-dimensional (2D) gel electrophoresis and stained with SyproRuby or Coomassie blue. Proteins of interest were further identified by mass spectrometry. 2D gel electrophoresis by isoelectric focusing and SDS-PAGE consistently showed a protein spot that shifted to a more basic isoelectric point after the MPT. This shift was prevented by CsA but did not occur after protonophoric uncoupling. Mass spectrometry identified this protein as the Rieske iron-sulfur protein (RISP) of ubiquinol-cytochrome c reductase (Complex III). Phosphatase treatment of sonicated mitochondria caused the same shift in RISP as occurred in MPT inducer-treated mitochondria. 2D gel electrophoresis by blue-native-PAGE and SDS-PAGE showed that RISP existed as an apparent monomer in mitochondrial membranes in addition to forming a complex with ubiquinol-cytochrome c reductase. These findings suggest that RISP may be part of MPT pores and that dephosphorylation of RISP may play a role in regulation of the MPT.     

Crystal structure of methylenetetrahydromethanopterin reductase (Mer) in complex with coenzyme F420: Architecture of the F420/FMN binding site of enzymes within the nonprolyl cis-peptide containing bacterial luciferase family

Methylenetetratetrahydromethanopterin reductase (Mer) is involved in CO(2) reduction to methane in methanogenic archaea and catalyses the reversible reduction of methylenetetrahydromethanopterin (methylene-H(4)MPT) to methyl-H(4)MPT with coenzyme F(420)H(2), which is a reduced 5'-deazaflavin. Mer was recently established as a TIM barrel structure containing a nonprolyl cis-peptide bond but the binding site of the substrates remained elusive. We report here on the crystal structure of Mer in complex with F(420) at 2.6 A resolution. The isoalloxazine ring is present in a pronounced butterfly conformation, being induced from the Re-face of F(420) by a bulge that contains the non-prolyl cis-peptide bond. The bindingmode of F(420) is very similar to that in F(420)-dependent alcohol dehydrogenase Adf despite the low sequence identity of 21%. Moreover, binding of F(420) to the apoenzyme was only associated with minor conformational changes of the polypeptide chain. These findings allowed us to build an improved model of FMN into its binding site in bacterial luciferase, which belongs to the same structural family as Mer and Adf and also contains a nonprolyl cis-peptide bond in an equivalent position.     

N 5,N 10-Methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus

Methylene-H₄MPT reductase was found to be present in Archaeoglobus fulgidus in a specific activity of 1 U/mg. The reductase was purified 410-fold. The native enzyme showed an apparent molecular mass of approximately 200 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only 1 polypeptide of apparent molecular mass 35 kDa. The ultraviolet/visible spectrum of the reductase was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was dependent on reduced coenzyme F₄₂₀ as electron donor. Neither NADH, NADPH, nor reduced viologen dyes could substitute for the reduced deazaflavin. From reciprocal plots, which showed an intersecting patter, a K _m for methylene-H₄MPT of 16 M, a K _m for F₄₂₀H₂ of 4 M, and a V _max of 450 U/mg (K_cat=265 s^-1) were obtained. The enzyme was found to be rapidly inactivated when incubated at 80°C in 100 mM Tris/HCl pH 7. The rate of inactivation, however, decreased to essentially zero in the presence of either F₄₂₀ (0.2 mM), methylene-H₄MPT (0.2 mM), albumin (1 mg/ml), or KCl (0.5 M). The N-terminal amino acid sequence was determined and found to be similar to that of methylene-H₄MPT reductase (F₄₂₀-dependent) from the methanogens Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Methanopyrus kandleri. The purification and some properties of formylmethanofuran dehydrogenase from A. fulgidus are also described.Abbreviations H₄MPT tetrahydromethanopterin - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - CHH₄MPT methenyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - MFR methanofuran - CHO-MFR formyl-MFR - 1 U 1 mol/min     

Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

The sodium cycle in methanogenesis. CO2 reduction to the formaldehyde level in methanogenic bacteria is driven by a primary electrochemical potential of Na+ generated by formaldehyde reduction to CH4

CH4 formation from CO2 and H2 rather than from formaldehyde and H2 in methanogenic bacteria is inhibited by uncouplers, indicating that CO2 reduction to the formaldehyde level is energy-driven. We report here that in Methanosarcina barkeri the driving force is a primary electrochemical sodium potential (delta mu Na+) generated by formaldehyde reduction to CH4. This is concluded from the following findings. 1. CO2 reduction to CH4 was insensitive towards protonophores, when the Na+/H+ antiporter was inhibited; under these conditions delta mu Na+ was 120 mV (inside negative), whereas both delta mu H+ and the cellular ATP content were low. 2. CO2 reduction to CH4, rather than formaldehyde reduction, was sensitive towards Na+ ionophores, which dissipated delta mu Na+. 3. CO2 reduction to CH4, in the presence of protonophores and Na+/H+ antiport inhibitors, was coupled with the extrusion of 1-2 mol Na+/mol CH4, and formaldehyde reduction to CH4 was coupled with the extrusion of 3-4 mol Na+/mol CH4. Thus during CO2 reduction to the formaldehyde level 2-3 mol Na+ were consumed.     

Anaerobic oxidation of methane with sulfate: on the reversibility of the reactions that are catalyzed by enzymes also involved in methanogenesis from CO2

Anaerobic oxidation of methane (AOM) with sulfate is apparently catalyzed by an association of methanotrophic archaea (ANME) and sulfate-reducing bacteria. In many habitats, the free energy change (ΔG) available through this process is only -20 kJ/mol and therefore AOM with sulfate reduction generating life-supporting ATP is predicted to operate near thermodynamic equilibrium (ΔG=0 kJ/mol). On the basis of meta-genome sequencing and enzyme studies, it has been proposed that AOM in ANME is catalyzed by the same enzymes that catalyze CO2 reduction to CH4 in methanogenic archaea. Here, this proposal is reviewed and evaluated in terms of the process thermodynamics, kinetics, and enzyme reversibilities. Currently, there is no evidence for the presence of the gene that encodes methylene-tetrahydromethanopterin reductase in ANME, one of the central enzymes in the CO2 to CH4 pathway. However, all of the remaining enzymes do appear to be present and, with the exception of a coenzyme M-S-S-coenzyme B heterodisulfide reductase, all of these enzymes have been confirmed to catalyze reversible reactions.     

Proton-motive-force-driven formation of CO from CO2 and H2 in methanogenic bacteria

Cell suspensions of methanogenic bacteria (Methanosarcina barkeri, Methanospirillum hungatei, Methano-brevibacter arboriphilus, and Methanobacterium thermoautotrophicum) were found to form CO from CO2 and H2 according to the reaction: CO2 + H2----CO + H2O; delta G0 = +20 kJ/mol. Up to 15,000 ppm CO in the gas phase were reached which is significantly higher than the equilibrium concentration calculated from delta G0 (95 ppm under the experimental conditions). This indicated that CO2 reduction with H2 to CO is energy-driven and indeed the cells only generated CO when forming CH4. The coupling of the two reactions was studied in more detail with acetate-grown cells of M. barkeri using methanogenic substrates. The effects of the protonophore tetrachlorosalicylanilide (TCS) and of the proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide (cHxN)2C were determined. TCS completely inhibited CO formation from CO2 and H2 without affecting methanogenesis from CH3OH and H2. In the presence of the protonophore the proton motive force delta p and the intracellular ATP concentration were very low. (cHxN)2C, which partially inhibited methanogenesis from CH3OH and H2, had no effect on CO2 reduction to CO. In the presence of (cHxN)2C delta p was high and the intracellular ATP content was low. These findings suggest that the endergonic formation of CO from CO2 and H2 is coupled to the exergonic formation of CH4 from CH3OH and H2 via the proton motive force and not via ATP. CO formation was not stimulated by the addition of sodium ions.     

Methanofuran (carbon dioxide reduction factor), a formyl carrier in methane production from carbon dioxide in Methanobacterium

Methanofuran (carbon dioxide reduction factor) became labeled when incubated in cell extracts of Methanobacterium under hydrogen and 14CO2 in the absence of methanopterin. Proton NMR spectroscopy revealed that a formyl group was bound to the primary amine of methanofuran. [14C]Formylmethanofuran was enzymically converted to 14CH4 in the presence of CH3-S-CoM [2-(methylthio)ethanesulfonic acid], hydrogen, and methanopterin, establishing the formyl moiety as an intermediate in methanogenesis. In the absence of methanopterin, a substantial portion of the formyl label was oxidized to 14CO2 rather than reduced to 14CH4, consistent with a model in which the C1 intermediate is first bound to methanofuran and then to methanopterin, during its reduction. When CH3-S-CoM was replaced by HS-CoM (2-mercaptoethanesulfonic acid), most of the formyl label was oxidized to 14CO2, indicating that methyl group reduction by the CH3-S-CoM methylreductase is required for the conversion of formylmethanofuran to methane.     

Catalytic coupling of the active sites in acetyl-CoA synthase, a bifunctional CO-channeling enzyme.

Acetogenic bacteria contain acetyl-CoA synthase (ACS), an enzyme with two distinct nickel-iron-sulfur active sites connected by a tunnel through which CO migrates. One site reduces CO2 to CO, while the other synthesizes acetyl-CoA from CO, CoA, and the methyl group of another protein (CH3-CP). Rapid binding of CO2 and a two-electron reduction activates ACS. When CoA and CH3-CP bind ACS, CO is rerouted through the tunnel to the synthase site, and kinetic parameters at the reductase site are altered. Under these conditions, the rates of CO2 reduction and acetyl-CoA synthesis are synchronized by an ordered catalytic mechanism.     

Tetrahydromethanopterin, a carbon carrier in methanogenesis

Evidence obtained by 13C NMR spectroscopy indicates that tetrahydromethanopterin (H4MPT) serves as a carbon carrier for C1 units at the methine, methylene, and methyl levels of oxidation. All three derivatives of H4MPT served as substrates for methanogenesis by cell extracts under a hydrogen atmosphere; in each instance, methane evolved at a rate comparable to that obtained when 2-(methylthio)ethanesulfonic acid was used as the substrate. Each C1 derivative of H4MPT stimulated the reduction of CO2 as efficiently as 2-(methylthio)ethanesulfonic acid. High resolution fast atom bombardment mass spectrometry indicated that the product of the spontaneous reaction of formaldehyde with H4MPT was methylene-H4MPT, with the molecular formula C31H45N6O16P. 13C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methylene-H4MPT was bound to two nitrogen atoms. Molecular formulas of C31H44N6O16P and C31H47N6O16P were assigned to methenyl-H4MPT+, and methyl-H4MPT, by high resolution fast atom bombardment mass spectrometry. 1H NMR spectroscopy of methyl-H4MPT indicated that the methyl group was bound to a nitrogen atom. Sensitivity of each derivative to oxygen was noted. Apparent extinction coefficients of H4MPT and its derivatives were recorded. Evidence for the enzymatic synthesis of methylene-H4MPT from methenyl-H4MPT+ is presented.     

Characterization of a CO: heterodisulfide oxidoreductase system from acetate-grown Methanosarcina thermophila.

During the methanogenic fermentation of acetate by Methanosarcina thermophila, the CO dehydrogenase complex cleaves acetyl coenzyme A and oxidizes the carbonyl group (or CO) to CO2, followed by electron transfer to coenzyme M (CoM)-S-S-coenzyme B (CoB) and reduction of this heterodisulfide to HS-CoM and HS-CoB (A. P. Clements, R. H. White, and J. G. Ferry, Arch. Microbiol. 159:296-300, 1993). The majority of heterodisulfide reductase activity was present in the soluble protein fraction after French pressure cell lysis. A CO:CoM-S-S-CoB oxidoreductase system from acetate-grown cells was reconstituted with purified CO dehydrogenase enzyme complex, ferredoxin, membranes, and partially purified heterodisulfide reductase. Coenzyme F420 (F420) was not required, and CO:F420 oxidoreductase activity was not detected in cell extracts. The membranes contained cytochrome b that was reduced with CO and oxidized with CoM-S-S-CoB. The results suggest that a novel CoM-S-S-CoB reducing system operates during acetate conversion to CH4 and CO2. In this system, ferredoxin transfers electrons from the CO dehydrogenase complex to membrane-bound electron carriers, including cytochrome b, that are required for electron transfer to the heterodisulfide reductase. The cytochrome b was purified from solubilized membrane proteins in a complex with six other polypeptides. The cytochrome was not reduced when the complex was incubated with H2 or CO, and H2 uptake hydrogenase activity was not detected; however, the addition of CO dehydrogenase enzyme complex and ferredoxin enabled the CO-dependent reduction of cytochrome b.     

Consumption of methane and CO2 by methanotrophic microbial mats from gas seeps of the anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH(4) and CO(2) assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO(2) reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average delta(13)C carbon isotopic signature of -67.1 per thousand, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (-66.4 per thousand +/- 3.9 per thousand [mean +/- standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (-72.9 per thousand +/- 2.2 per thousand; n = 7). Incorporation of (14)C from radiolabeled CH(4) or CO(2) revealed one hot spot for methanotrophy and CO(2) fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with (14)CH(4) or (14)CO(2) revealed that there was interconversion of CH(4) and CO(2.) The level of CO(2) reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.     

Novel methylotrophy genes of Methylobacterium extorquens AM1 identified by using transposon mutagenesis including a putative dihydromethanopterin reductase

Ten novel methylotrophy genes of the facultative methylotroph Methylobacterium extorquens AM1 were identified from a transposon mutagenesis screen. One of these genes encodes a product having identity with dihydrofolate reductase (DHFR). This mutant has a C(1)-defective and methanol-sensitive phenotype that has previously only been observed for strains defective in tetrahydromethanopterin (H(4)MPT)-dependent formaldehyde oxidation. These results suggest that this gene, dmrA, may encode dihydromethanopterin reductase, an activity analogous to that of DHFR that is required for the final step of H(4)MPT biosynthesis.     

Novel reactions involved in energy conservation by methanogenic archaea.

Methanogenic archaea of the order Methanosarcinales which utilize C(1) compounds such as methanol, methylamines or H(2)+CO(2), employ two novel membrane-bound electron transport systems generating an electrochemical proton gradient: the H(2):heterodisulfide oxidoreductase and the F(420)H(2):heterodisulfide oxidoreductase. The systems are composed of the heterodisulfide reductase and either a membrane-bound hydrogenase or a F(420)H(2) dehydrogenase which is functionally homologous to the proton-translocating NADH dehydrogenase. Cytochromes and the novel electron carrier methanophenazine are also involved. In addition, the methyl-H(4)MPT:HS-CoM methyltransferase is bioenergetically relevant. The enzyme couples methyl group transfer with the translocation of sodium ions and seems to be present in all methanogens. The proton-translocating systems with the participation of cytochromes and methanophenazine have been found so far only in the Methanosarcinales.     

Photocatalytic reduction of carbon dioxide using sol-gel derived titania-supported CoPc catalysts.

CoPc loaded titania was synthesized by an improved sol-gel method using a homogeneous hydrolysis technique. The grain size of TiO2 and CoPc-TiO2 was uniform and average diameters were less than 20 nm. Both catalysts had two crystal phases, of which rutile accounted for about 28%. When they were used for the photoreduction of CO2 in NaOH solution under ambient conditions, the formate yield was significantly higher than that of the others, such as formaldehyde, methanol and so on. In order to increase the activity of the photocatalysts some factors were optimized, including the quantity of CoPc-TiO2 catalyst, concentration of Na(2)SO(3) hole scavenger, concentration of the NaOH solution and irradiation time. Under the optimal conditions, the gross photocatalytic reduction of CO2 was 1032 micromol (g cat)(-1). Furthermore, the yield of CH4 did not increase in the presence of CH(3)OH or HCHO, showing that the generation of CH4 from CO2 did not proceed via methanol as an intermediate under these conditions.