Lin, Y.-H.; Peng, J.-H.; Huang, S.-Y.; Wang, P.-Y.; Huang, C.-C.

Several metabolites within the reductive tricarboxylic acid (rTCA) cycle have been found to form prebiotically. However, how these metabolites connect to each other and form rTCA cycle remains unresolved. The rTCA cycle is an ancient route and is considered significant for the emergence of life, since it connects to the routes of amino acids and nucleobases synthesis. A major challenge to complete the rTCA cycle under prebiotic conditions is the thermodynamically unfavorable reductive carboxylation of succinate to alpha-ketoglutarate. Here, we address this challenge by using the nature of energy: nonequilibrium conditions. By calculating the changes in free energy of succinate to alpha-ketoglutarate, and its downstream reactions: alpha-ketoglutarate to glutamate and alpha-ketoglutarate to isocitrate under different nonequilibrium conditions, we find that these two-step reactions are exergonic under nonequilibrium conditions at a 10000:1 reactant-to-product ratio at 1.013 bar, pH 10 and 70 celcius degree. To prove the concept, we catalyze succinate to glutamate at a 10000:1 reactant-to-product ratio, with NH2OH and sodium dithionite. The process is catalyzed by Fe(0), Fe3O4, and artificial proto-[4Fe4S] clusters in 1M NaCl at pH 10 and 70 celcius degree under 1 atm of 13CO2 for 48 hours. This nonequilibrium condition and one-pot system successfully promote the formation of alpha-ketoglutarate through carbon fixation with succinate and its subsequent conversion to glutamate. These findings demonstrate nonequilibrium states enable alpha-ketoglutarate formation through succinate and CO2, and suggest that a tendency toward natural thermodynamics may serve as a driving force for autocatalysis in the origin of life.