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 characterised. 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 groups, 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 hydroxyl 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.
Albury
Mary S
MS
Department of Chemistry & Biochemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
Elliott
Catherine
C
Moore
Anthony L
AL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000968:Antimycin A / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D010940:Plant Proteins; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism; D000099188:Alternative Oxidase
D000968:Antimycin A; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D029702:Schizosaccharomyces pombe Proteins; D014451:Ubiquinone; D000099188:Alternative Oxidase; C432586:ubiquinol oxidase; C003741:ubiquinol