Chemiosmotic energy conversion and the membrane ATPase of Methanolobus tindarius

Abstract

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 Ay. Under the influence of N,N'-dicyclohexylcarbodiimide [(cHxN),C] 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)~C and protonophores, decreased the intracellular ATP concentration.Labeling experiments with ( C H X N )~'~C showed membranes to contain a proteolipid, with a molecular mass of 5.5 kDa, that resembles known (cHxN),C-binding proteins of Fo-F1 ATPases. The (cHxN)2-sensitive membrane ATPase hydrolysed Mg . ATP at a pH optimum of 5.0 with a K , (ATP) of 2.5 mM (V = 77 mujmg). 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 Al.The ATPase was extractable with EDTA at low salt concentrations. The purified enzyme consists of four different subunits, CI (67 kDa), , h' (52 kDa), y (20 kDa) and 6 ( < 10 kDa), as determined from SDS gel electrophoresis.Among archaebacteria the methanogens represent a diverse group of strictly anaerobic organisms producing methane from CO,, acetate, formate, methanol and methylamines by disproportionation or by reduction with molecular hydrogen (for a review see [l]). As reported by Blaut and Gottschalk [2], methanogens can use the energy from substrate reduction for ATP synthesis by an electron-transport-driven mechanism. Moreover, proton extrusion coupled to methanogenesis has been demonstrated [3]. The methylreductase system has been discussed as a candidate for a membrane-associated proton pump which catalyses the exergonic reduction of a coenzymebound methyl group to methane [4, 51.ATPases as possible catalysts of ATP synthesis have been found in the membranes of several methanogens [6-101; one of these has been identified as being vanadate-sensitive [8].Ion-transducing membrane ATPases can be divided into three classes, the P (or El-E2) type of plasma membranes, the V type of vacuolar membranes and the F type of chloroplasts, mitochondria and bacterial membranes [I I]. Members of these classes differ in their physiological role and their sensitivity against ionic [12] and antibiotic [13] inhibitors.

Hydrogen is a preferred intermediate in the energy-conserving electron transport chain of Methanosarcina barkeri

“…The situation probably is similar in M. mazei because it has functional Frh, Vht, and Fpo enzymes as well (11). However, methylotrophic species such as M. acetivorans (10,21,28), Methanolobus tindarius (40,41), and Methanococcoides burtonii (42) do not encode functional hydrogenases (22). Hence, these organisms probably rely exclusively on the F 420 H 2 :heterodisulfide oxidoreductase system for energy conservation.…”

Subunit Structure and Organization of the Genes of the A1A0 ATPase from the Archaeon Methanosarcina mazei Gö1

“…The archaeal A 1 A 0 ATPase 2 shares properties with both, bacterial F 1 F 0 and eucaryal V 1 V 0 ATPases (3,4). It clearly functions as an ATP synthase, which is in accordance with F 1 F 0 but in sharp contrast to V 1 V 0 ATPases; the structure of the proteolipid (one of the subunits of the A 0 domain), which is in the range of 6 -8 kDa in A 1 A 0 and F 1 F 0 ATPases but 16 kDa in V 1 V 0 ATPases, was suggested to be at least one of the reasons for this difference (5)(6)(7)(8). On the other hand, the primary sequences of the subunits A and B of the catalytic A 1 domain are clearly more closely related to vacuolar V 1 V 0 ATPases (9 -12).…”

“…It is generally accepted that the A 1 domain contains at least two large subunits of 62-80 kDa (subunit A) and 49 -55 kDa (subunit B) and the A 0 domain contains a least a 7-kDa subunit, the so-called proteolipid. Minor subunits co-purified with the enzyme (5,8,(13)(14)(15)(16)(17), but it has not been established whether these are genuine constituents of the ATPase. Furthermore, little is known about the genetic organization of the A 1 A 0 ATPases.…”

Potassium extrusion by the moderately halophilic and alkaliphilic methanogen methanolobus taylorii GS-16 and homeostasis of cytosolic pH

“…The intracellular ATP content was determined by the luciferase assay (41). Samples (0.5 ml) of the cell suspension were placed in 2.0 ml of boiling glycine (pH 7.2) buffer to inactivate enzymatic activity, and then 0.15 ml of each sample was mixed with 50 RI of luciferaseluciferin agent (Sigma).…”