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

Type II NADH dehydrogenases (NDH-2s) are accessory enzymes of the bacterial electron transport chain (ETC). While they functionally overlap with Complex I, their main role is not proton translocation but maintaining the intracellular NADH/NAD+ 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-2s catalyze electron transfer from NADH to quinones, yet two distinct catalytic mechanisms have been described for members of the family: a classical ping-pong mechanism and an atypical ternary mechanism involving the formation of a charge transfer complex (CTC). The molecular basis of these mechanisms remains unclear. Also, their occurrence among NDH-2s from different bacterial lineages in unknown. Here we combined molecular phylogenetics, ancestral sequence reconstruction, expression and biochemical characterization of ancestral and modern enzymes and, molecular dynamics simulations to explore the mechanistic versatility of NDH-2s across Bacteria. Our results show the atypical ternary mechanism is restricted to the Firmicutes (Bacillota) lineage and it is defined by the presence of a single substitution located at the bottom of the active site. This work provides an evolutionary framework for understanding NDH-2 mechanistic versatility. Besides, it establishes a basis for drug discovery targeting pathogenic strains and opens avenues to develop innovative strategies to complement dysfunctional mitochondria.