Inorganic pyrophosphate synthesis during methanogenesis from methylcoenzyme M by cell-free extracts of Methanobacterium thermoautotrophicum (strain delta H)

Abstract

Cell-free extracts of Methanobacterium thermoautotrophicum (strain delta H) were found to contain high concentrations of inorganic pyrophosphate (up to 40 mM). The compound was accumulated by the organism despite high activity of inorganic pyrophosphatase which was found to be present in the cell extracts (1-2 mumol min-1 mg protein-1). This activity was strongly inhibited at [PPi] greater than 1.0 mM. It was demonstrated that PPi synthesis occurred during methylcoenzyme M reduction under hydrogen atmosphere: in the first stage of the reaction for each mole of methane formed one mole of PPi was produced. Inhibition of the methylcoenzyme M reduction by 2-bromoethanesulfonic acid or by high concentrations (greater than 3 microM) of tetrachlorosalicylanilide also inhibited PPi synthesis. In contrast, low concentrations (1.3 microM) of tetrachlorosalicylanilide only inhibited PPi synthesis to the same extent as the methylcoenzyme M reduction was affected. In a later stage of the methylcoenzyme M reduction, PPi synthesis dropped and a second, as yet unidentified, unstable compound was formed. Synthesis of this compound also paralleled methane formation in a stoichiometric way and was affected by the inhibiting substances in a similar way as PPi synthesis.