COVALENT AND NONCOVALENT DIMERS OF THE CYANIDE-RESISTANT ALTERNATIVE OXIDASE PROTEIN IN HIGHER-PLANT MITOCHONDRIA AND THEIR RELATIONSHIP TOENZYME-ACTIVITY

Evidence for a mixed population of covalently and noncovalently associ ated dimers of the cyanide-resistant alternative oxidase protein in pl ant mitochondria is presented. High molecular mass (oxidized) species of the alternative oxidase protein, having masses predicted for homodi mers, appeared on immunoblots when the sulfhydryl reductant, dithiothr eitol (DTT), was omitted from sodium dodecyl sulfate-polyacrylamide ge l sample buffer. These oxidized species were observed in mitochondria from soybean (Glycine max [L.] Merr. cv Ransom), Sauromatum guttatum S chott, and mung bean (Vigna radiata [L.] R. Wilcz). Reduced species of the alternative oxidase were also present in the same mitochondrial s amples. The reduced and oxidized species in isolated soybean cotyledon mitochondria could be interconverted by incubation with the sulfhydry l reagents DTT and azodicarboxylic acid bis(dimethylamide) (diamide). Treatment with chemical cross-linkers resulted in cross-linking of the reduced species, indicating a noncovalent dimeric association among t he reduced alternative oxidase molecules. Alternative pathway activity of soybean mitochondria increased following reduction of the alternat ive oxidase protein with DTT and decreased following oxidation with di amide, indicating that electron flow through the alternative pathway i s sensitive to the sulfhydryl/disulfide redox poise. In mitochondria f rom S. guttatum floral appendix tissue, the proportion of the reduced species increased as development progressed through thermogenesis.