THE REACTION OF THE SOYBEAN COTYLEDON MITOCHONDRIAL CYANIDE-RESISTANTOXIDASE WITH SULFHYDRYL-REAGENTS SUGGESTS THAT ALPHA-KETO ACID ACTIVATION INVOLVES THE FORMATION OF A THIOHEMIACETAL

The cyanide-resistant alternative oxidase of plant mitochondria is kno wn to be activated by alpha-keto acids, such as pyruvate, and by the r eduction of a disulfide bond that bridges the two subunits of the enzy me homodimer. When the regulatory cysteines are oxidized, the inactiva ted enzyme is much less responsive to pyruvate than when these groups are reduced. When soybean cotyledon mitochondria were isolated in the presence of iodoacetate or N-ethylmaleimide, the intermolecular disulf ide bond did not form and the alternative oxidase was present only as a noncovalently associated dimer. N-Ethylmaleimide inhibited alternati ve oxidase activity, but iodoacetate was found to stimulate activity m uch like pyruvate, including enhancing the enzyme's apparent affinity for reduced ubiquinone. The presence of pyruvate or iodoacetate blocke d inhibition of the enzyme by N-ethylmaleimide, indicating that all th ree compounds acted at the same sulfhydryl group on the alternative ox idase protein, The site of pyruvate and iodoacetate action was shown t o be a different sulfhydryl than that involved in the redox-active reg ulatory disulfide bond, because iodoacetate bound to the alternative o xidase at the activating site even when the redox-active regulatory su lfhydryls were oxidized. Given the nature of the covalent adduct forme d by the reaction of iodoacetate with sulfhydryls, the activation of t he alternative oxidase by alpha-keto acids appears to involve the form ation of a thiohemiacetal.