title: Glutathionylation of the iron superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis.
abstract: Our previous work showed that the adduct between beta-mercaptoethanol and the single cysteine residue (Cys57) in superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis (PhSOD) reduces the enzyme inactivation by peroxynitrite. In this work, immunoblotting experiments prove that peroxynitrite inactivation of PhSOD involves formation of nitrotyrosine residue(s). In order to study the role of Cys57 as a redox-sensor residue modifiable by cellular thiols, a recombinant PhSOD and two Cys57 mutants were produced and characterized. Recombinant and mutant enzymes share similar activity and peroxynitrite inactivation, but different reactivity towards three glutathione forms. Indeed, oxidized glutathione and S-nitrosoglutathione, but reduced glutathione, lead to S-glutathionylation of recombinant PhSOD. This new covalent modification for a Fe-SOD does not occur in both Cys57 mutants, thus indicating that its target is Cys57. Moreover, mass spectrometry analysis confirmed that S-glutathionylation of Cys57 takes place also with endogenous PhSOD. Formation of this mixed disulfide in PhSOD protects the enzyme from tyrosine nitration and peroxynitrite inactivation. PhSOD undergoes S-glutathionylation during its overproduction in E. coli cells and in a growing culture of P. haloplanktis. In both cases the extent of glutathionylated PhSOD is enhanced upon cell exposure to oxidative agents. We suggest that S-glutathionylation of PhSOD could represent a further cold-adaptation strategy to improve the antioxidant cellular defence mechanism.
authors.affiliation: Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy.
authors.forename: Immacolata
authors.initials: I
authors.lastname: Castellano
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Ruocco
authors.forename: Francesca
authors.initials: F
authors.lastname: Cecere
authors.forename: Antimo
authors.initials: A
authors.lastname: Di Maro
authors.forename: Angela
authors.initials: A
authors.lastname: Chambery
authors.forename: Andzelika
authors.initials: A
authors.lastname: Michniewicz
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Parlato
authors.forename: Mariorosario
authors.initials: M
authors.lastname: Masullo
authors.forename: Emmanuele
authors.initials: E
authors.lastname: De Vendittis
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli; D005051:Eubacterium / Q000201:enzymology*; D005978:Glutathione / Q000378:metabolism*; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism; D031161:Pseudoalteromonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D030421:Peroxynitrous Acid; D014443:Tyrosine; D013482:Superoxide Dismutase; D005978:Glutathione; D003545:Cysteine