Ubiquinol-binding site in the alternative oxidase Mutagenesis reveals features important for substrate binding and inhibition

The alternative oxidase (AOX) is a non protonmotive ubiquinol oxidase that is found in all plants some fungi green algae bacteria and pathogenic protozoa The lack of AOX in the mammalian host renders this protein an important potential therapeutic target in the treatment of pathogenic protozoan infections Bioinformatic searches revealed that within a putative ubiquinol-binding crevice in AOX Gln242 Asn247 Tyr253 Ser256 His261 and Arg262 were highly conserved To confirm that these amino-acid residues are important for ubiquinol-binding and hence activity substitution mutations were generated and char acterised Assessment of AOX activity in isolated Schizosaccharomyces pombe mitochondria revealed that mutation of either Gln242 Ser256 His261 and Arg262 resulted in >90% inhibition of antimycin A insensitive respiration suggesting that hydroxyl guanidino imidazole group polar and charged residues in addition to the size of the amino-acid chain are Important for ubiquinone binding Substitution of Asn247 with glutamine or Tyr253 with phenylalanine had little effect upon the respiratory rate indicating that these residues are not critical for AOX activity However replacement of Tyr253 by alanine resulted in a 72% loss of activity suggesting that the benzoquinone group and not hydro group is Important for quinol binding These results provide important new insights into the ubiquinol binding site of the alternative oxidase the identity of which maybe Important for future rational drug design (C) 2010 Elsevier BV All rights reserved