RESUMEN
Cytochrome c (cyt c) can undergo reversible conformational changes under biologically relevant conditions. Revealing these alternative cyt c conformers at the cell and tissue level is challenging. A monoclonal antibody (mAb) identifying a key conformational change in cyt c was previously reported, but the hybridoma was rendered nonviable. To resurrect the mAb in a recombinant form, the amino-acid sequences of the heavy and light chains were determined by peptide mapping-mass spectrometry-bioinformatic analysis and used to construct plasmids encoding the full-length chains. The recombinant mAb (R1D3) was shown to perform similarly to the original mAb in antigen-binding assays. The mAb bound to a variety of oxidatively modified cyt c species (e.g., nitrated at Tyr74 or oxidized at Met80), which lose the sixth heme ligation (Fe-Met80); it did not bind to several cyt c phospho- and acetyl-mimetics. Peptide competition assays together with molecular dynamic studies support that R1D3 binds a neoepitope within the loop 40-57. R1D3 was employed to identify alternative conformations of cyt c in cells under oxidant- or senescence-induced challenge as confirmed by immunocytochemistry and immunoaffinity studies. Alternative conformers translocated to the nuclei without causing apoptosis, an observation that was further confirmed after pinocytic loading of oxidatively modified cyt c to B16-F1 cells. Thus, alternative cyt c conformers, known to gain peroxidatic function, may represent redox messengers at the cell nuclei. The availability and properties of R1D3 open avenues of interrogation regarding the presence and biological functions of alternative conformations of cyt c in mammalian cells and tissues.
Asunto(s)
Citocromos c , Hemo , Animales , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Citocromos c/química , Hemo/química , Hibridomas , Oxidación-Reducción , Melanoma Experimental , RatonesRESUMEN
The interaction between cytochrome c and cardiolipin is a relevant process in the mitochondrial redox homeostasis, playing roles in the mechanism of electron transfer to cytochrome c oxidase and also modulating cytochrome c conformation, reactivity and function. Peroxynitrite is a widespread nitrating agent formed in mitochondria under oxidative stress conditions, and can result in the formation of tyrosine nitrated cytochrome c. Some of the nitro-cytochrome c species undergo conformational changes at physiological pH and increase its peroxidase activity. In this work we evaluated the influence of cardiolipin on peroxynitrite-mediated cytochrome c nitration yields and site-specificity. Our results show that cardiolipin enhances cytochrome c nitration by peroxynitrite and targets it to heme-adjacent Tyr67. Cytochrome c nitration also modifies the affinity of protein with cardiolipin. Using a combination of experimental techniques and computer modeling, it is concluded that structural modifications in the Tyr67 region are responsible for the observed changes in protein-derived radical and tyrosine nitration levels, distribution of nitrated proteoforms and affinity to cardiolipin. Increased nitration of cytochrome c in presence of cardiolipin within mitochondria and the gain of peroxidatic activity could then impact events such as the onset of apoptosis and other processes related to the disruption of mitochondrial redox homeostasis.
Asunto(s)
Cardiolipinas/metabolismo , Cardiolipinas/farmacología , Citocromos c/química , Citocromos c/metabolismo , Nitratos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Tirosina/metabolismo , Animales , Sitios de Unión , Caballos , Cinética , Modelos Moleculares , Ácido Peroxinitroso/metabolismo , Conformación Proteica/efectos de los fármacos , Especificidad por SustratoRESUMEN
Human MnSOD is a homotetramer and represents an essential mitochondrial antioxidant enzyme, which catalyzes the dismutation of superoxide radicals (O2Ë-) at near diffusion-controlled rates. Under a variety of disease conditions and in the process of aging, nitric oxide (ËNO) can outcompete MnSOD and react with O2Ë- to yield the potent oxidant peroxynitrite (ONOO-). Then, peroxynitrite can promote the regio-specific nitration of MnSOD at active site tyrosine 34, which turns the enzyme inactive. In this review we assess the kinetic aspects of the formation of peroxynitrite in the presence of MnSOD and the biochemical mechanisms of peroxynitrite-mediated MnSOD nitration. In particular, the central role of the Mn atom in the reaction of the enzyme with peroxynitrite (k = 1.0 × 105 M-1 s-1 per tetramer at pH = 7.4 and T = 37 °C) and the catalysis of nitration at the active site are disclosed. Then, we analyze at the atomic level of detail how a single oxidative post-translational modification in the enzyme, namely the nitration of tyrosine 34, results in enzyme inactivation. Herein, kinetic, molecular, structural biology and computational studies are integrated to rationalize the specificity and impact of peroxynitrite-dependent MnSOD tyrosine nitration in vitro and in vivo from both functional and structural perspectives.
Asunto(s)
Ácido Peroxinitroso/farmacología , Superóxido Dismutasa/antagonistas & inhibidores , Catálisis , Humanos , Metales/química , Modelos Moleculares , Óxido Nítrico/metabolismo , Superóxido Dismutasa/química , Tirosina/químicaRESUMEN
Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible structure, particularly in the ferric form, such that it is able to sample a broad conformational space. Depending on the specific conditions, interactions, and cellular localization, different conformations may be stabilized, which differ in structure, redox properties, binding affinities, and enzymatic activity. The primary function is electron shuttling in oxidative phosphorylation, and is exerted by the so-called native cyt c in the intermembrane mitochondrial space of healthy cells. Under pro-apoptotic conditions, however, cyt c gains cardiolipin peroxidase activity, translocates into the cytosol to engage in the intrinsic apoptotic pathway, and enters the nucleus where it impedes nucleosome assembly. Other reported functions include cytosolic redox sensing and involvement in the mitochondrial oxidative folding machinery. Moreover, post-translational modifications such as nitration, phosphorylation, and sulfoxidation of specific amino acids induce alternative conformations with differential properties, at least in vitro. Similar structural and functional alterations are elicited by biologically significant electric fields and by naturally occurring mutations of human cyt c that, along with mutations at the level of the maturation system, are associated with specific diseases. Here, we summarize current knowledge and recent advances in understanding the different structural, dynamic, and thermodynamic factors that regulate the primary electron transfer function, as well as alternative functions and conformations of cyt c. Finally, we present recent technological applications of this moonlighting protein.
Asunto(s)
Citocromos c/metabolismo , Animales , Técnicas Biosensibles , Transporte de Electrón , Humanos , Cinética , Mitocondrias/enzimología , Oxidación-Reducción , TermodinámicaRESUMEN
Tyrosine nitration is an oxidative post-translational modification that can occur in proteins associated to hydrophobic bio-structures such as membranes and lipoproteins. In this work, we have studied tyrosine nitration in membranes using a model system consisting of phosphatidylcholine liposomes with pre-incorporated tyrosine-containing 23 amino acid transmembrane peptides. Tyrosine residues were located at positions 4, 8 or 12 of the amino terminal, resulting in different depths in the bilayer. Tyrosine nitration was accomplished by exposure to peroxynitrite and a peroxyl radical donor or hemin in the presence of nitrite. In egg yolk phosphatidylcholine liposomes, nitration was highest for the peptide with tyrosine at position 8 and dramatically increased as a function of oxygen levels. Molecular dynamics studies support that the proximity of the tyrosine phenolic ring to the linoleic acid peroxyl radicals contributes to the efficiency of tyrosine oxidation. In turn, α-tocopherol inhibited both lipid peroxidation and tyrosine nitration. The mechanism of tyrosine nitration involves a "connecting reaction" by which lipid peroxyl radicals oxidize tyrosine to tyrosyl radical and was fully recapitulated by computer-assisted kinetic simulations. Altogether, this work underscores unique characteristics of the tyrosine oxidation and nitration process in lipid-rich milieu that is fueled via the lipid peroxidation process.
Asunto(s)
Membrana Celular/metabolismo , Radicales Libres/metabolismo , Peroxidación de Lípido , Péptidos/metabolismo , Ácido Peroxinitroso/metabolismo , Tirosina/metabolismo , Amidinas/metabolismo , Secuencia de Aminoácidos , Membrana Celular/química , Hemina/metabolismo , Liposomas/química , Liposomas/metabolismo , Simulación de Dinámica Molecular , Oxidación-Reducción , Oxígeno/metabolismo , Péptidos/química , Tirosina/químicaRESUMEN
SIGNIFICANCE: "Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function. CRITICAL ISSUES: Identification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation. FUTURE DIRECTIONS: The use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.
Asunto(s)
Nitratos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Tirosina/metabolismo , Animales , Humanos , Espectrometría de Masas/métodos , Espectrometría de Masas/normas , Nitratos/química , Nitrosación , Proteínas/química , Proteoma/metabolismo , Proteómica/métodos , Sensibilidad y Especificidad , Tirosina/análogos & derivados , Tirosina/químicaRESUMEN
The specific and sensitive detection of peroxynitrite (ONOO-/ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. Herein, we validated the advantages of using fluorescein-boronate (Fl-B) as a highly sensitive fluorescent probe for the direct detection of peroxynitrite under biologically-relevant conditions in two different cell models. The synthesis of Fl-B was achieved by a very simply two-step conversion synthetic route with high purity (>99%) and overall yield (â¼42%). Reactivity analysis of Fl-B with relevant biological oxidants including hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and peroxynitrite were performed. The rate constant for the reaction of peroxynitrite with Fl-B was 1.7×106M-1s-1, a million times faster than the rate constant measured for H2O2 (k=1.7M-1s-1) and 2,700 faster than HOCl (6.2×102M-1s-1) at 37°C and pH 7.4. The reaction of Fl-B with peroxynitrite was significant even in the presence of physiological concentrations of CO2, a well-known peroxynitrite reactant. Experimental and simulated kinetic analyses confirm that the main oxidation process of Fl-B takes place with peroxynitrite itself via a direct bimolecular reaction and not with peroxynitrite-derived radicals. Fl-B was successfully applied for the detection of endogenously-generated peroxynitrite by endothelial cells and in macrophage-phagocyted parasites. Moreover, the generated data allowed estimating the actual intracellular flux of peroxynitrite. For instance, ionomycin-stimulated endothelial cells generated peroxynitrite at a rate of â¼ 0.1µMs-1, while immunostimulated macrophages do so in the order of â¼1µMs-1 inside T. cruzi-infected phagosomes. Fl-B revealed not to be toxic in concentrations up to 1mM for 24h. Cellular peroxynitrite detection was achieved by conventional laboratory fluorescence-based methods including flow cytometry and epi-fluorescence microscopy. Fl-B was shown to be more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite.
Asunto(s)
Ácidos Borónicos/química , Fluoresceínas/química , Colorantes Fluorescentes/síntesis química , Macrófagos/metabolismo , Ácido Peroxinitroso/análisis , Animales , Aorta/citología , Aorta/metabolismo , Bovinos , Línea Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Peróxido de Hidrógeno/metabolismo , Ácido Hipocloroso/metabolismo , Cinética , Macrófagos/citología , Macrófagos/parasitología , Ratones , Oxidación-Reducción , Ácido Peroxinitroso/biosíntesis , Fagocitosis/fisiología , Cultivo Primario de Células , Sensibilidad y Especificidad , Trypanosoma cruziRESUMEN
Human Mn-containing superoxide dismutase (hMnSOD) is a mitochondrial enzyme that metabolizes superoxide radical (O2(â¢-)). O2(â¢-) reacts at diffusional rates with nitric oxide to yield a potent nitrating species, peroxynitrite anion (ONOO(-)). MnSOD is nitrated and inactivated in vivo, with active site Tyr34 as the key oxidatively modified residue. We previously reported a k of â¼1.0 × 10(5) M(-1) s(-1) for the reaction of hMnSOD with ONOO(-) by direct stopped-flow spectroscopy and the critical role of Mn in the nitration process. In this study, we further established the mechanism of the reaction of hMnSOD with ONOO(-), including the necessary re-examination of the second-order rate constant by an independent method and the delineation of the microscopic steps that lead to the regio-specific nitration of Tyr34. The redetermination of k was performed by competition kinetics utilizing coumarin boronic acid, which reacts with ONOO(-) at a rate of â¼1 × 10(6) M(-1) s(-1) to yield the fluorescence product, 7-hydroxycoumarin. Time-resolved fluorescence studies in the presence of increasing concentrations of hMnSOD provided a k of â¼1.0 × 10(5) M(-1) s(-1), fully consistent with the direct method. Proteomic analysis indicated that ONOO(-), but not other nitrating agents, mediates the selective modification of active site Tyr34. Hybrid quantum-classical (quantum mechanics/molecular mechanics) simulations supported a series of steps that involve the initial reaction of ONOO(-) with Mn(III) to yield Mn(IV) and intermediates that ultimately culminate in 3-nitroTyr34. The data reported herein provide a kinetic and mechanistic basis for rationalizing how MnSOD constitutes an intramitochondrial target for ONOO(-) and the microscopic events, with atomic level resolution, that lead to selective and efficient nitration of critical Tyr34.
Asunto(s)
Nitratos/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Superóxido Dismutasa/metabolismo , Tirosina/metabolismo , Western Blotting , Dominio Catalítico , Humanos , Cinética , Modelos Moleculares , Oxidación-Reducción , Proteómica , Umbeliferonas/metabolismoRESUMEN
Cytochrome c (cyt c) is a cationic hemoprotein of â¼100 amino acid residues that exhibits exceptional functional versatility. While its primary function is electron transfer in the respiratory chain, cyt c is also recognized as a key component of the intrinsic apoptotic pathway, the mitochondrial oxidative protein folding machinery, and presumably as a redox sensor in the cytosol, along with other reported functions. Transition to alternative conformations and gain-of-peroxidase activity are thought to further enable the multiple functions of cyt c and its translocation across cellular compartments. In vitro, direct interactions of cyt c with cardiolipin, post-translational modifications such as tyrosine nitration, phosphorylation, methionine sulfoxidation, mutations, and even fine changes in electrical fields lead to a variety of conformational states that may be of biological relevance. The identification of these alternative conformations and the elucidation of their functions in vivo continue to be a major challenge. Here, we unify the knowledge of the structural flexibility of cyt c that supports functional moonlighting and review biochemical and immunochemical evidence confirming that cyt c undergoes conformational changes during normal and altered cellular homeostasis.
Asunto(s)
Citocromos c/química , Citocromos c/metabolismo , Animales , Cardiolipinas/química , Electricidad , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Espacio Intracelular/metabolismo , Fosfolípidos/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformación Proteica , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Transporte de ProteínasRESUMEN
We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are â¼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein.
Asunto(s)
Cardiolipinas/química , Citocromos c/química , Animales , Dominio Catalítico , Caballos , Conformación Proteica , Espectrofotometría Ultravioleta , Espectrometría RamanRESUMEN
Cytochrome c (Cyt-c) has been previously shown to participate in cardiolipin (CL) oxidation and, therefore, in mitochondrial membrane permeabilization during the early events of apoptosis. The gain in this function has been ascribed to specific CL/Cyt-c interactions. Here we report that the cationic protein Cyt-c is also able to interact electrostatically with the main lipid components of the mitochondrial membranes, the zwitterionic lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE), through the mediation of phosphate anions that bind specifically to amino groups in the surfaces of protein and model membranes. In these complexes, Cyt-c reacts efficiently with H2O2 at submillimolar levels, which oxidizes the sulfur atom of the axial ligand Met80. The modified protein is stable and presents significantly enhanced peroxidatic activity. Based on these results, we postulate that the rise of H2O2 concentrations to the submillimolar levels registered during initiation of the apoptotic program may represent one signaling event that triggers the gain in peroxidatic function of the Cyt-c molecules bound to the abundant PE and PC membrane components. As the activated protein is a chemically stable species, it can potentially bind and oxidize important targets, such as CL.
RESUMEN
Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10(4) M(-1) s(-1) and 4.3 ± 0.4 × 10(4) M(-1) s(-1) at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr(35). Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys(83) mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys(83) present in Fe-SODB acts as an electron donor that repairs Tyr(35) radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.
Asunto(s)
Proteínas Protozoarias/metabolismo , Superóxido Dismutasa/metabolismo , Trypanosoma cruzi/enzimología , Animales , Sitios de Unión/genética , Western Blotting , Dominio Catalítico , Enfermedad de Chagas/parasitología , Cristalografía por Rayos X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Activación Enzimática/efectos de los fármacos , Interacciones Huésped-Parásitos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Nitratos/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/farmacología , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiología , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMEN
Here we report a spectroscopic, electrochemical and computational study of cytochrome c showing that nitration of Tyr74 induces Tyr deprotonation, which is coupled to Met/Lys axial ligand exchange, and results in concomitant gain of peroxidatic activity at physiological pH.
Asunto(s)
Citocromos c/química , Nitratos/química , Protones , Tirosina/química , Simulación por Computador , Concentración de Iones de Hidrógeno , Ligandos , Modelos Moleculares , Oxidación-Reducción , TermodinámicaRESUMEN
Formation of cytochrome c (cyt c)/cardiolipin (CL) peroxidase complex selective toward peroxidation of polyunsaturated CLs is a pre-requisite for mitochondrial membrane permeabilization. Tyrosine residues - via the generation of tyrosyl radicals (Tyr) - are likely reactive intermediates of the peroxidase cycle leading to CL peroxidation. We used mutants of horse heart cyt c in which each of the four Tyr residues was substituted for Phe and assessed their contribution to the peroxidase catalysis. Tyr67Phe mutation was associated with a partial loss of the oxygenase function of the cyt c/CL complex and the lowest concentration of H(2)O(2)-induced Tyr radicals in electron paramagnetic resonance (EPR) spectra. Our MS experiments directly demonstrated decreased production of CL-hydroperoxides (CL-OOH) by Tyr67Phe mutant. Similarly, oxidation of a phenolic substrate, Amplex Red, was affected to a greater extent in Tyr67Phe than in three other mutants. Tyr67Phe mutant exerted high resistance to H(2)O(2)-induced oligomerization. Measurements of Tyr fluorescence, hetero-nuclear magnetic resonance (NMR) and computer simulations position Tyr67 in close proximity to the porphyrin ring heme iron and one of the two axial heme-iron ligand residues, Met80. Thus, the highly conserved Tyr67 is a likely electron-donor (radical acceptor) in the oxygenase half-reaction of the cyt c/CL peroxidase complex.
Asunto(s)
Cardiolipinas/química , Citocromos c/química , Peroxidasas/química , Tirosina/química , Animales , Simulación por Computador , Espectroscopía de Resonancia por Spin del Electrón , Hemo/química , Caballos , Peróxido de Hidrógeno/química , Hierro/química , Espectroscopía de Resonancia Magnética/métodos , Membranas Mitocondriales/metabolismo , Mutación , Miocardio/metabolismo , Oxígeno/química , Oxigenasas/química , Peroxidasa/química , Fenilalanina/químicaRESUMEN
Manganese Superoxide Dismutase (MnSOD) is an essential mitochondrial antioxidant enzyme that protects organisms against oxidative damage, dismutating superoxide radical (O2(.)â») into H2O2 and O2. The active site of the protein presents a Mn ion in a distorted trigonal-bipyramidal environment, coordinated by H26, H74, H163, D159 and one â»OH ion or H2O molecule. The catalytic cycle of the enzyme is a "ping-pong" mechanism involving Mn³+/Mn²+. It is known that nitration of Y34 is responsible for enzyme inactivation, and that this protein oxidative modification is found in tissues undergoing inflammatory and degenerative processes. However, the molecular basis about MnSOD tyrosine nitration affects the protein catalytic function is mostly unknown. In this work we strongly suggest, using computer simulation tools, that Y34 nitration affects protein function by restricting ligand access to the active site. In particular, deprotonation of 3-nitrotyrosine increases drastically the energetic barrier for ligand entry due to the absence of the proton. Our results for the WT and selected mutant proteins confirm that the phenolic moiety of Y34 plays a key role in assisting superoxide migration.
Asunto(s)
Nitrocompuestos/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Tirosina/metabolismo , Dominio Catalítico , Activación Enzimática , Humanos , Simulación de Dinámica Molecular , Mutación , Superóxido Dismutasa/genética , Superóxidos/metabolismo , TermodinámicaRESUMEN
Mitochondria are primary loci for the intracellular formation and reactions of reactive oxygen and nitrogen species including superoxide (O2â¢â»), hydrogen peroxide (H2O2) and peroxynitrite (ONOOâ»). Depending on formation rates and steady-state levels, the mitochondrial-derived short-lived reactive species contribute to signalling events and/or mitochondrial dysfunction through oxidation reactions. Among relevant oxidative modifications in mitochondria, the nitration of the amino acid tyrosine to 3-nitrotyrosine has been recognized in vitro and in vivo. This post-translational modification in mitochondria is promoted by peroxynitrite and other nitrating species and can disturb organelle homeostasis. This study assesses the biochemical mechanisms of protein tyrosine nitration within mitochondria, the main nitration protein targets and the impact of 3-nitrotyrosine formation in the structure, function and fate of modified mitochondrial proteins. Finally, the inhibition of mitochondrial protein tyrosine nitration by endogenous and mitochondrial-targeted antioxidants and their physiological or pharmacological relevance to preserve mitochondrial functions is analysed.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Nitrocompuestos/metabolismo , Tirosina/metabolismo , HumanosRESUMEN
We previously described a coding mutation (L60F) in the mitochondrial superoxide dismutase (SOD2) gene of the human T cell leukemia-derived cell line Jurkat. In cell extracts the L60F mutant enzyme showed unusual inhibition by thiol reagents not seen in wild-type enzyme. Here we compare the properties of purified recombinant L60F SOD2 with a previously described SOD2 mutant, I58T. Both mutant proteins display a weakened dimer-dimer interaction and thermal instability at 55 degrees C. Both I58T and L60F lose activity at 37 degrees C in the presence of 5 mM N-ethylmaleimide, whereas the wild-type SOD2 does not. Each subunit contains one exposed, reactive cysteine residue at position 196 and a second cysteine residue at 140, which is buried and unreactive in the wild-type tetramer. We propose that the mutant enzymes, which exist largely as dimers, allow both cysteine residues to react with thiol reagents. When the cysteine residue at 140 was changed to serine by site-directed mutagenesis, both double mutants I58T/C140S and L60F/C140S lost their increased thiol sensitivity. The evolutionary significance of Cys140 is discussed.
Asunto(s)
Cisteína/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Cisteína/química , Humanos , Células Jurkat , Mutagénesis Sitio-Dirigida , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Compuestos de Sulfhidrilo , Superóxido Dismutasa/genéticaRESUMEN
Human recombinant MnSOD and CuZnSOD were both inactivated when exposed to simultaneous fluxes of superoxide (JO(2)(*-)) and nitric oxide (J*NO). The inactivation was also observed with varying J*NO/JO(2)(*-) ratios. Protein-derived radicals were detected in both CuZn and MnSOD by immuno-spin trapping. The formation of protein radicals was followed by tyrosine nitration in the case of MnSOD. When MnSOD was exposed to J*NO and JO(2)(*-) in the presence of uric acid, a scavenger of peroxynitrite-derived free radicals, nitration was decreased but inactivation was not prevented. On the other hand, glutathione, known to react with both peroxynitrite and nitrogen dioxide, totally protected MnSOD from inactivation and nitration on addition of authentic peroxynitrite but, notably, it was only partially inhibitory in the presence of the more biologically relevant J*NO and JO(2)(*-). The data are consistent with the direct reaction of peroxynitrite with the Mn center and a metal-catalyzed nitration of Tyr-34 in MnSOD. In this context, we propose that inactivation is also occurring through a *NO-dependent nitration mechanism. Our results help to rationalize MnSOD tyrosine nitration observed in inflammatory conditions in vivo in the presence of low molecular weight scavengers such as glutathione that otherwise would completely consume nitrogen dioxide and prevent nitration reactions.