Reactions of nitric oxide with mitochondrial cytochrome c: a novel mechanism for the formation of nitroxyl anion and peroxynitrite (original) (raw)
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International Journal of Cell Biology, 2012
Nitric oxide (NO) reacts with Complex I and cytochrome c oxidase (CcOX, Complex IV), inducing detrimental or cytoprotective effects. Two alternative reaction pathways (PWs) have been described whereby NO reacts with CcOX, producing either a relatively labile nitrite-bound derivative (CcOX-NO 2 − , PW1) or a more stable nitrosyl-derivative (CcOX-NO, PW2). The two derivatives are both inhibited, displaying different persistency and O 2 competitiveness. In the mitochondrion, during turnover with O 2 , one pathway prevails over the other one depending on NO, cytochrome c 2+ and O 2 concentration. High cytochrome c 2+ , and low O 2 proved to be crucial in favoring CcOX nitrosylation, whereas under-standard cell-culture conditions formation of the nitrite derivative prevails. All together, these findings suggest that NO can modulate physiologically the mitochondrial respiratory/OXPHOS efficiency, eventually being converted to nitrite by CcOX, without cell detrimental effects. It is worthy to point out that nitrite, far from being a simple oxidation byproduct, represents a source of NO particularly important in view of the NO cell homeostasis, the NO production depends on the NO synthases whose activity is controlled by different stimuli/effectors; relevant to its bioavailability, NO is also produced by recycling cell/body nitrite. Bioenergetic parameters, such as mitochondrial ΔΨ, lactate, and ATP production, have been assayed in several cell lines, in the presence of endogenous or exogenous NO and the evidence collected suggests a crucial interplay between CcOX and NO with important energetic implications.
Cytochrome c Nitration by Peroxynitrite
Journal of Biological Chemistry, 2000
and nitric oxide (⅐NO) reaction, inhibits mitochondrial respiration and can stimulate apoptosis. Cytochrome c, a mediator of these two aspects of mitochondrial function, thus represents an important potential target of ONOO ؊ during conditions involving accelerated rates of oxygen radical and ⅐NO generation. Horse heart cytochrome c 3؉ was nitrated by ONOO ؊ , as indicated by spectral changes, Western blot analysis, and mass spectrometry. A dose-dependent loss of cytochrome c 3؉ 695 nm absorption occurred, inferring that nitration of a critical heme-vicinal tyrosine (Tyr-67) promoted a conformational change, displacing the Met-80 heme ligand. Nitration was confirmed by cross-reactivity with a specific antibody against 3-nitrotyrosine and by increased molecular mass compatible with the addition of a nitro-(-NO 2 ) group. Mass analysis of tryptic digests indicated the preferential nitration of Tyr-67 among the four conserved tyrosine residues in cytochrome c. Cytochrome c 3؉ was more extensively nitrated than cytochrome c 2؉ because of the preferential oxidation of the reduced heme by ONOO ؊ . Similar protein nitration patterns were obtained by ONOO ؊ reaction in the presence of carbon dioxide, whereupon secondary nitrating species arise from the decomposition of the nitroso-peroxocarboxylate (ONOOCO 2 ؊ ) intermediate. Peroxynitrite-nitrated cytochrome c displayed significant changes in redox properties, including (a) increased peroxidatic activity, (b) resistance to reduction by ascorbate, and (c) impaired support of state 4-dependent respiration in intact rat heart mitochondria. These results indicate that cytochrome c nitration may represent both oxidative and signaling events occurring during ⅐NOand ONOO ؊ -mediated cell injury.
doi:10.1155/2012/571067 Review Article The Chemical Interplay between Nitric Oxide and Mitochondrial
2015
Copyright © 2012 Paolo Sarti et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Nitric oxide (NO) reacts with Complex I and cytochrome c oxidase (CcOX, Complex IV), inducing detrimental or cytoprotective eects. Two alternative reaction pathways (PWs) have been described whereby NO reacts with CcOX, producing either a relatively labile nitrite-bound derivative (CcOX-NO2 −, PW1) or a more stable nitrosyl-derivative (CcOX-NO, PW2). The two derivatives are both inhibited, displaying dierent persistency and O2 competitiveness. In the mitochondrion, during turnover with O2, one pathway prevails over the other one depending on NO, cytochrome c2+ and O2 concentration. High cytochrome c2+, and low O2 proved to be crucial in favoring CcOX nitrosylation, whereas under-standard cell-culture conditions formation of the nitrite ...
Nitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell
Free Radical Biology and Medicine, 2003
The aim of this work is to review the information available on the molecular mechanisms by which the NO radical reversibly downregulates the function of cytochrome c oxidase (CcOX). The mechanisms of the reactions with NO elucidated over the past few years are described and discussed in the context of the inhibitory effects on the enzyme activity. Two alternative reaction pathways are presented whereby NO reacts with the catalytic intermediates of CcOX populated during turnover. The central idea is that at "cellular" concentrations of NO (Յ M), the redox state of the respiratory chain results in the formation of either the nitrosyl-or the nitrite-derivative of CcOX, with potentially different metabolic implications for the cell. In particular, the role played by CcOX in protecting the cell from excess NO, potentially toxic for mitochondria, is also reviewed highlighting the mechanistic differences between eukaryotes and some prokaryotes.
Mechanisms of the interaction of nitroxyl with mitochondria
Biochemical Journal, 2004
It is now thought that NO • (nitric oxide) and its redox congeners may play a role in the physiological regulation of mitochondrial function. The inhibition of cytochrome c oxidase by NO • is characterized as being reversible and oxygen dependent. In contrast, peroxynitrite, the product of the reaction of NO • with superoxide, irreversibly inhibits several of the respiratory complexes. However, little is known about the effects of HNO (nitroxyl) on mitochondrial function. This is especially important, since HNO has been shown to be more cytotoxic than NO • , may potentially be generated in vivo, and elicits biological responses with some of the characteristics of NO and peroxynitrite. In the present study, we present evidence that isolated mitochondria, in the absence or presence of substrate, convert HNO into NO • by a process that is dependent on mitochondrial concentration as well as the concentration of the HNO donor Angeli's salt. In addition, HNO is able to inhibit mitochondrial respiration through the inhibition of complexes I and II, most probably via modification of specific cysteine residues in the proteins. Using a proteomics approach, extensive modification of mitochondrial protein thiols was demonstrated. From these data it is evident that HNO interacts with mitochondria through mechanisms distinct from those of either NO • or peroxynitrite, including the generation of NO • , the modification of thiols and the inhibition of complexes I and II.
Cytochrome c: a catalyst and target of nitrite-hydrogen peroxide-dependent protein nitration
Archives of Biochemistry and Biophysics, 2004
Nitration of protein tyrosine residues to 3-nitrotyrosine (NO 2 Tyr) serves as both a marker and mediator of pathogenic reactions of nitric oxide ( Å NO), with peroxynitrite (ONOO À ) and leukocyte peroxidase-derived nitrogen dioxide ( Å NO 2 ) being proximal mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO À 2 ) and hydrogen peroxide (H 2 O 2 )-mediated nitration reactions. Cytochrome c catalyzes both self-and adjacent-molecule (hydroxyphenylacetic acid, Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and Å NO 2 production, as for phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive tyrosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H 2 O 2 and NO À 2 , with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to biomolecule nitration reactions.
Nitric Oxide and Mitochondrial Complex IV
2003
Micromolar nitric oxide (NO) rapidly (ms) inhibits cytochrome c oxidase in turnover with physiological substrates. Two reaction mechanisms have been identified leading, respectively, to formation of a nitrosyl-[a 3 2+ -NO] or a nitrite-[a 3 3+ -NO 2 7 ] derivative of the enzyme. In the presence of O 2 , the nitrosyl adduct recovers activity slowly, following NO displacement at k'&0.01 s 71 (378C); the recovery of the nitrite adduct is much faster. Relevant to pathophysiology, the enzyme does not degrade NO by following the first mechanism, whereas by following the second one it promotes NO oxidation and disposal as nitrite/nitrate. The reaction between NO and cytochrome c oxidase has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. Oxygraphic measurements show that cultured cells or isolated state 4 mitochondria exposed to micromolar (or less) NO recover from NO inhibition rapidly, as if the nitrite reaction was predominant. Mitochondria in state 3 display a slightly more persistent inhibition than in state 4, possibly due to a higher accumulation of the nitrosyl adduct. Among a number of parameters that appear to control the switch over between the two mechanisms, the concentration of reductants (reduced cytochrome c) at the cytochrome c oxidase site has been proved to be the most relevant one. IUBMB Life, 55: 605-611, 2003
Mechanism and Biological Role of Nitric Oxide Binding to Cytochrome c‘
Biochemistry, 2002
The binding of nitric oxide to ferric and ferrous Chromatium Vinosum cytochrome c′ was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c′ data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c′ was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c′ was found to be in the range typical of receptors and carriers. In addition, cytochrome c′ was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c′ functioning as a nitric oxide carrier was proposed. Cytochrome c′ is a homodimeric hemoprotein of 14 kD molecular mass (per subunit) that is found in many gramnegative species including photosynthetic (1), denitrifying (2), nitrogen-fixing (3), and sulfur-reducing bacteria (4). Cytochrome c′ has been shown to consist of a four R-helical bundle structure (5-9) possessing low overall sequence conservation (10). Unlike mitochondrial cytochrome c, the heme group is attached at the C-terminus. The heme binding sequence is CKXCH. The histidine following the second cysteine is the proximal ligand, while the sixth coordination site is vacant. The heme iron is pentacoordinate in cytochrome c′, similar to that of hemoglobin, myoglobin, catalase, cytochrome P450, and other heme proteins for which ligand/ substrate binding is the primary function. In contrast, a hexacoordinate structure is observed for purely electron transport hemoproteins, of which cytochrome c is one example. Recent cytochrome c′ knockout mutant experiments with Rhodobacter capsulatus (11) have suggested that cytochrome c′ may play a role in NO binding. Since cytochrome c′ has