Abstract
In Methanobacterium thermoautotrophicum, the protonmotive force for the H+-translocating ATPase consists mainly of a transmembrane electrical gradient (Deltapsi). These cells do not establish a significant transmembrane pH gradient (inside alkaline) and, in fact, if the suspending medium is of pH >/= 7.0, the pH gradient may be reversed-i.e., inside acid with respect to the extracellular pH. These studies show by both 23Na NMR and 22Na+ distribution that Na+ extrusion with the generation of Deltapsi precedes methanogenesis in Mb. thermoautotrophicum. It is calculated that the newly established Na+ gradients increase Deltapsi by approximately 50 mV (inside negative). There is no detectable H+ extrusion during methane synthesis; instead there is a high rate of H+ consumption for methane synthesis and an increase in internal pH. This was supported by 31P NMR experiments, which showed an internal pH shift from 6.8 to 7.6. With the cells maintained at an external pH of 7.2, the initial transmembrane pH gradient of -0.4 (inside acid) at 60 degrees C is equivalent to Deltapsi of + 27 mV (inside positive); after 20 min of incubation, the transmembrane pH gradient is + 0.4 (inside alkaline), which at 60 degrees C is equivalent to Deltapsi of -27 mV (inside negative). Actively respiring cells generated a protonmotive force of -198 mV. It is proposed that energy for CO2 reduction to the level of formaldehyde (the first step in methane synthesis) in Mb. thermoautotrophicum is derived from the Deltapsi generated by electrogenic Na+ extrusion. The protonmotive force required for ATP synthesis consists primarily of Deltapsi and appears to be the result of both an electrogenic Na+ extrusion and a pH gradient (inside alkaline) which develops during methanogenesis.