RESUMO
Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.
Assuntos
Citocromos c/química , Citocromos c/metabolismo , Ácido Peroxinitroso/química , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Transporte de Elétrons , Cavalos , Mitocôndrias Cardíacas/enzimologia , Dados de Sequência Molecular , Peroxidase/química , Ácido Peroxinitroso/metabolismo , Cianeto de Potássio/química , Ratos , Cianeto de Sódio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tetranitrometano/química , Fatores de TempoRESUMO
Nitration of ovine placental lactogen (oPL) with a 10-fold molar excess of tetranitromethane over protein content resulted in the modification of 0.8 tyrosine residue. No conformational changes were observed by either fourth-derivative spectral analysis or circular dichroism. Nitration significantly decreased the binding capacity of the hormone to lactogenic and somatogenic rat liver receptors. This binding capacity was not restored by reduction of the nitro groups, thus indicating that the decrease was not due to the difference in pK between tyrosine and nitrotyrosine. The nitrotyrosine-containing peptide was isolated from a tryptic digest by HPLC and its modification extent was of 67%, which is consistent with the decrease in binding capacities (65% and 70%). Its amino acid sequence was determined and the modified tyrosine residue was identified as Tyr-46. These results provide the first evidence of the involvement of a tyrosine residue in the binding of oPL to both lactogenic and somatogenic receptors. This tyrosine appears to be a shared binding epitope between oPL and the prolactins.
Assuntos
Lactogênio Placentário/química , Lactogênio Placentário/metabolismo , Receptores de Peptídeos/metabolismo , Receptores da Somatotropina/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Feminino , Hormônio do Crescimento/antagonistas & inibidores , Hormônio do Crescimento/metabolismo , Fígado/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Lactogênio Placentário/antagonistas & inibidores , Ligação Proteica , Conformação Proteica , Ratos , Ovinos , Espectrofotometria , Tetranitrometano/química , Tripsina/metabolismoRESUMO
The bovine growth hormone dimeric form covalently stabilized by cross-linking with dimethyl suberimidate (DMS) and the hormone modified by DMS without forming covalent links with other hormone molecules (DMS-bGH) were oxidized with chloramine-T at molar-ratios of 2 and 50 with respect to methionine content. The extent of oxidation undergone by each methionine residue, estimated on the purified tryptic peptides, closely resembled that obtained for the native hormone, thus suggesting that methionine residues are not involved in the protomers interaction area. Evaluation of the reactivity of tyrosine residues toward tetranitromethane indicated that, in both the covalent dimer and DMS-bGH, tyrosine residues 35, 174 and 142 are the more susceptible to undergo reaction. Net charges can be induced in the iodotyrosine residues in the iodinated hormone, by setting the pH at 10.5. At this pH, dissociation of a fraction of uniformly iodinated hormone was observed in the derivatives containing 2 or more iodine atoms, indicating that tyrosine residues might integrate the contact area between protomers.