Regulation of plant alternative oxidase activity: A tale of two cysteines

Two Cys residues, Cys_I and Cys_II, are present in most plant alternative oxidases (AOXs). Cys_I inactivates AOX by forming a disulfide bond with the corresponding Cys_I residue on the adjacent subunit of the AOX homodimer. When reduced, Cys_I associates with α-keto acids, such as pyruvate, to activate AOX, an effect mimicked by charged amino acid substitutions at the Cys_I site. Cys_II may also be a site of AOX activity regulation, through interaction with the small α-keto acid, glyoxylate. Comparison of Arabidopsis AOX1a (AtAOX1a) mutants with single or double substitutions at Cys_I and Cys_II confirmed that glyoxylate interacted with either Cys, while the effect of pyruvate (or succinate for AtAOX1a substituted with Ala at Cys_I) was limited to Cys_I. A variety of Cys_II substitutions constitutively activated AtAOX1a, indicating that neither the catalytic site nor, unlike at Cys_I, charge repulsion is involved. Independent effects at each Cys were suggested by lack of Cys_II substitution interference with pyruvate stimulation at Cys_I, and close to additive activation at the two sites. However, results obtained using diamide treatment to covalently link the AtAOX1a subunits by the disulfide bond indicated that Cys_I must be in the reduced state for activation at Cys_II to occur.