Chemiosmotic energy conversion and the membrane ATPase of Methanolobus tindarius

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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 ΔΨ. 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)214C 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, α (67 kDa), β (52 kDa), γ (20 kDa) and δ (< 10 kDa), as determined from SDS gel electrophoresis.