Abstract
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.
Highlights
Plant mitochondria possess a terminal cyanide-resistant alternative oxidase in the electron transport system of the inner membrane [1, 2]
When either iodoacetate or NEM was present in the buffers used to isolate mitochondria from soybean cotyledons, no oxidized alternative oxidase dimers were found in the purified mitochondria, whereas mitochondria isolated without the sulfhydryl reagents had pronounced levels of covalently linked dimers (Fig. 2A)
The oxidant diamide markedly enhanced the relative level of covalently crosslinked alternative oxidase dimers, but failed to oxidize the sulfhydryl reagent-reacted alternative oxidase to the covalently linked dimeric form (Fig. 2B), indicating that both sulfhydryl reagents were taken up by the mitochondria during the course of the isolation procedure and had reacted with the sulfhydryl groups involved in the intermolecular disulfide linkage
Summary
Plant Material—Soybean seedlings (Glycine max (L.) Merr. cv Ransom, Essex, or FF583) were grown in a greenhouse as described previously [11]. When used during the course of activity assays, sulfhydryl reagents were added to a final concentration of 5 mM from stocks made on the day of the experiment. For experiments in which the effects of diamide and DTT on alternative oxidase activity were determined, mitochondria isolated with or without iodoacetate were treated with these reagents (3 and 20 mM final concentrations, respectively) and washed twice in excess volume of wash buffer before any subsequent assays or procedures, as described previously [11]. The NADH dehydrogenase activity and the level of ubiquinone pool reduction (Qr) were increased by titrating with incremental additions of CaCl2, added from a stock of 100 mM CaCl2 in 20 mM MOPS, pH 7.4, up to a final concentration of 2 mM. Ϫ I-Ac mitochondriaa Uninhibited ϩ 6 M myxothiazol ϩ 6 M myxothiazol ϩ 5 mM pyruvate ϩ I-Ac mitochondriaa Uninhibited ϩ 6 M myxothiazol ϩ 6 M myxothiazol ϩ 5 mM pyruvate ϩ NEM mitochondriab Uninhibited ϩ 6 M myxothiazol ϩ 6 M myxothiazol ϩ 5 mM pyruvate
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