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 h omodimeric protein whose activity can be regulated by a redox-sensitiv e intersubunit sulfhydryl/disulfide system and by alpha-keto acids. Af ter determining that the Arabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesi s of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala. Using diamide oxi dation and chemical cross-linking of the protein expressed in Escheric hia coli, Cys-78 was shown to be: 1) the Cys residue involved in the s ulfhydryl/disulfide system; and 2) not required for subunit dimerizati on. 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 insensitiv e to pyruvate, consistent with alpha-keto acid activation occurring th rough a thiohemiacetal, These results indicate that Cys-78 serves as b oth the regulatory sulfhydryl/disulfide and the site of activation by alpha-keto acids. In light of these results, the previously observed e ffects of sulfhydryl reagents on the alternative oxidase of isolated s oybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of alpha-keto ac id activation and of the regulatory sulfhydryl/disulfide system.