Abstract
Mitochondrial alternative oxidase (AOX) in plants is a non-proton-motive ubiquinol oxidase that is activated by redox mechanisms and 2-oxo acids. A comparative analysis of the AOX isoenzymes AOX1A, AOX1C, and AOX1D from Arabidopsis (Arabidopsis thaliana) revealed that cysteine residues, CysI and CysII, are both involved in 2-oxo acid activation, with AOX1A activity being more increased by 2-oxo acids than that of AOX1C and AOX1D. Substitution of cysteine in AOX1A by glutamate mimicked its activation by pyruvate or glyoxylate, but not in AOX1C and AOX1D. CysIII, only present in AOX1A, is not involved in activation by reduction or metabolites, but substitutions at this position affected activity. AOX1A carrying a serine residue at position CysI was activated by succinate, while correspondingly substituted variants of AOX1C and AOX1D were insensitive. Activation by glutamate at CysI and CysII is consistent with the formation of the thiohemiacetal, while succinate activation after changing CysI to serine suggests hemiacetal formation. Surprisingly, in AOX1A, replacement of CysI by alanine, which cannot form a (thio)hemiacetal, led to even higher activities, pointing to an alternative mechanism of activation. Taken together, our results demonstrate that AOX isoforms are differentially activated and that activation at CysI and CysII is additive.
Highlights
Mitochondrial alternative oxidase (AOX) in plants is a non-proton-motive ubiquinol oxidase that is activated by redox mechanisms and 2-oxo acids
Each AOX isoform was recombinantly expressed in E. coli strain BHH8, and membrane vesicles enriched in individual AOX isoenzymes were isolated (Selinski et al, 2016)
The activation pattern of AOX1D-CCS and -CCE is consistent with this hypothesis. Both mutant proteins are activated by pyruvate or glyoxylate to the same extent as the wild-type protein (2- to 3-fold increase), the basal activity of AOX1D-CCS is much lower than that of wild-type AOX1D-CCL (Figs. 3 and 4; Supplemental Fig. S4). These results demonstrate that both Cys residues at positions 64 (CysI) and CysII contribute to the activation of AOX1A, AOX1C, and AOX1D by pyruvate and glyoxylate, with CysI appearing to be strongly involved in metabolite activation while CysII represents a secondary, less effective activation site
Summary
Mitochondrial alternative oxidase (AOX) in plants is a non-proton-motive ubiquinol oxidase that is activated by redox mechanisms and 2-oxo acids. The gene expression of these isoforms is tissue specific, depends on the developmental stage of the plant, changes in cellular metabolism as well as various abiotic and biotic stress conditions, and is highly responsive to dysfunctions in the mitochondrial respiratory metabolism (Juszczuk and Rychter, 2003; Millenaar and Lambers, 2003; Zarkovic et al, 2005; Clifton et al, 2006; Van Aken et al, 2009; Feng et al, 2013; Vanlerberghe, 2013). When electron transport through the cytochrome c respiratory chain is limited using a surrogate mutant approach, an additional inactivation of aox1a leads to a more severe growth phenotype, even though AOX1D is highly expressed at the transcript and protein levels (Kühn et al, 2015). The balance between oxidized and reduced AOX1A protein depends on the redox state of the NAD(P)H pool, which itself is linked to mitochondrial metabolism (Vanlerberghe et al, 1995; Juszczuk and Rychter, 2003)
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