diamide and glyoxylic-acid

Sacred lotus (Nelumbo nucifera) regulates temperature in its floral chamber to 32 degrees C to 35 degrees C across ambient temperatures of 8 degrees C to 40 degrees C with heating achieved through high alternative pathway fluxes. In most alternative oxidase (AOX) isoforms, two cysteine residues, Cys(1) and Cys(2), are highly conserved and play a role in posttranslational regulation of AOX. Further control occurs via interaction of reduced Cys(1) with alpha-keto acids, such as pyruvate. Here, we report on the in vitro regulation of AOX isolated from thermogenic receptacle tissues of sacred lotus. AOX protein was mostly present in the reduced form, and only a small fraction could be oxidized with diamide. Cyanide-resistant respiration in isolated mitochondria was stimulated 4-fold by succinate but not pyruvate or glyoxylate. Insensitivity of the alternative pathway of respiration to pyruvate and the inability of AOX protein to be oxidized by diamide suggested that AOX in these tissues may lack Cys(1). Subsequently, we isolated two novel cDNAs for AOX from thermogenic tissues of sacred lotus, designated as NnAOX1a and NnAOX1b. Deduced amino acid sequences of both confirmed that Cys(1) had been replaced by serine; however, Cys(2) was present. This contrasts with AOXs from thermogenic Aroids, which contain both Cys(1) and Cys(2). An additional cysteine was present at position 193 in NnAOX1b. The significance of the sequence data for regulation of the AOX protein in thermogenic sacred lotus is discussed and compared with AOXs from other thermogenic and nonthermogenic species.

Topics: Amino Acid Sequence; Cysteine; Diamide; Disulfides; Glyoxylates; Immunoblotting; Isoenzymes; Mitochondria; Mitochondrial Proteins; Molecular Sequence Data; Nelumbo; Oxidoreductases; Oxygen; Phylogeny; Plant Proteins; Protein Multimerization; Pyruvic Acid; Succinates; Temperature

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 alpha-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 alpha-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.

Topics: Amino Acid Sequence; Amino Acid Substitution; Arabidopsis; Arabidopsis Proteins; Cysteine; Diamide; Disulfides; Glyoxylates; Mitochondrial Proteins; Mutagenesis, Site-Directed; Oxidoreductases; Plant Proteins; Pyruvic Acid