Regulation of plant alternative oxidase activity: a tale of two cysteines.

Two Cys residues, CysI and CysII, are present in most plant alternative oxidases (AOXs). CysI inactivates AOX by forming a disulfide bond with the corresponding CysI residue on the adjacent subunit of the AOX homodimer. When reduced, CysI associates with α-keto acids, such as pyruvate, to activate AOX, an effect mimicked by charged amino acid substitutions at the CysI site. CysII 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 CysI and CysII confirmed that glyoxylate interacted with either Cys, while the effect of pyruvate (or succinate for AtAOX1a substituted with Ala at CysI) was limited to CysI. A variety of CysII substitutions constitutively activated AtAOX1a, indicating that neither the catalytic site nor, unlike at CysI, charge repulsion is involved. Independent effects at each Cys were suggested by lack of CysII substitution interference with pyruvate stimulation at CysI, 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 CysI must be in the reduced state for activation at CysII to occur.