Genetic, biochemical, and molecular characterization ofMethanosarcina barkerimutants lacking three distinct classes of hydrogenase

Abstract

17The methanogenic archaeon Methanosarcina barkeri encodes three distinct 18 types of hydrogenase, whose functions vary depending on the growth substrate. 19These include the F420-dependent (Frh), methanophenazine-dependent (Vht), and 20 ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, 21we characterized a series of mutants lacking each hydrogenase in various 22 40 IMPORTANCE 41Methanogenic archaea are key players in the global carbon cycle due to their 42 ability to facilitate the remineralization of organic substrates in many anaerobic 43 environments. The consequences of biological methanogenesis are far reaching, 44 with impacts on atmospheric methane and CO2 concentrations, agriculture, energy 45 production, waste treatment and human health. The data presented here clarify the 46 in vivo function of hydrogenases during methanogenesis, which in turn deepens our 47 understanding of this unique form of metabolism. This knowledge is critical for a 48 variety of important issues ranging from atmospheric composition to human health. 49 50 Methanogens without cytochromes produce at least four types of 62 hydrogenase; including (i) the electron-bifurcating Mvh hydrogenase, which couples 63 oxidation of hydrogen to reduction of ferredoxin and a mixed coenzyme M-64 coenzyme B disulfide, (ii) the coenzyme F420-dependent hydrogenase, (iii) the [Fe] 65 hydrogenase, which couples hydrogen oxidation to reduction of 66 methenyltetrahydromethanopterin, and (iv) the ferredoxin-dependent, energy-67 converting hydrogenases (4). The first three are cytoplasmic enzymes, which supply 68 the electrons needed to reduce CO2 to methane. The last is a membrane bound 69 multi-subunit complex that couples hydrogenase activity to the 70 production/consumption of the ion-motive force across the cell membrane (hence 71 the designation as "energy-converting"). In non-cytochrome-containing 72 methanogens, these energy-converting hydrogenases are believed to provide low-73 potential electrons, in the form of reduced ferredoxin, needed for anaplerotic 74 reactions (6). 75Methanogens with cytochromes, typified by Methanosarcina barkeri, encode 76 a different set of hydrogenases that includes one cytoplasmic and two membrane-77 bound enzymes (Fig 1) (7). Like the non-cytochrome containing methanogens, M. 78 barkeri produces a cytoplasmic, three-subunit F420-dependent hydrogenase known 79 as Frh (for F420-reducing hydrogenase). Frh is encoded by the four-gene frhADGB 80 operon, which encodes the α, β and γ subunits (FrhA, FrhB and FrhG, respectively), 81 along with the maturation protease FrhD (8). A second locus, freAEGB, encodes 82 proteins that are 86-88% identical to FrhA, FrhB and FrhG, but lacks the gene for the 83 maturation protease FrhD, instead encoding a small protein of unknown function 84 (FrhE). It is not known whether the fre operon is capable of producing an active 85 hydrogenase (8)(9)(10)(11). A membrane-bound hydrogenase linked to the quinone-like 86 electron carrier methanophenazine has, to dat...

Redox cycling of Fe(II) and Fe(III) in magnetite accelerates aceticlastic methanogenesis by Methanosarcina mazei

“…The electrons released from the oxidation of CO moiety end up in the reduction of methyl moiety thus completing the aceticlastic methanogenesis. This H 2 ‐dependent pathway depends very much on the efficiency of Vho to recapture H 2 that may escape the oxidation due to the easy diffusion away from the cell (Welte and Deppenmeier, ; Kulkarni et al ., ; Mand et al ., ).…”

Energy Conservation via Hydrogen Cycling in the Methanogenic Archaeon Methanosarcina barkeri

“…Like Frh, Vht is a three-subunit enzyme encoded by a four-gene operon ( vhtGACD ) that includes a maturation protease, VhtD ( 19 ). M. barkeri also contains genes that encode homologs of both the frh and vht operons (the freAEGB and vhxGAC operons, respectively); however, multiple lines of evidence suggest that these genes are incapable of producing active hydrogenases ( 16 , 22 ). Thus, the presence of these genes has no bearing on the results presented herein.…”

Perspectives on Potential Applications of Nanometal Derivatives in Gaseous Bioenergy Pathways: Mechanisms, Life Cycle, and Toxicity

“…Mph is responsible for delivering the electrons to Hdr. Under MIET conditions, H 2 diffuses inside the cell and can be oxidized to the sake of Mph reduction. − n-Fe 3 O 4 was found to mimic the Mph role, and therefore a reduction for n-Fe 3 O 4 can occur on the surface of the cell (Figure ). This mechanism was suggested by Fu et al, who used n-Fe 3 O 4 for accelerating the acetotrophic methanogenesis by M. barkeri and noticed the presence of hematite on the surface of M. barkeri .…”

“…Many previous studies highlighted that the electron transfer from NMDs to the electrogenic microbes (e.g., Geobacter sp., Shewanella sp., Desulfovibrio sp., and Syntrophobacter sp.) can be demonstrated by an observed remarkable change in the expression of genes encoding certain enzymes, and cofactors, e.g., F 420 , ,, Pil A, and omcS . , F 420 cofactor is generated from the oxidation of F 420 H 2 , coupled with methanophenazine reduction, by membrane-bound Fpo complex. ,, The fundamental role of the F 420 cofactor is to transport electrons required for the reduction of CO 2 (Figure ). The electron donation by F 420 is expected to increase the interaction of NMDs with microorganisms. , Zhuang et al showed that increased conversion of acetate to CH 4 correlated with the concentration of the cofactor F 420 when Fe 3 O 4 was loaded on the water hyacinth-derived biochars.…”

“…19,165 F 420 cofactor is generated from the oxidation of F 420 H 2 , coupled with methanophenazine reduction, by membrane-bound Fpo complex. 59,61,166 The fundamental role of the F 420 cofactor is to transport electrons required for the reduction of CO 2 (Figure 1). 166 The electron donation by F 420 is expected to increase the interaction of NMDs with microorganisms.…”