Regulation of the cyanide-resistant alternative oxidase of plant mitochondria. Identification of the cysteine residue involved in alpha-keto acid stimulation and intersubunit disulfide bond formation.
The cyanide-resistant alternative oxidase of plant mitochondria is a homodimeric protein whose activity can be regulated by a redox-sensitive intersubunit sulfhydryl/disulfide system and by alpha-keto acids. After determining that the Arabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesis of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala. Using diamide oxidation and chemical cross-linking of the protein expressed in Escherichia coli, Cys-78 was shown to be: 1) the Cys residue involved in the sulfhydryl/disulfide system; and 2) not required for subunit dimerization. The C128A mutant was stimulated by pyruvate, while the C78A mutant protein had little activity and displayed no stimulation by pyruvate. Mutating Cys-78 to Glu produced an active enzyme which was insensitive to pyruvate, consistent with alpha-keto acid activation occurring through a thiohemiacetal. These results indicate that Cys-78 serves as both the regulatory sulfhydryl/disulfide and the site of activation by alpha-keto acids. In light of these results, the previously observed effects of sulfhydryl reagents on the alternative oxidase of isolated soybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of alpha-keto acid activation and of the regulatory sulfhydryl/disulfide system.
Rhoads
D M
DM
Developmental, Cell, and Molecular Biology/Botany Department, Duke University, Durham, North Carolina 27708-1000, USA.
Umbach
A L
AL
Sweet
C R
CR
Lennon
A M
AM
Rauch
G S
GS
Siedow
J N
JN
D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology*; D003486:Cyanides / Q000494:pharmacology*; D003545:Cysteine / Q000032:analysis* / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D007461:Iodoacetates / Q000378:metabolism; D007651:Keto Acids / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D024101:Mitochondrial Proteins; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010940:Plant Proteins; D013025:Glycine max / Q000201:enzymology; D000099188:Alternative Oxidase
D003486:Cyanides; D003545:Cysteine; D004220:Disulfides; D007461:Iodoacetates; D007651:Keto Acids; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D000099188:Alternative Oxidase