To determine the cellular localization of components of the methyltransferase system, we separated cell extracts of Methanosarcina strain Gol into cytoplasmic and inverted-vesicle fractions. Measurements demonstrated that 83% of the methylene-tetrahydromethanopterin reductase activity resided in the cytoplasm whereas 88% of the methyl-tetrahydromethanopterin:coenzyme M methyltransferase (methyltransferase) was associated with the vesicles. The activity of the methyltransferase was stimulated 4.6-fold by ATP and 10-fold by ATP plus a reducing agent [e.g., Ti(III)J. In addition, methyltransferase activity depended on the presence of Na+ (apparent Km = 0.7 mM) and Na+ was pumped into the lumen of the vesicles in the course of methyl transfer from methyl-tetrahydromethanopterin not only to coenzyme M but also to hydroxycobalamin. Both methyl transfer reactions were inhibited by 1-iodopropane and reconstituted by illumination. A model for the methyl transfer reactions is presented.
Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.
The N'-methyltetrahydomethanopterin (H,MPT) :coenzyme M methyltransferase is a membrane associated, corrinoid-containing protein that uses the methylation of coenzyme M (HS-CoM) by methyltetrahydromethanopterin to drive an energy-conserving sodium ion pump. The enzyme was purified from acetate-grown Methanosarcina mazei GO1 by a two-step solubilization with s-octyl-P-glucoside, chromatography on hydroxyapatite, and by gel filtration on Superdex 200 or Sepharose CL-6B. The highly purified protein was apparently composed of six different subunits of 34, 28, 20, 13, 12, and 9 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native enzyme exhibited an apparent molecular mass of about 380 kDa. During purification, the enzyme was stabilized with 10 pM hydroxocobalamin. The highest specific activity reached during purification was 10.4 U/mg. The purified enzyme was reconstituted in monolayer liposomes prepared from ether lipids of M. mazei GO1. In experiments with radioactive sodium ions, it was shown that the methyltransferase catalyzes the vectorial translocation of sodium ions across the membrane. Methyltransferase activity was stimulated by sodium ions. 1.7 mol Na+/mol methyl groups transferred were translocated. Methyltetrahydrofolate and methylcobalamin could substitute for methyl-H,MPT.
Washed invested vesicle preparations of Methanosarcina strain Gö1 catalyzed the formation of methyl‐CoM from formaldehyde, H2 and CoM in the presence of tetrahydromethanopterin and 2‐bromoethanesulfonate. The reaction was associated with the translocation of sodium ions into the lumen of the vesicles. This translocation was abolished by the Na+ ionophore ETH 157 but it was insensitive to the addition of the uncoupler SF6847 and the Na+/H+ antiport inhibitor amiloride and, therefore, is the result of a primary Na+ pump. Since the translocation of Na+ was also observed when formaldehyde + tetrahydromethanopterin was replaced by methyl‐tetrahydromethanopterin, it follows that the methyl transfer from tetrahydromethanopterin to CoM is the sodium‐motive reaction. Methyl‐tetrahydromethanopterin could be replaced by methyl‐tetrahydrofolate.
Methane is a major terminal product of the anaerobic degradation of biomass. It is produced by methanogenic archaea from substrates such as acetate, H 2 ϩ CO 2 , methanol, methylamines, and formate. Two reactions involved in methanogenesis from H 2 ϩ CO 2 are coupled to energy-conserving ion translocations: the H 2 -heterodisulfide reductase functions as a primary proton pump (6) and the corrinoid enzyme N 5
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