Methanolobus Tindarius Research Articles

Development of an efficient dimethylallyl diphosphate regeneration system by a co-immobilization of multi-enzyme cascade for the one-pot synthesis of prenylated flavonoids

Prenylated flavonoids are the primary modification of flavonoids and exhibit a diverse range of physiological activities. In this study, a co-immobilization of two-enzyme cascade was developed to regenerate dimethylallyl diphosphate (DMAPP). Shigella flexneri promiscuous kinase (SfPK) and Methanolobus tindarius isopentenyl phosphate kinase (MtIPK) were immobilized onto carboxymethyl cellulose magnetic nanoparticles (CMN) with a maximum load of 0.35 mg/mg and 0.28 mg/mg, respectively. The optimal activity of CMN-SfPK and CMN-MtIPK were at pH 9.5 and 55°C, and pH 7.0 and 35 °C, respectively. CMN-SfPK and CMN-MtIPK exhibited superior catalytic efficiency compared to free enzymes. CMN-SfPK was coupled with CMN-MtIPK to develop an efficient DMAPP regeneration system from prenol. Subsequently, SfPK, MtIPK and Aspergillus fumigatus prenyltransferase (AfPT) were co-immobilized on CMN to form CMN-SfPK-MtIPK-AfPT (CSMA) according to the optimal ratio. The 3’-C-prenylnaringenin production rate in CSMA reached 0.37 mmol/L/h, which was 1.85 times that of single-immobilized enzymes. Finally, the total production and production rate of 3’-C-prenylnaringenin in CSMA reached 2.55 mM and 0.255 mmol/L/h with 10 cycles. Therefore, the method described herein for efficient production of DMAPP and 3’-C-prenylnaringenin by using co-immobilized enzymes can be widely used for the prenylation of flavonoids.

Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade.

Dimethylallyl diphosphate (DMAPP) is a key intermediate metabolite in the synthesis of isoprenoids and is also the prenyl donor for biosynthesizing prenylated flavonoids. However, it is difficult to prepare DMAPP via chemical and enzymatic methods. In this study, three promiscuous kinases from Shigella flexneri (SfPK), Escherichia coli (EcPK), and Saccharomyces cerevisiae (ScPK) and three isopentenyl phosphate kinases from Methanolobus tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and Arabidopsis thaliana (AtIPK) were cloned and expressed in Escherichia coli. The enzymatic properties of recombinant enzymes were determined. The Kcat/Km value of SfPK for DMA was 6875 s-1 M-1, which was significantly higher than those of EcPK and ScPK. The Kcat/Km value of MtIPK for DMAP was 402.9 s-1 M-1, which was ~400% of that of MthIPK. SfPK was stable at pH 7.0-9.5 and had a 1 h half-life at 65 °C. MtIPK was stable at pH 6.0-8.5 and had a 1 h half-life at 50 °C. The stability of SfPK and MtIPK was better than that of the other enzymes. Thus, SfPK and MtIPK were chosen to develop a one-pot enzymatic cascade for producing DMAPP from DMA because of their catalytic efficiency and stability. The optimal ratio between SfPK and MtIPK was 1:8. The optimal pH and temperature for the one-pot enzymatic cascade were 7.0 and 35 °C, respectively. The optimal concentrations of ATP and DMA were 10 and 80 mM, respectively. Finally, maximum DMAPP production reached 1.23 mM at 1 h under optimal conditions. Therefore, the enzymatic method described herein for the biosynthesis of DMAPP from DMA can be widely used for the synthesis of isoprenoids and prenylated flavonoids.

Methanolobus profundi sp. nov., a methylotrophic methanogen isolated from deep subsurface sediments in a natural gas field

A mesophilic, methylotrophic methanogen, strain MobM(T), was isolated from a natural gas field in Japan. Strain MobM(T) grew on methanol and methylamines, but not on H(2)/CO(2), formate, acetate or dimethyl sulfide. The cells were motile, irregular cocci (diameter, 0.9-1.2 microm) and occurred singly, in pairs, as tetracocci or (occasionally) as aggregates. Strain MobM(T) grew at 9-37 degrees C (optimally at 30 degrees C) and at pH 6.1-7.8 (optimally at pH 6.5). Sodium and magnesium were required for growth, at 0.1-1.0 M Na(+) (optimally at 0.35 M) and 10-400 mM Mg(2+) (optimally at 15-25 mM). The G+C content of the genomic DNA was 42.4 mol%. 16S rRNA gene sequencing revealed that the isolate is a member of the genus Methanolobus, but distinct from its closest neighbours, Methanolobus tindarius DSM 2278(T) (sequence similarity, 98.0 %) and Methanolobus vulcani DSM 3029(T) (98.1 %). On the basis of phenotypic and phylogenetic features of MobM(T), it is clear that this strain represents a novel species of the genus Methanolobus, for which the name Methanolobus profundi sp. nov. is proposed. The type strain is MobM(T) (=DSM 21213(T)=NBRC 104158(T)).

Purification and properties of an F420H2 dehydrogenase from Methanosarcina mazei Gö1

Abstract The F420H2 dehydrogenase is part of the energy-conserving F420H2:heterodisulfide oxidoreductase system in Methanosarcina mazei Go1. The enzyme was purified 75-fold to apparent homogeneity from washed membranes. The molecular mass as determined by both native gel electrophoresis and gel filtration was 115 000. The purified enzyme was composed of five different polypeptides with molecular masses of 40, 37, 22, 20, and 16 kDa and contained 7 mol S2− and 7 mol Fe. The specific activity was 17 U mg protein−1 (apparent Vmax) using F420H2 as electron donor (Km=7 μM) and methylviologen and metronidazole as electron acceptors at pH 8.5 at a temperature of 39°C. The enzyme also catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone and tetramethyl-p-benzoquinone. The protein did not exhibit hydrogenase activity since F420 was not reduced by hydrogen and H2 production from F420H2 was not observed. In contrast to the F420H2 dehydrogenase from Archaeoglobus fulgidus, the enzyme from Methanosarcina mazei Go1 was similar to the corresponding protein of Methanolobus tindarius with respect to molecular mass and subunit composition. However, the proteins of the methanogenic organisms are different in cofactor content, since evidence is presented that the enzyme from Methanosarcina mazei Go1 contains FAD.

The F420H2 dehydrogenase from Methanosarcina mazei is a Redox-driven proton pump closely related to NADH dehydrogenases.

The F(420)H(2) dehydrogenase is part of the energy conserving electron transport system of the methanogenic archaeon Methanosarcina mazei Gö1. Here it is shown that cofactor F(420)H(2)-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H(+) translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + P(i). The gene cluster encoding the F(420)H(2) dehydrogenase of M. mazei Gö1 comprises 12 genes that are referred to as fpoA, B, C, D, H, I, J, K, L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F(420)H(2) dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei Gö1. Instead, the gene product FpoF may be responsible for F(420)H(2) oxidation and may function as the electron input part. Thus, the F(420)H(2) dehydrogenase from M. mazei Gö1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the methanogenic archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F(420) and a modified output module adopted to the reduction of methanophenazine.

The F 420H 2-dehydrogenase from Methanolobus tindarius: cloning of the ffd operon and expression of the genes in Escherichia coli

The membrane-bound F 420H 2-dehydrogenase from the methylotrophic methanogen Methanolobus tindarius oxidizes reduced coenzyme F 420 and feeds the electrons into an energy-conserving electron transport chain. Based on the N-terminal amino acid sequence of the 40-kDa subunit of F 420H 2-dehydrogenase the corresponding gene ffdB was detected in chromosomal DNA of M. tindarius. Sequence analysis, primer extension, and RT-PCR experiments indicated that ffdB is part of an operon harboring three additional open reading frames ( ffdA, ffdC, ffdD). The corresponding mRNA transcript and transcription start sites were determined. All four genes could be heterologously expressed in Escherichia coli.

The F420H2-dehydrogenase from Methanolobus tindarius: cloning of the ffd operon and expression of the genes in Escherichia coli.

The membrane-bound F420H2-dehydrogenase from the methylotrophic methanogen Methanolobus tindarius oxidizes reduced coenzyme F420 and feeds the electrons into an energy-conserving electron transport chain. Based on the N-terminal amino acid sequence of the 40-kDa subunit of F420H2-dehydrogenase the corresponding gene ffdB was detected in chromosomal DNA of M. tindarius. Sequence analysis, primer extension, and RT-PCR experiments indicated that ffdB is part of an operon harboring three additional open reading frames (ffdA, ffdC, ffdD). The corresponding mRNA transcript and transcription start sites were determined. All four genes could be heterologously expressed in Escherichia coli.

Investigation of serine hydroxymethyltransferase in methanogens

The cofactor specificity of serine hydroxymethyltransferase (SHMT) activities was tested in extracts of several methanogens using tetrahydromethanopterin (H4MPt) from Methanobacterium thermoautotrophicum Marburg, tetrahydrosarcinapterin (H4SPt) from Methanosarcina barkeri, and tetrahydrofolate (H4folate) as the potential C1 carrier. In Methanosphaera stadtmanae and Methanococcus thermolithotrophicus, the activities were H4MPt dependent. In Methanospirillum hungatei GP1, Methanosaeta concilii, Methanolobus tindarius, and Methanosarcina barkeri Fusaro, the activities were strictly H4folate dependent. H4SPt was reactive with the SHMT of Methanosphaera stadtmanae but not with that of Methanosarcina barkeri. In both Methanosarcina barkeri and Methanospirillum hungatei, pyridoxal phosphate stimulated SHMT activity. The apparent Km values for H4folate and L-serine were 0.086 and 0.29 mM in Methanosarcina barkeri and 0.065 and 0.31 mM in Methanospirillum hungatei, respectively. Key words: tetrahydromethanopterin, tetrahydrofolate, Archaea, serine hydroxymethyltransferase, methanogen.

Investigation of serine hydroxymethyltransferase in methanogens.

The cofactor specificity of serine hydroxymethyltransferase (SHMT) activities was tested in extracts of several methanogens using tetrahydromethanopterin (H4MPt) from Methanobacterium thermoautotrophicum Marburg, tetrahydrosarcinapterin (H4SPt) from Methanosarcina barkeri, and tetrahydrofolate (H4folate) as the potential C1 carrier. In Methanosphaera stadtmanae and Methanococcus thermolithotrophicus, the activities were H4MPt dependent. In Methanospirillum hungatei GP1, Methanosaeta concilii, Methanolobus tindarius, and Methanosarcina barkeri Fusaro, the activities were strictly H4folate dependent. H4SPt was reactive with the SHMT of Methanosphaera stadtmanae but not with that of Methanosarcina barkeri. In both Methanosarcina barkeri and Methanospirillum hungatei, pyridoxal phosphate stimulated SHMT activity. The apparent K(m) values for H4folate and L-serine were 0.086 and 0.29 mM in Methanosarcina barkeri and 0.065 and 0.31 mM in Methanospirillum hungatei, respectively.

Purification and properties of an F 420H 2 dehydrogenase from Methanosarcina mazei Gö1

The F 420H 2 dehydrogenase is part of the energy-conserving F 420H 2:heterodisulfide oxidoreductase system in Methanosarcina mazei Gö1. The enzyme was purified 75-fold to apparent homogeneity from washed membranes. The molecular mass as determined by both native gel electrophoresis and gel filtration was 115 000. The purified enzyme was composed of five different polypeptides with molecular masses of 40, 37, 22, 20, and 16 kDa and contained 7 mol S 2− and 7 mol Fe. The specific activity was 17 U mg protein −1 (apparent V max) using F 420H 2 as electron donor ( K m=7 μM) and methylviologen and metronidazole as electron acceptors at pH 8.5 at a temperature of 39°C. The enzyme also catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone and tetramethyl- p-benzoquinone. The protein did not exhibit hydrogenase activity since F 420 was not reduced by hydrogen and H 2 production from F 420H 2 was not observed. In contrast to the F 420H 2 dehydrogenase from Archaeoglobus fulgidus, the enzyme from Methanosarcina mazei Gö1 was similar to the corresponding protein of Methanolobus tindarius with respect to molecular mass and subunit composition. However, the proteins of the methanogenic organisms are different in cofactor content, since evidence is presented that the enzyme from Methanosarcina mazei Gö1 contains FAD.

Physiological Characterization and Emended Description of Methanolobus vulcani

Methanolobus vulcani PL-12/MTT(T = type strain) grew on methylamines and methanol but not on dimethyl sulfide, formate, acetate, or H2-CO2. The cells grew rapidly at mesophilic temperatures, at neutral pH values (pH 6 to 7.5), and in medium supplemented with 0.1 to 1.2 M NaCI and 13 mM Mg2+. The cells grew in mineral medium containing biotin and a catabolic substrate, but growth was stimulated by yeast extract and peptones. M. vulcani was physiologically similar to Methanolobus tindarius and had a similar 16S rRNA sequence, although the results of DNA-DNA hybridization experiments indicated that these organisms should be considered separate species.

Transformation of tetrachloroethylene to trichloroethylene by homoacetogenic bacteria.

Eight homoacetogenic strains of the genera Acetobacterium, Clostridium and Sporomusa were tested for their ability to dechlorinate tetrachloroethylene (perchloroethene, PCE). Of the organisms tested only Sporomusa ovata was able to reductively dechlorinate PCE with methanol as an electron donor. Resting cells of S. ovata reductively dechlorinated PCE at a rate of 9.8 nmol h-1 (mg protein)-1 to trichloroethylene (TCE) as the sole product. The dechlorination activity depended on concomitant acetogenesis from methanol and CO2. Cell-free extracts of S. ovata, Clostridium formicoaceticum, Acetobacterium woodii, and the methanogenic bacterium Methanolobus tindarius transformed PCE to TCE with Ti(III) or carbon monoxide as electron donors. Corrinoids were shown in S. ovata to be involved in the dechlorination reaction of PCE to TCE as evident from the reversible inhibition with propyl iodide. Rates of dechlorination followed a pseudo-first-order kinetic.

A chemical method for estimating methanogenic biomass

Methane-forming bacteria belong to the archaebacterial kingdom and as such possess unique membrane lipids in that phytanyl ether linked phospholipids replace the more usual ester linked analogues. A common methanogenic membrane ether lipid (Di-Phytanyl Glycerol Ether; DPGE) can be extracted using solvents, chemically broken down, derivatized, purified using thin layer chromatography, and finally analysed quantitatively by capillary gas chromatography. In order to evaluate the concentration of DPGE as a means of estimating the biomass of methanogens, this membrane lipid was compared with cell numbers, methane production and turbidity at 578 nm in a controlled growth experiment of a marine methanogenic monoculture of Methanolobus tindarius. It was found that the DPGE lipid data produced similar growth curves to the other measured parameters. All the parameters used to monitor the growth experiment showed an interesting change at a point in the growth cycle of M. tindarius where cell division slowed down and the growth of individual cells appeared to be the major mechanism of increasing the biomass. Preliminary environmental samples taken from both a marine inter-tidal and a freshwater site were analysed for the DPGE lipid and the results are discussed.

Purification and characterization of F420H2‐dehydrogenase from Methanolobus tindarius

The oxidation of F420H2 (reduced coenzyme F420) is a key reaction in the final step of methanogenesis. This step is catalyzed in Methanolobus tindarius by the membrane-bound F420H2-dehydrogenase which was purified 31-fold to apparent homogeneity. The apparent molecular mass of the native enzyme was 120 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of five different subunits of apparent molecular masses of 45 kDa, 40 kDa, 22 kDa, 18 kDa and 17 kDa. The purified F420H2-dehydrogenase, which was yellowish, contained 16 +/- 2 mol iron and 16 +/- 3 mol acid-labile sulfur/mol enzyme. No flavin could be detected. The oxygen-stable enzyme catalyzed the oxidation of F420H2 (apparent Km = 5.4 microM) with methylviologen and metronidazole as electron acceptors at a specific rate of 13 mumol.min-1.mg-1 (kcat = 25.5 s-1). The isoelectric point was at pH 5.0. The temperature optimum was at 37 degrees C and the pH optimum at 6.8.

Structure determination of two new amino acid-containing derivatives of adenosine from tRNA of thermophilic bacteria and archaea.

Two new nucleosides have been identified in unfractionated transfer RNA of two thermophilic bacteria, Thermodesulfobacterium commune, and Thermotoga maritima, six hyperthermophilic archaea, including Pyrobaculum islandicum, Pyrococcus furiosus and Thermococcus sp. and two mesophilic archaea, Methanococcus vannielii and Methanolobus tindarius. Structures were determined primarily by mass spectrometry, as 3-hydroxy-N-[[(9-beta-D-ribofuranosyl-9H-purin-6- yl)amino]carbonyl]norvaline, (hn6A), structure 1, and 3-hydroxy-N-[[(9-beta-D-ribofuranosyl-9H-2-methylthiopurin-6- yl)amino]carbonyl]norvaline (ms2hn6A), 2. The amino acid side chain was characterized as 3-hydroxynorvaline (3) by gas chromatography-mass spectrometry of the trimethylsilyl derivative after cleavage from 1 and 2 by alkaline hydrolysis. Evidence for the amino acid-purine carbamoyl linkage was obtained from the collision-induced dissociation mass spectrum of trimethylsilylated 1, and the total structure was confirmed by chemical synthesis of 1.

H2: heterodisulfide oxidoreductase, a second energy-conserving system in the methanogenic strain G�1

Washed everted vesicles of the methanogenic bacterium strain Go1 catalyzed an H2-dependent reduction of the heterodisulfide of HS-CoM (2-mercaptoethanesulfonate) and HS-HTP (7-mercaptoheptanoylthreonine phosphate) (CoM-S-S-HTP). This process was independent of coenzyme F420 and was coupled to proton translocation across the cytoplasmic membrane into the lumen of the everted vesicles. The maximal H+/CoM-S-S-HTP ratio was 2. The tranmembrane electrochemical gradient thereby generated was shown to induce ATP synthesis from ADP+Pi, exhibiting a stoichiometry of 1 ATP synthesized per 2 CoM-S-S-HTP reduced (H+/ATP=4). ATP formation was inhibited by the uncoupler 3,5-di-tert-butyl-4-hydroxy-benzylidene-malononitrile (SF 6847) and by the ATP synthase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD). This energy-conserving system showed a stringent coupling. The addition of HS-CoM and HS-HTP at 1 mM each decreased the heterodisulfide reductase activity to 50% of the control. Membranes from Methanolobus tindarius showed F420H2-dependent but no H2-dependent heterodisulfide oxidoreductase activity. Neither of these activities was detectable in membranes of Methanococcus thermolithotrophicus.

An immunological study of corrinoid proteins from bacteria revealed homologous antigenic determinants of a soluble corrinoid-dependent methyltransferase and corrinoid-containing membrane proteins from Methanobacterium species

The hypothesis of common epitopes in corrinoid-dependent enzymes was tested by a monospecific polyclonal antiserum against the 33 kDa corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum Marburg. Cross-reaction was detected with the 33 kDa and the 31 kDa subunits of the corrinoid-containing enriched 5-methyl-H4MPT: 5-hydroxybenzimidazolyl cobamide methyltransferase from the cytoplasmic fraction and a 33 kDa protein from the membrane fraction of Methanobacterium thermoauto-trophicum ΔH. This indicates that both proteins have similar antigenic determinants and that they may have similar function as methyltransfer proteins. Also a soluble 20 kDa protein of yet unknown function from Clostridium barkeri cross-reacted with the antiserum. No cross-reactions were observed with the purified corrinoid-containing 2-methyleneglutarate mutase from C. barkeri, the corrinoid/iron-sulfur protein from C. thermoaceticum, the carbon monoxide dehydrogenases from C. thermoaceticum and Methanothrix soehngenii, and the corrinoid-binding protein intrinsic factor from porcine gastric mucosa. Also cell extracts from the corrinoid-rich bacteria Sporomusa ovata, Methanolobus tindarius, Chloroflexus aurantiacus, Propionibacterium shermanii, the membrane fraction and the cytoplasmic fraction of Methanococcus voltae or extracts from human liver, contained no antibody combining sites others than with the preimmunological serum. These findings indicate, that many corrinoid-containing proteins from bacteria have no common antigenic determinants.

Membrane-bound F 420H 2-dependent heterodisulfide reductase in methanogenic bacterium strain Göl and Methanolobus tindarius

Washed membrane or cytoplasmic fractions of the methanogenic bacterium strain Göl catalyzed the oxidation of coenzyme F 420H 2 with a variety of electron acceptors. The F 420H 2-oxidizing activity of the cytoplasmic fraction could be assigned to a NADP +:F 420 oxidoreductase. The membrane fraction but not the cytoplasmic fraction catalyzed the oxidation of F 420H 2 with the concomitant reduction of the heterodisulfide of 2-mercapto-ethanesulfonate and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) at a rate of 100 nmol min-mg protein according to the following equation: F 420H 2+CoM-S-S-HTP → F 420 + CoM + HTP-SH. The activity depended linearly on the membrane protein up to a concentration of 60 μg ml The physiological electron acceptor CoM-S-S-HTP could not be replaced by the corresponding homodisulfides CoM-S-S-CoM and HTP-S-S-HTP or by NADP +. A membrane-bound F 420H 2-dependent CoM-S-S-HTP reductase was also detected in Methanolobus tindarius exhibiting a specific activity of 75 nmol min m ̇ g protein. The absence of a F 420-dependent hydrogenase in this organism excludes the involvement of this enzyme in electron transfer from H 420H 2 to CoM-S-S-HTP.

Chemiosmotic energy conversion and the membrane ATPase of Methanolobus tindarius

Electron transport phosphorylation has been demonstrated to drive ATP synthesis for the methanogenic archaebacterium Methanolobus tindarius: Protonophores evoked uncoupler effects and lowered the membrane potential delta psi. Under the influence of N,N'-dicyclohexylcarbodiimide [(cHxN)2C] the membrane potential increased while methanol turnover was inhibited. 2-Bromoethanesulfonate, an inhibitor of methanogenesis, had no effect on the membrane potential but, like (cHxN)2C and protonophores, decreased the intracellular ATP concentration. Labeling experiments with (cHxN)2(14)C showed membranes to contain a proteolipid, with a molecular mass of 5.5 kDa, that resembles known (cHxN)2C-binding proteins of F0-F1 ATPases. The (cHxN)2-sensitive membrane ATPase hydrolysed Mg.ATP at a pH optimum of 5.0 with a Km (ATP) of 2.5 mM (V = 77 mU/mg). It was inhibited competitively by ADP; Ki (ADP) = 0.65 mM. Azide or vanadate caused no significant loss in ATPase activity, but millimolar concentrations of nitrate showed an inhibitory effect, suggesting a relationship to ATPases from vacuolar membranes. In contrast, no inhibition occurred in the presence of bafilomycin A1. The ATPase was extractable with EDTA at low salt concentrations. The purified enzyme consists of four different subunits, alpha (67 kDa), beta (52 kDa), gamma (20 kDa) and beta (less than 10 kDa), as determined from SDS gel electrophoresis.

Further studies on the distribution of cytochromes in methanogenic bacteria

Methanolobus tindarius and Methanococcoides methylutens were shown to contain a cytochrome c in concentrations of 0.189 and 0.306 μ mol/g membrane protein, respectively. A cytochrome b was present in much smaller amounts. Methanoplanus limicola was found to be devoid of cytochromes.