The kinetic investigation of the uncatalyzed rTCA cycle required decomposition into mechanistically tractable elementary steps. The break-two-form-two bonds (b2f2) rule was selected because it represents the simplest mechanistic pathway capable of describing all transformations in the cycle when complex reagents and co-enzymes are replaced by prebiotic proxies such H2, H2O, and CO2. A comprehensive description of the enzymatic rTCA cycle is provided by Steffens et al. which was the starting point for developing our uncatalyzed enzymatic cycle. The resulting uncatalyzed cycle (Fig. ) is similar to that studied by Zubarev et al. but provides additional mechanistic details necessary for transition state analysis. The rTCA cycle can be started from any point along the cycle but as per the numbering conventions used in this study, the cycle commences with the reduction of oxaloacetate (1) to form malate (2) through carbonyl reduction. This enzymatic step is facilitated by nicotinamide adenine dinucleotide (NADH) and is substituted here by H2. The rationale for using H2 is that it is a plausible prebiotic reductant with sufficient reducing power to drive the cycle under ambient conditions. The resulting malate (2) undergoes dehydration to yield fumarate (3), which then undergoes alkene reduction with the addition of an H2 to generate succinate. The conversion of succinate (4) to 2-oxoglutarate (7) represents a key transformation that differs significantly between enzymatic and uncatalyzed pathways. While the modern enzymatic pathway employs coenzyme A (CoA) and adenosine triphosphate (ATP), the primitive uncatalyzed pathway must proceed through simpler intermediates. By substituting the CoA group with a hydrogen atom (-H), the reaction can proceed through three elementary b2f2 steps. First, H2 addition reduces succinate (4) to 4-dihydroxybutanoic acid (5), followed by dehydration to form succinic semialdehyde (6), and finally CO2 addition generates 2-oxoglutarate (7). This decomposition illustrates the substitutions necessary still effect the non-catalyzed cycle in a prebiotic environment. 2oxoglutarate (7) then undergoes a subsequent two-step reaction of carboxylation by CO2 addition to form oxalosuccinic acid (8) and carbonyl reduction by H2 addition to form isocitrate (9) in the uncatalyzed cycle. In the enzymatic counterpart, this reaction uses nicotinamide adenine dinucleotide phosphate (NADPH) and a proton. In the uncatalyzed cycle, the isocitrate (9) intermediate is converted to citrate (11) through an isomerization reaction, which is decomposed into a two-step elementary b2f2 mechanism involving dehydration to form aconitic acid (10) followed by hydration step to form citrate (11). The final step in the cycle involves C-C bond cleavage resulting in the formation of acetate and oxaloacetate (1), thereby completing the rTCA cycle.