Mutagenesis of the Sauromatum guttatum alternative oxidase reveals features important for oxygen binding and catalysis.
The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in mitochondria of all higher plants studied to date. To investigate the role of highly conserved amino acid residues in catalysis we have expressed site-directed mutants of Cys-172, Thr-179, Trp-206, Tyr-253, and Tyr-299 in AOX in the yeast Schizosaccharomyces pombe. Assessment of AOX activity in isolated yeast mitochondria reveals that mutagenesis of Trp-206 to phenylalanine or tyrosine abolishes activity, in contrast to that observed with either Tyr-253 or 299 both mutants of which retained activity. None of the mutants exhibited sensitivity to Q-like inhibitors that differed significantly from the wild type AOX. Interestingly, however, mutagenesis of Thr-179 or Cys-172 (a residue implicated in AOX regulation by alpha-keto acids) to alanine not only resulted in a decrease of maximum AOX activity but also caused a significant increase in the enzyme's affinity for oxygen (4- and 2-fold, respectively). These results provide important new insights in the mechanism of AOX catalysis and regulation by pyruvate.
Crichton
Paul G
PG
Department of Biochemistry and Biomedical Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
Albury
Mary S
MS
Affourtit
Charles
C
Moore
Anthony L
AL
D019943:Amino Acid Substitution; D029064:Araceae / Q000201:enzymology* / Q000235:genetics*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics; D000099188:Alternative Oxidase
D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010100:Oxygen; D010940:Plant Proteins; D011994:Recombinant Proteins; D000099188:Alternative Oxidase