The cyanide-resistant alternative oxidases from the fungi Pichia stipitis and Neurospora crassa are monomeric and lack regulatory features of the plant enzyme.
Both plant and fungal mitochondria have cyanide-resistant alternative oxidases that use reductant from the mitochondrial ubiquinone pool to reduce oxygen to water in a reaction that conserves no energy for ATP synthesis. The dimeric plant alternative oxidase is relatively inactive when its subunits are linked by a disulfide bond. When this bond is reduced, the enzyme can then be stimulated by its activators, alpha-keto acids. A Cys in the N-terminal section of the protein is responsible for both of these features. We examined the alternative oxidases in mitochondria isolated from two fungi Neurospora crassa and Pichia stipitis for dimeric structure, ability to form an intermolecular disulfide, and sensitivity to alpha-keto acids. Neither of the two fungal alternative oxidases could be covalently linked by diamide, which induces disulfide bond formation between nearby Cys residues, nor could they be cross-linked by a Lys-specific reagent or glutaraldehyde at concentrations which cross-link the plant alternative oxidase dimer completely. Alternative oxidase activity in fungal mitochondria was not stimulated by the alpha-keto acids pyruvate and glyoxylate. Pyruvate did stimulate activity when succinate was the respiratory substrate, but this was not a direct effect on the alternative oxidase. In contrast, added GMP was a strong activator of fungal alternative oxidase activity. Analysis of plant and fungal alternative oxidase protein sequences revealed a unique domain of about 40 amino acids surrounding the regulatory Cys in the plant sequences that is not present in the fungal sequences. This domain may be where dimerization of the plant enzymes occurs. In contrast to plant enzymes, the fungal alternative oxidases studied here are monomeric and their activities are independent of alpha-keto acids.
Umbach
A L
AL
DCMB Group/Botany Department, Duke University, Durham, North Carolina 27708, USA.
Siedow
J N
JN
D000143:Acids / Q000378:metabolism; D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000378:metabolism; D003486:Cyanides / Q000494:pharmacology*; D019281:Dimerization; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D005615:Freezing; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D018506:Gene Expression Regulation, Plant; D006873:Hydroquinones / Q000494:pharmacology; D015151:Immunoblotting; D008928:Mitochondria / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009243:NAD / Q000494:pharmacology; D009492:Neurospora crassa / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D010101:Oxygen Consumption; D010843:Pichia / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019289:Pyruvic Acid / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology; D019802:Succinic Acid / Q000494:pharmacology
D000143:Acids; D003432:Cross-Linking Reagents; D003486:Cyanides; D004220:Disulfides; D005656:Fungal Proteins; D006873:Hydroquinones; D010940:Plant Proteins; D009243:NAD; D019289:Pyruvic Acid; D019802:Succinic Acid; D010088:Oxidoreductases; C016421:duroquinol