The evolution and mechanistic versatility of the bacterial NADH dehydrogenases type II

balance. Although often non-essential, NDH-2 become crucial in species lacking complex I, serving as the primary electron entry point into the ETC. Their virtual absence in mammals makes these enzymes attractive targets for antimicrobial drug development and mitochondrial functional restoration. NDH-2 catalyse electron transfer from NADH to quinones, yet two distinct catalytic mechanisms have been described for members of the family: a classical ping-pong one or an atypical ternary mechanism involving the formation of a charge transfer complex (CTC). The molecular basis of these mechanisms remains unclear. Additionally, their occurrence among NDH-2 from different bacterial lineages is unknown. Here we combined molecular phylogenetics, ancestral sequence reconstruction, expression and biochemical characterisation of ancestral and modern enzymes, and molecular dynamics simulations to explore the mechanistic versatility of NDH-2 across Bacteria. Our results show the atypical ternary mechanism is restricted to the Firmicutes (Bacillota) lineage and correlates with the presence of a single substitution located at the bottom of the active site. This work provides an evolutionary framework for understanding the mechanistic versatility of NDH-2. Furthermore, it establishes a basis for drug discovery targeting pathogenic strains and opens avenues for developing innovative strategies to complement dysfunctional mitochondria.