Structure of the plant alternative oxidase. Site-directed mutagenesis provides new information on the active site and membrane topology.
All higher plants and many fungi contain an alternative oxidase (AOX), which branches from the cytochrome pathway at the level of the quinone pool. In an attempt, first, to distinguish between two proposed structural models of this di-iron protein, and, second, to examine the roles of two highly conserved tyrosine residues, we have expressed an array of site-specific mutants in Schizosaccharomyces pombe. Mitochondrial respiratory analysis reveals that S. pombe cells expressing AOX proteins in which Glu-217 or Glu-270 were mutated, no longer exhibit antimycin-resistant oxygen uptake, indicating that these residues are essential for AOX activity. Although such data corroborate a model that describes the AOX as an interfacial membrane protein, they are not in full agreement with the most recently proposed ligation sphere of its di-iron center. We furthermore show that upon mutation of Tyr-253 and Tyr-275 to phenylalanines, AOX activity is fully maintained or abolished, respectively. These data are discussed in reference to the importance of both residues in the catalytic cycle of the AOX.
Albury
Mary S
MS
Biochemistry Department, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom. M.S.Albury@sussex.ac.uk
Affourtit
Charles
C
Crichton
Paul G
PG
Moore
Anthony L
AL
D000935:Antifungal Agents / Q000494:pharmacology; D000968:Antimycin A / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D019069:Cell Respiration / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D024101:Mitochondrial Proteins; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000494:pharmacology; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010101:Oxygen Consumption; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology; D000099188:Alternative Oxidase
D000935:Antifungal Agents; D000968:Antimycin A; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D024101:Mitochondrial Proteins; D009243:NAD; D010088:Oxidoreductases; D010940:Plant Proteins; D014475:Uncoupling Agents; D000099188:Alternative Oxidase