Carbon isotopic heterogeneity of coenzyme F430 and membrane lipids in methane‐oxidizing archaea

Archaeal ANaerobic MEthanotrophs (ANME) facilitate the anaerobic oxidation of methane (AOM), a process that is believed to proceed via the reversal of the methanogenesis pathway. Carbon isotopic composition studies indicate that ANME are metabolically diverse and able to assimilate metabolites including methane, methanol, acetate, and dissolved inorganic carbon (DIC). Our data support the interpretation that ANME in marine sediments at methane seeps assimilate both methane and DIC, and the carbon isotopic compositions of the tetrapyrrole coenzyme F430 and the membrane lipids archaeol and hydroxy‐archaeol reflect their relative proportions of carbon from these substrates. Methane is assimilated via the methyl group of CH₃‐tetrahydromethanopterin (H₄MPT) and DIC from carboxylation reactions that incorporate free intracellular DIC. F430 was enriched in ¹³C (mean δ¹³C = ?27‰ for Hydrate Ridge and ?80‰ for the Santa Monica Basin) compared to the archaeal lipids (mean δ¹³C = ?97‰ for Hydrate Ridge and ?122‰ for the Santa Monica Basin). We propose that depending on the side of the tricarboxylic acid (TCA) cycle used to synthesize F430, its carbon was derived from 76% DIC and 24% methane via the reductive side or 57% DIC and 43% methane via the oxidative side. ANME lipids are predicted to contain 42% DIC and 58% methane, reflecting the amount of each assimilated into acetyl‐CoA. With isotope models that include variable fractionation during biosynthesis for different carbon substrates, we show the estimated amounts of DIC and methane can result in carbon isotopic compositions of ? 73‰ to ? 77‰ for F430 and ? 105‰ for archaeal lipids, values close to those for Santa Monica Basin. The F430 δ¹³C value for Hydrate Ridge was ¹³C‐enriched compared with the modeled value, suggesting there is divergence from the predicted two carbon source models.     

Nickel dependence of factor F430 content in Methanobacterium thermoautotrophicum

Factor F₄₃₀ is a yellow compound of unknown structure present in methanogenic bacteria. It has recently been shown to contain nickel. In this communication the influence of the nickel concentration in the growth medium on the factor F₄₃₀ content of Methanobacterium thermoautotrophicum and on the nickel content of factor F₄₃₀ was studied. It was found: (1) The content of factor F₄₃₀ in the cells was strongly dependent on the nickel concentration of the growth medium. Cells grown on media with 2.5 M NiCl₂ contained 28 times as much factor F₄₃₀ per g as those grown on media with 0.075 M NiCl₂; (2) factor F₄₃₀ was synthesized in nickel deprived cells only upon the addition of nickel Nickel uptake paralleled factor F₄₃₀ synthesis; (3) independent of the nickel concentration in the growth medium, the extinction coefficient at 430 nm of factor F₄₃₀ per mol nickel was always near 22,500 cm^-1 (mol Ni)^-1. These findings indicate that nickel is an essential component of factor F₄₃₀.Dedicated to Professor Otto Kandler on the occasion of his 60th birthday     

Interconversion of F430 derivatives of methanogenic bacteria

F₄₃₀ is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F₄₃₀ is thermolabile and in the presence of acetonitrile or C10 _in4 ^sup- two epimerization products are obtained upon heating; in the absence of these compounds F₄₃₀ is oxidized to 12, 13-didehydro-F₄₃₀. The latter is stereoselectively reduced under H₂ atmosphere to F₄₃₀ by cell-free extracts of M. barkeri or M. thermoautotrophicum. H₂ may be replaced by the reduced methanogenic electron carrier coenzyme F₄₂₀.Abbreviations CH₃S-CoM methylcoenzyme M, 2-methylthioethanesulfonic acid - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid - F₄₃₀ Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton - 13-epi-F₄₃₀ and 12,13-di-epi-F₄₃₀ the 12, 13- and 12, 13-derivatives of F₄₃₀ - 12, 13-didehydro-F₄₃₀ F₄₃₀ oxidized at C-12 and C-13 - coenzyme F₄₂₀ 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative - coenzyme F₄₂₀H₂ reduced coenzyme F₄₂₀ - MV⁺ methylviologen semiquinone - HPLC high-performance liquid chromatography     

Sirohydrochlorin,a precursor of factor F430 biosynthesis in Methanobacterium thermoautotrophicum

Factor F₄₃₀ is a nickel-containing coenzyme of methanogenic bacteria with porphinoid structure which is derived from uroporphyrinogen III. It is shown that sirohydrochlorin is metabolized by cell free extracts of Methanobacterium thermoautotrophicum to factor F₄₃₀ demonstrating that this compound, or a reduced form of it, is an intermediate in the biosynthesis of F₄₃₀, and not only of vitamin B₁₂ and siroheme.     

Nickel,a component of factor F 430 from Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum, growing on medium supplemented with 2 mol ⁶³NiCl₂/l, was found to take up 1.2 mol ⁶³Ni per g cells (dry weight). More than 70% of the radioisotope was incorporated into a compound, which dissociated from the protein fraction after heat treatment, was soluble in 70% acetone, and could be purified by chromatography on QAE-Sephadex A-25, Sephadex G-25, and DEAE cellulose. The purified ⁶³Ni labelled compound had an absorption spectrum and properties identical to those of factor F ₄₃₀ and is therefore considered to be identical with factor F ₄₃₀.Factor F ₄₃₀, a compound of molecular weight higher than 1000 with an absorbance maximum at 430 nm, has recently been purified from Methanobacterium thermoautotrophicum (Gunsalus and Wolfe, 1978). The structure and function of this compound are not yet known.     

New PCR primers based on mcrA gene for retrieving more anaerobic methanotrophic archaea from coastal reedbed sediments

Two pairs of PCR primes ANMEallF/R and ANME23F/R were designed by Codehop method based on sequences available to retrieve more anaerobic methanotrophic (ANME) archaea mcrA gene sequences and ANME 2 and 3 subtypes from reedbed in two seasons. Overall, the PCR primers showed slightly favor for ANME group mcrA gene sequences. Due to the predominance of methanogens mainly affiliated to Methanomicrobiales in the samples, a large portion of mcrA gene sequences amplified in the clone libraries belonged to methanogens. Differences in PCR primers and performance affected the mcrA gene-PCR-amplified community composition to a minor extent. PCR primers targeting ANME mcrA group g–h were designed to apply real-time PCR for quantifying more groups of mcrA gene-affiliated ANME archaea and tested with these same samples, and the most abundant group in the whole ANME mcrA community was ANME group g–h. In addition, a stable mcrA gene-harboring archaeal community pattern was detected in the reedbed sediment samples collected from two distinctively different seasons. The PCR and qPCR primers designed in this study can expand our knowledge on the distribution of ANME mcrA genes and community composition in the ecosystem to better understand the carbon cycle.     

Incorporation of 8 succinate per mol nickel into factors F430 by Methanobacterium thermoautotrophicum

Factors F₄₃₀ from methanogenic bacteria have recently been shown to contain nickel and it has been speculated that they may have a nickel tetrapyrrole structure. This assumption was tested by determining whether succinate is incorporated by growing Methanobacterium thermoautotrophicum into three factors F₄₃₀. Succinate is assimilated by Methanobacterium thermoautotrophicum into the amino acids glutamate, arginine and proline and into tetrapyrroles rather than other cell components. It was found that per mol nickel 8–9 mol of succinate were incorporated into the three factors F₄₃₀ which is the amount predicted for a tetrapyrrole structure. Since the three factors F₄₃₀ only contained significant amounts of glutamate rather than arginine or proline, the incorporation data suggest that factors F₄₃₀ are nickel tetrapyrrole compounds. Spectral properties of the three factors F₄₃₀, apparent molecular weights, and the absence of phosphor in these compounds are also described.     

甲烷代谢古菌分离培养研究进展

全球变暖是全人类面临的一个巨大挑战,而温室气体排放持续上升是全球变暖的关键因素,并引发一系列生态环境问题。甲烷是第二温室气体,对全球变暖的贡献达20%。然而,在甲烷代谢中发挥重要作用的产甲烷古菌和厌氧甲烷氧化古菌(anaerobic methanotroph,ANME)较难培养,极大地限制了人们对甲烷代谢及其影响碳源-汇关系与机制的研究。本文综述了最新产甲烷古菌和ANME富集、分离和培养方法,包括富集培养、原位培养、共培养、微流控技术、稀释分离和固体分离技术、ANME反应器和培养瓶富集培养,以及宏基因组预测和反向基因组学,并对这些方法的优缺点进行了评估,对未来甲烷代谢古菌的富集、分离和培养提出新的建议。     

Proteins containing the factor F430 from methanosarcina barkeri and methanobacterium thermoautotrophicum: Isolation and properties

The unique nickel-containing cofactor (compound F₄₃₀) has for the first time been shown to exist in a protein-bound form. The proteins isolated from Methanosarcina barkeri and Methanobacterium thermoautotrophicum were designated P_425(445) on the basis of their spectral characteristics. The proteins contained three different subunits and had molecular weights of approx. 300000. Heat treatment or acidification of the proteins releases an F₄₃₀ cofactor identical to the one isolated in Methanobacterium bryantii (Whitman, W.B. and Wolfe, R.S. (1980) Biochem. Biophys. Res. Commun. 92, 1196–1201), and Methanobacterium thermoautotrophicum (Gunsalus, R.P. and Wolfe, R.S. (1978) FEMS Lett. 3, 191–193, Diekert, G., Klee, B. and Thauer, R.K. (1980) Arch. Microbiol. 124, 103–106 and Ellefson, W.L. and Wolfe, R.S. (1981) J. Biol. Chem. 256, 4259–4262).     

Relationship of Intracellular Coenzyme F420 Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri

The use of F₄₂₀ as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H₂ and CO₂, and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F₄₂₀ was followed by high-resolution fluorescence spectroscopy. F₄₂₀ concentration in M. bryantii ranged from 1.84 to 3.65 μmol · g of protein⁻¹; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 μmol · g⁻¹ depending on growth conditions. The content of F₄₂₀ in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F₄₂₀ content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F₄₂₀ approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F₄₂₀ content. However, qCH₄(F₄₂₀), calculated by dividing the methane production rate by the coenzyme F₄₂₀ concentration, almost paralleled qCH₄(protein). These results suggest that F₄₂₀ may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH₄(F₄₂₀) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.     

A method for estimating phosphate in the presence of phytate and its application to the determination of phytase

Quantification of coenzymes and related compounds from methanogens was performed in extracts obtained from whole cells with aqueous ethanol at 80°C. By means of high-performance liquid chromatography the following compounds could be detected and quantified in extracts from Methanobacterium thermoautotrophicum: coenzyme MF₄₃₀, the prosthetic group of methylcoenzyme M reductase, F₅₆₀, an oxidation product of this compound, coenzyme F₄₂₀, F₃₄₂, methanopterin, and carboxytetrahydromethanopterin, previously known as YFC. Coenzyme MF₄₃₀, coenzyme F₄₂₀, and methanopterin could be determined in extracts from Methanosarcina barkeri. Structural differences were noticed between the coenzymes from the methanogenic bacteria studied.     

Integrated metagenomic and metaproteomic analyses of an ANME-1-dominated community in marine cold seep sediments

Sulfate‐reducing methanotrophy by anaerobic methanotrophic archaea (ANME) and sulfate‐reducing bacteria (SRB) is a major biological sink of methane in anoxic methane‐enriched marine sediments. The physiology of a microbial community dominated by free‐living ANME‐1 at 14–16 cm below the seafloor in the G11 pockmark at Nyegga was investigated by integrated metagenomic and metaproteomic approaches. Total DNA was subjected to 454‐pyrosequencing (829 527 reads), and 16.6 Mbp of sequence information was assembled into 27352 contigs. Taxonomic analysis supported a high abundance of Euryarchaea (70%) with 66% of the assembled metagenome belonging to ANME‐1. Extracted sediment proteins were separated in two dimensions and subjected to mass spectrometry (LTQ‐Orbitrap XL). Of 356 identified proteins, 245 were expressed by ANME‐1. These included proteins for cold‐adaptation and production of gas vesicles, reflecting both the adaptation of the ANME‐1 community to a permanently cold environment and its potential for positioning in specific sediment depths respectively. In addition, key metabolic enzymes including the enzymes in the reverse methanogenesis pathway (except N⁵,N¹⁰‐methylene‐tetrahydromethanopterin reductase), heterodisulfide reductases and the F₄₂₀H₂:quinone oxidoreductase (Fqo) complex were identified. A complete dissimilatory sulfate reduction pathway was expressed by sulfate‐reducing Deltaproteobacteria. Interestingly, an APS‐reductase comprising Gram‐positive SRB and related sequences were identified in the proteome. Overall, the results demonstrated that our approach was effective in assessing in situ metabolic processes in cold seep sediments.     

Function of coenzyme F420-dependent NADP reductase in methanogenic archaea containing an NADP-dependent alcohol dehydrogenase

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO₂ reduction to CH₄ contain either an NADP-dependent or a coenzyme F₄₂₀-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase and the N ⁵, N ¹⁰-methylenetetrahydromethanopterin reductase, two enzymes involved in CO₂ reduction to CH₄, are specific for F₄₂₀. This raised the question how F₄₂₀H₂ is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F₄₂₀ with NADPH. The F₄₂₀-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced. Received: 12 May 1997 / Accepted: 1 July 1997     

Spectroscopic investigation of the nickel-containing porphinoid cofactor F<Subscript>430</Subscript>. Comparison of the free cofactor in the +1, +2 and +3 oxidation states with the cofactor bound to methyl-coenzyme M reductase in the silent,red and ox forms

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. It contains the nickel porphinoid F₄₃₀, a prosthetic group that has been proposed to be directly involved in the catalytic cycle by the direct binding and subsequent reduction of the substrate methyl-coenzyme M. The active enzyme (MCRred1) can be generated in vivo and in vitro by reduction from MCRox1, which is an inactive form of the enzyme. Both the MCRred1 and MCRox1 forms have been proposed to contain F₄₃₀ in the Ni(I) oxidation state on the basis of EPR and ENDOR data. In order to further address the oxidation state of the Ni center in F₄₃₀, variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), coupled with parallel absorption and EPR studies, have been used to compare the electronic and magnetic properties of MCRred1, MCRox1, and various EPR silent forms of MCR, with those of the isolated penta-methylated cofactor (F₄₃₀M) in the +1, +2 and +3 oxidation states. The results confirm Ni(I) assignments for MCRred1 and MCRred2 forms of MCR and reveal charge transfer transitions involving the Ni d orbitals and the macrocycle orbitals that are unique to Ni(I) forms of F₄₃₀. Ligand field transitions associated with S=1 Ni(II) centers are assigned in the near-IR MCD spectra of MCRox1-silent and MCR-silent, and the splitting in the lowest energy d–d transition is shown to correlate qualitatively with assessments of the zero-field splitting parameters determined by analysis of VTVH MCD saturation magnetization data. The MCD studies also support rationalization of MCRox1 as a tetragonally compressed Ni(III) center with an axial thiolate ligand or a coupled Ni(II)-thiyl radical species, with the reality probably lying between these two extremes. The reinterpretation of MCRox1 as a formal Ni(III) species rather than an Ni(I) species obviates the need to invoke a two-electron reduction of the F₄₃₀ macrocyclic ligand on reductive activation of MCRox1 to yield MCRred1.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0549-9Abbreviations F₄₃₀ cofactor 430 - F₄₃₀M penta-methylated form of cofactor 430 - Ni(I)F₄₃₀M F₄₃₀M with the nickel atom in the +1 oxidation state - Ni(II)F₄₃₀M F₄₃₀M with the nickel atom in the +2 oxidation state - Ni(III)F₄₃₀M F₄₃₀M with the nickel atom in the +3 oxidation state - MCR methyl-coenzyme M reductase - MCRox1 MCR exhibiting the MCR-ox1 EPR signal - MCRox1-silent EPR silent form of MCR obtained from the MCRox1 form - MCRred1 MCR exhibiting the EPR signals red1c and/or red1m - MCRred1c MCRred1 in the presence of coenzyme M - MCRred1m MCRred1 in the presence of methyl-coenzyme M - MCRred2 MCR exhibiting both the red1 and red2 EPR signals - MCRred1-silent EPR silent form of MCR obtained from the MCRred1 form - MCRsilent EPR silent form of MCR     

Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na⁺ F₁F_O ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F_OV_O hybrid rotor that contains nine copies of a F_O-like c subunit and one copy of a V_O-like c ₁ subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na⁺ F₁F_O ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na⁺ F₁F_O ATP synthase that was purified in a single step after inserting a His₆-tag to its β subunit. The purified enzyme was competent in Na⁺ transport and contained the F_OV_O hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na⁺ F₁F_O ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F_OV_O hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.     

Single-molecule Analysis of F0F1-ATP Synthase Inhibited by N,N-Dicyclohexylcarbodiimide

N,N-Dicyclohexylcarbodiimide (DCCD) is a classical inhibitor of the F₀F₁-ATP synthase (F₀F₁), which covalently binds to the highly conserved carboxylic acid of the proteolipid subunit (c subunit) in F₀. Although it is well known that DCCD modification of the c subunit blocks proton translocation in F₀ and the coupled ATP hydrolysis activity of F₁, how DCCD inhibits the rotary dynamics of F₀F₁ remains elusive. Here, we carried out single-molecule rotation assays to characterize the DCCD inhibition of Escherichia coli F₀F₁. Upon the injection of DCCD, rotations irreversibly terminated with first order reaction kinetics, suggesting that the incorporation of a single DCCD moiety is sufficient to block the rotary catalysis of the F₀F₁. Individual molecules terminated at different angles relative to the three catalytic angles of F₁, suggesting that DCCD randomly reacts with one of the 10 c subunits. DCCD-inhibited F₀F₁ sometimes showed transient activation; molecules abruptly rotated and stopped after one revolution at the original termination angle, suggesting that hindrance by the DCCD moiety is released due to thermal fluctuation. To explore the mechanical activation of DCCD-inhibited molecules, we perturbed inhibited molecules using magnetic tweezers. The probability of transient activation increased upon a forward forcible rotation. Interestingly, during the termination F₀F₁, showed multiple positional shifts, which implies that F₁ stochastically changes the angular position of its rotor upon a catalytic reaction. This effect could be caused by balancing the angular positions of the F₁ and the F₀ rotors, which are connected via elastic elements.     

Evidence of Active Methanogen Communities in Shallow Sediments of the Sonora Margin Cold Seeps

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.     

Methanogenic capabilities of ANME‐archaea deduced from 13C‐labelling approaches

Anaerobic methanotrophic archaea (ANME) are ubiquitous in marine sediments where sulfate dependent anaerobic oxidation of methane (AOM) occurs. Despite considerable progress in the understanding of AOM, physiological details are still widely unresolved. We investigated two distinct microbial mat samples from the Black Sea that were dominated by either ANME‐1 or ANME‐2. The ¹³C lipid stable isotope probing (SIP) method using labelled substances, namely methane, bicarbonate, acetate, and methanol, was applied, and the substrate‐dependent methanogenic capabilities were tested. Our data provide strong evidence for a versatile physiology of both, ANME‐1 and ANME‐2. Considerable methane production rates (MPRs) from CO₂‐reduction were observed, particularly from ANME‐2 dominated samples and in the presence of methane, which supports the hypothesis of a co‐occurrence of methanotrophy and methanogenesis in the AOM systems (AOM/MPR up to 2:1). The experiments also revealed strong methylotrophic capabilities through ¹³C‐assimilation from labelled methanol, which was independent of the presence of methane. Additionally, high MPRs from methanol were detected in both of the mat samples. As demonstrated by the ¹³C‐uptake into lipids, ANME‐1 was found to thrive also under methane free conditions. Finally, C₃₅‐isoprenoid hydrocarbons were identified as new lipid biomarkers for ANME‐1, most likely functioning as a hydrogen sink during methanogenesis.     

Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens

Methane-forming archaea are strictly anaerobic microbes and are essential for global carbon fluxes since they perform the terminal step in breakdown of organic matter in the absence of oxygen. Major part of methane produced in nature derives from the methyl group of acetate. Only members of the genera Methanosarcina and Methanosaeta are able to use this substrate for methane formation and growth. Since the free energy change coupled to methanogenesis from acetate is only − 36 kJ/mol CH₄, aceticlastic methanogens developed efficient energy-conserving systems to handle this thermodynamic limitation. The membrane bound electron transport system of aceticlastic methanogens is a complex branched respiratory chain that can accept electrons from hydrogen, reduced coenzyme F₄₂₀ or reduced ferredoxin. The terminal electron acceptor of this anaerobic respiration is a mixed disulfide composed of coenzyme M and coenzyme B. Reduced ferredoxin has an important function under aceticlastic growth conditions and novel and well-established membrane complexes oxidizing ferredoxin will be discussed in depth. Membrane bound electron transport is connected to energy conservation by proton or sodium ion translocating enzymes (F₄₂₀H₂ dehydrogenase, Rnf complex, Ech hydrogenase, methanophenazine-reducing hydrogenase and heterodisulfide reductase). The resulting electrochemical ion gradient constitutes the driving force for adenosine triphosphate synthesis. Methanogenesis, electron transport, and the structure of key enzymes are discussed in this review leading to a concept of how aceticlastic methanogens make a living. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.