Activation of the plant mitochondrial alternative oxidase: insights from site-directed mutagenesis

• Published in: Biochimica et biophysica acta Vol. 1554; no. 1; pp. 118 - 128
• Main Authors: Umbach, Ann L., Gonzàlez-Meler, Miquel A., Sweet, Charles R., Siedow, James N.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 22-04-2002
• Subjects: Activity regulation; Adenosine Triphosphate - metabolism; Alternative oxidase; Amino Acid Substitution; Arabidopsis - enzymology; Arabidopsis - ultrastructure; Catalysis; Cyanide-resistant respiration; Cysteine - metabolism; Dimerization; Enzyme Activation; Escherichia coli; Glutamic Acid - metabolism; Glyoxylates - pharmacology; Mitochondria - enzymology; Mitochondrial Proteins; Mutagenesis, Site-Directed; Oxidoreductases - genetics; Oxidoreductases - metabolism; Oxygen - metabolism; Plant mitochondria; Plant Proteins; Protein Conformation; Pyruvic Acid - pharmacology; Regulatory cysteine; Sequence Analysis, DNA; Serine - metabolism; Structure-Activity Relationship; Succinic Acid - metabolism; Water - metabolism; α-Keto acid; Plant mitochondria; Activity regulation; DTT; Diamide; SHAM; Alternative oxidase; Regulatory cysteine; EGS; DU; LDH; SDS-PAGE; Cyanide-resistant respiration; SDH; IOA; α-Keto acid
• ISSN: 0005-2728; 0006-3002; 1879-2650
• DOI: 10.1016/S0005-2728(02)00219-0

The homodimeric cyanide-resistant alternative oxidase of plant mitochondria reduces oxygen to water without forming ATP.
Arabidopsis thaliana alternative oxidase AOX1a is stimulated by pyruvate or other α-keto acids associating with a regulatory cysteine at position 78, by succinate in a serine-78 mutant, and by site-directed mutation of position 78 to glutamate. The mechanism of activation was explored with additional amino acid substitutions made at Cys-78 in AOX1a, which was functionally expressed in
Escherichia coli. Oxidases with positively charged substitutions (Lys and Arg) were insensitive to pyruvate or succinate but were more active than the wild type without pyruvate. Uncharged substitutions (Gln, Leu) produced an inactive enzyme. These results indicate that activation may be due to conformational changes caused by charge repulsion between the dimer subunits and not through a direct role of α-keto acids in catalysis. Oxygen isotope fractionation experiments suggest that the charge of the amino acid at position 78 also affects the entry of oxygen into the active site. Therefore, the N-terminal portion of the protein containing residue 78 can indirectly affect both catalysis at the diiron active site and the path of oxygen to that site. In addition, both positively and negatively substituted alternative oxidases were stimulated by glyoxylate, suggesting the presence of a second activation site, possibly Cys-128.