Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials

Abstract

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (.DELTA..mu.H+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 .mu.mol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 .mu.mol min-1 (mg protein)-1, the intracellular ATP content was 20 nmol/mg protein and the .DELTA..mu.H+ was 200 mV (inside negative). When K+ was added to the suspensions the measured .DELTA..mu.H+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of .DELTA..mu.H+ adjustment, it was found that lowering .DELTA..mu.H+ from 200 mV ([K+]in/[K+]out = 1000) to 100 mV ([K+]in/[K+]out = 40) had no effect on the rate of methane formation and on the intracellular ATP content. At .DELTA..mu.H+ values below 100 mV ([K+]in/[K+]out < 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when .DELTA..mu.H+ was adjusted to values lower 50 mV ([K+]in/[K+]out < 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri. Protonophoric uncouplers like 3,5,3'',4''-tetrachlorosalicylanilide (TCS) or 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) were found not to dissipate .DELTA..mu.H+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.