Bioengineering the Ice Worm Complex V ATP6 subunit to Increase Energy Production

Abstract. Glacier ice worms, Mesenchytraeus solifugus, are among a few animals that complete their life cycle in hydrated glacier ice. These worms are distinguished from congener species by relatively high intracellular ATP levels, likely associated with a lateral gene transfer event adding 18 amino acids (aa) to the carboxy terminal of the Complex V F_O ATP6 subunit, a major regulator of ATP synthesis. By examining the kinetic profiles of respective 13/18 aa natural variants fused with E. coli AtpB (prokaryotic counterpart of ATP6) in the context of ice worm evolution, we conclude that the 18 aa variant predates the 13 aa variant, but the latter likely outcompetes the former in natural populations. Utilizing the 13 aa variant as a template, a panel of genetically engineered extension mutants were constructed and tested by Michaelis–Menten kinetics as a function of increasing [ADP]. Our data show that V_max can be increased significantly over natural 13/18 aa variants, and suggest a functional mechanism by which a dynamic tether transfers protons from the F_O exit pore to the mitochondrial lumen via the protonation/deprotonation of distal histidine residue(s).