Kinetics overcome thermodynamics in primitive analogs of the reverse tricarboxylic acid cycle

The reverse/reductive tricarboxylic acid (rTCA) cycle is a metabolic pathway that facilitates CO₂ fixation in certain anaerobic bacteria and archaea. Its presence in phylogenetically ancient organisms has led to hypotheses about its role in the early evolution of CO₂ fixation pathways. While the thermodynamics of the pathway is well studied, the kinetic feasibility of uncatalyzed rTCA cycle reactions remains uncertain. In this study, we report a systematic, mechanistic, and kinetic characterization of the uncatalyzed rTCA cycle and its side reactions. A primitive, uncatalyzed rTCA reaction network is elucidated for carbon dioxide fixation that includes all transition states and water-catalyzed reaction channels. The thermodynamics and kinetics of competing off-cycle reactions and potential cycles that are parallel to the uncatalyzed rTCA cycle are also investigated using a newly developed chemical reaction network exploration method. While previous work examined overall thermodynamics and possible pathways, our work focuses on kinetic bottlenecks, which guide where in nature to search for primordial catalysts (clays, minerals, etc.) that could lower the major transition state barriers. This exploration reveals that the uncatalyzed rTCA pathway lies in a reaction neighborhood that is thermodynamically favored, but several key steps are kinetically challenging in the absence of catalysis owing to competitive intramolecular side-reactions and the absence of favorable parallel cycles. The kinetic modeling also provides intermediates that would accumulate in an uncatalyzed environment, serving as prebiotic signposts.