The reaction of the soybean cotyledon mitochondrial cyanide-resistant oxidase with sulfhydryl reagents suggests that alpha-keto acid activation involves the formation of a thiohemiacetal.
The cyanide-resistant alternative oxidase of plant mitochondria is known to be activated by alpha-keto acids, such as pyruvate, and by the reduction of a disulfide bond that bridges the two subunits of the enzyme homodimer. When the regulatory cysteines are oxidized, the inactivated 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 disulfide bond did not form and the alternative oxidase was present only as a noncovalently associated dimer. N-Ethylmaleimide inhibited alternative oxidase activity, but iodoacetate was found to stimulate activity much like pyruvate, including enhancing the enzyme's apparent affinity for reduced ubiquinone. The presence of pyruvate or iodoacetate blocked inhibition of the enzyme by N-ethylmaleimide, indicating that all three compounds acted at the same sulfhydryl group on the alternative oxidase protein. The site of pyruvate and iodoacetate action was shown to be a different sulfhydryl than that involved in the redox-active regulatory disulfide bond, because iodoacetate bound to the alternative oxidase at the activating site even when the redox-active regulatory sulfhydryls were oxidized. Given the nature of the covalent adduct formed by the reaction of iodoacetate with sulfhydryls, the activation of the alternative oxidase by alpha-keto acids appears to involve the formation of a thiohemiacetal.
Umbach
A L
AL
Developmental, Cell, and Molecular Biology Group/Botany Department, Duke University, Durham, North Carolina 27708-1000, USA.
Siedow
J N
JN
D000080:Acetals / Q000378:metabolism*; D018548:Cotyledon / Q000201:enzymology*; D005033:Ethylmaleimide / Q000378:metabolism; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D007651:Keto Acids / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D013025:Glycine max / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000378:metabolism*
D000080:Acetals; D007461:Iodoacetates; D007651:Keto Acids; D013439:Sulfhydryl Reagents; D010088:Oxidoreductases; C020094:duroquinol oxidase; D005033:Ethylmaleimide; D019807:Iodoacetic Acid