Regulation of the Cyanide-resistant Alternative Oxidase of Plant Mitochondria

Abstract

The cyanide-resistant alternative oxidase of plant mitochondria is a homodimeric protein whose activity can be regulated by a redox-sensitive intersubunit sulfhydryl/disulfide system and by ␣-keto acids. After determining that the Arabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesis of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala. Using diamide oxidation and chemical cross-linking of the protein expressed in Escherichia coli, Cys-78 was shown to be: 1) the Cys residue involved in the sulfhydryl/disulfide system; and 2) not required for subunit dimerization. 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 insensitive to pyruvate, consistent with ␣-keto acid activation occurring through a thiohemiacetal. These results indicate that Cys-78 serves as both the regulatory sulfhydryl/disulfide and the site of activation by ␣-keto acids. In light of these results, the previously observed effects of sulfhydryl reagents on the alternative oxidase of isolated soybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of ␣-keto acid activation and of the regulatory sulfhydryl/disulfide system.In all plants and many fungi, a cyanide-resistant, alternative respiratory pathway branches from the cytochrome pathway at the ubiquinone pool in mitochondria (1). Electron flow through this alternative pathway is not coupled to ATP synthesis at two of the three proton translocation sites. In specific thermogenic floral tissues of some plants, a high rate of electron flow through the alternative pathway generates heat and volatilizes compounds that attract insect pollinators (2). In non-thermogenic plant tissues, the alternative pathway may allow continued turnover of carbon skeletons through glycolysis and the tricarboxylic acid cycle when the ATP concentration is high and inhibits ATP synthase activity (3) so that the pathway can function as an energy overflow mechanism (4). However, because the alternative pathway can be active even when the cytochrome pathway is not saturated (5-7), it is likely that the two pathways are regulated in concert to balance ubiquinone pool oxidation/reduction and carbon skeleton turnover in response to cytosolic ATP levels (8, 9).In recent years, a great deal of progress has been made in understanding the post-translational regulation of the alternative oxidase, the terminal oxidase that comprises the alternative respiratory pathway. The alternative oxidase is a homodimeric protein in the inner mitochondrial membrane and the oxidation/reduction state of an intersubunit disulfide bond has been demonstrated to regulate its activity (10, 11). A second mechanism of activation of the alternative oxidase involves ␣-keto acids, notably pyruvate (12), which significan...

A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation

“…The C99S m u t a n t of G m A O X 3 , on the other hand, was not covalently linked upon oxidation with diamide ( Fig. 1), as shown previously for C to A mutants of A O X [20,21]. We consistently observed a higher level of expression of the C99S m u t a n t than of wild-type G m A O X 3 ( Fig.…”

“…(Fig. 2F), as observed for C to A mutants of AOX in other systems [20,21]. The slow rate of 02 uptake observed after addition of myxothiazol was unlikely to be due to the presence of endogenous tobacco AOX, since it was not stimulated by pyruvate (Fig.…”

“…We have expressed mutated soybean AOX3, in which C99 has been converted to serine, in tobacco plants, and have confirmed the results of [20,21]. However, while this mutated A O X protein cannot be activated by pyruvate, it is activated by succinate and cannot be dimerised by oxidation of sulphydryl groups.…”

“…Recently, site-directed mutagenesis has been used to identify a particular cysteine residue near the N-terminus of AOX 1 which is responsible for the disulphide linkage of subunits and also participates in the activation by c~-ketoacids [20,21]. Expression in Escherichia coli of Arabidopsis AOX sequences in which the cysteine residue had been converted to alanine resulted in an enzyme which could not be covalently linked but was poorly active and did not respond to pyruvate [20].…”

“…Expression in Escherichia coli of Arabidopsis AOX sequences in which the cysteine residue had been converted to alanine resulted in an enzyme which could not be covalently linked but was poorly active and did not respond to pyruvate [20]. Conversion of the same cysteine to glutamate resulted in a permanently active enzyme even in the absence of pyruvate, although the activity was somewhat less than that of the wild-type enzyme [20]. Homologous expression of the corresponding tobacco C to A mutant yielded plants whose mitochondria contained AOX which could not be oxidised but remained largely inactive in isolated organelles [21].…”