Inhibition of F1 -ATPase from Trypanosoma brucei by its regulatory protein inhibitor TbIF1

Abstract

Hydrolysis of ATP by the mitochondrial F-ATPase is inhibited by a protein called IF₁ . In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF₁ , is expressed exclusively in the procyclic stage, where the F-ATPase is synthesizing ATP. In the bloodstream stage, where TbIF₁ is absent, the F-ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF₁ that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F₁ -ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C-terminal portion of TbIF₁ facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF₁ , the inhibitory capacity of the C-terminally truncated TbIF₁ diminishes with decreasing pH, similar to full length TbIF₁ . This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C-terminally truncated TbIF₁ form dimers and monomers, respectively. TbIF₁ has no effect on bovine F₁ -ATPase, and this difference in the mechanism of regulation of the F-ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases.

Keywords: IF 1; Trypanosoma brucei; ATP synthase; F-ATPase; F1Fo-ATPase; TbIF1; enzyme inhibitor; trypanosome.