We report here a new mode of ATP synthesis in living cells. The anaerobic bacterium Propionigenium modestum gains its total energy for growth from the conversion of succinate to propionate according to: succinate + H2O → propionate + HCO3‐ (△Go’ = ‐20.6 kJ/mol). The small free energy change of this reaction does not allow a substrate‐linked phosphorylation mechanism, and no electron transport phosphorylation takes place. Succinate was degraded by cell‐free extracts to propionate and CO2 via succinyl‐CoA, methyl‐malonyl‐CoA and propionyl‐CoA. This pathway involves a membrane‐bound methylmalonyl‐CoA decarboxylase which couples the exergonic decarboxylation with a Na+ ion transport across the membrane. The organism also contained a membrane‐bound ATPase which was specifically activated by Na+ ions and catalyzed and transport of Na+ ions into inverted bacterial vesicles upon ATP hydrolysis. The transport was abolished by monensin but not by the uncoupler carbonylcyanide‐p‐trifluoromethoxy phenylhydrazone. Isolated membrane vesicles catalyzed the synthesis of ATP from ADP and inorganic phosphate when malonyl‐CoA was decarboxylated and malonyl‐CoA synthesis from acetyl‐CoA when ATP was hydrolyzed. These syntheses were sensitive to monensin which indicates that Na+ functions as the coupling ion. We conclude from these results that ATP synthesis in P. modestum is driven by a Na+ ion gradient which is generated upon decarboxylation of methylmalonyl‐CoA.

Upon resolution of the particulate cell fraction of Veilionella aclcalescens by chromatography, membranes and ribosomes were celarly resolved. Methylmalony‐CoA decarboxylase was bound to the membranes and not to ribosomes as reported earler. Membrane vesicels containing mentylmalonyl‐CoA decarboxylase were prepared by disrupting V. alcalescens cells with French pressure chdmaber. About 64% of the decaroxylase was oriented in these vesicles with the sbstrated binding site facing to the outside. The vesicels perforemd a rapid acdumulation of Na+ ions in respon se to the decarboylatino of methylmalonyl‐CoA.Decarbosylation and transport wer highly uncouped. The effciency of the transport was considerably increased if methylmalonyl‐CoA cecarbosylation was retared by using a low temperature of by slowaly genrating the substrate enzymically form priopionyl‐CoA. Under optimized coditions Na+ was concentrated inside the inverted vesicles eight‐times than in the incuabaion medium. Mehtylmalonyl‐CoA decarboxylase was solubilized formt eh membranes with Tritom X‐100 and purified about 20‐fp;d nu affinity chromatography on monomeric avidin‐Sepharose coulmns. The decarboxylase was specifically activated by Na+ ions (apparend Km∼ 0.6 mM). Wherase (S)‐methylmalonyl‐CoA was the superior substrte. (apparent km∼ 7 μM). The decarboxylation of methylmalonyl‐CoA yielded CO2 and not HCO3− as th primary reaction product. Analysis of the prurified enzyme by dodecylsulfate gel electrophoresis inducated the presence of four differnet polypetides α, β, γ, δ with Mr 60 0000, 33 0000, 18 500 and 14000. The letter of these polypetides was elearly visible only after stiaing but not after staining with Coomassie brilliant blue. A low moleualr weight polypetide wit similar staaining properties also found in oxaloacetate decarbvoxylase Methylmalonyl‐CoA decarboxlase contained about 1 mol convalently bound biotin per 125 500 g protien which was localized exculsively in the γ‐subunit. This subunit therfore represents th biotin carboysl carier protein of methylmalonyl‐CoA decarboxylase. A new very senstivitymethod for the dtection of biotin containg proteins is described.

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Veillonella alcalescens during lactate degradation developed an Na+ concentration gradient with 7–8 times higher external than internal Na+ concentrations in the logarithmic growth phase. The gradient declined to a factor of 1.9 in the late stationary phase. Methylmalonyl‐CoA decarboxylase reconstituted into proteoliposomes performed an active electrogenic Na+ transport, creating Ψ of 60 mV, pNa+ of 50 mV, and of 110 mV. In the initial phase of the transport, the decarboxylase catalyzed the uptake of 2 Na+ ions/malonyl‐CoA molecule decarboxylated. During further development of the electrochemical Na+ gradient, this ratio gradually declined to zero, when decarboxylation continued without further increase of the internal Na+ concentration. The rate of malonyl‐CoA decarboxylation declined initially during development of the membrane potential, but remained unchanged later on. Monensin abolished the Na+ gradient and increased the malonyl‐CoA decarboxylation rate 2.8‐fold. On dissipating the membrane potential with valinomycin, the internal Na+ concentration reached three times higher values than in its absence, and the decarboxylation rate increased 2.8‐fold. Methylmalonyl‐CoA decarboxylase catalyzed an exchange of internal and external Na+ ions in addition to net Na+ accumulation. The initial rate of Na+ influx was double that of malonyl‐CoA decarboxylation. In the following, both rates decreased about twofold in parallel to values which remained constant during further development of the electrochemical Na+ gradient. Thus, Na+ influx and malonyl‐CoA decarboxylation follow a stoichiometry of approximately 2:1, independent of the magnitude of the electrochemical Na+ gradient and are thus highly coupled events.