Nitric oxide and peroxynitrite cause irreversible increases in the Km for oxygen of mitochondrial cytochrome oxidase: in vitro and in vivo studies (original) (raw)
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IUBMB Life, 2007
The present study shows that nitric oxide (NO) irreversibly inhibits purified cytochrome oxidase in a reverse oxygen concentration-dependent manner. The inhibition is dramatically protected by a peroxynitrite scavenger, suggesting that peroxynitrite is formed from the reaction of NO with cytochrome oxidase at low oxygen concentration, and that peroxynitrite is involved in irreversible cytochrome oxidase inactivation. Production of nitroxyl anion or superoxide was tested as potential mechanisms underlying the conversion of NO to peroxynitrite. A nitroxyl anion scavenger potently protected the irreversible inhibition, whereas a superoxide dismutase did not provide protective effect, suggesting that the peroxynitrite was formed from nitroxyl anion rather than the reaction of NO with superoxide.
Cytochrome c oxidase maintains mitochondrial respiration during partial inhibition by nitric oxide
Journal of Cell Science, 2006
Nitric oxide (NO), generated endogenously in NO-synthase-transfected cells, increases the reduction of mitochondrial cytochrome c oxidase (CcO) at O2 concentrations ([O2]) above those at which it inhibits cell respiration. Thus, in cells respiring to anoxia, the addition of 2.5 μM L-arginine at 70 μM O2 resulted in reduction of CcO and inhibition of respiration at [O2] of 64.0±0.8 and 24.8±0.8 μM, respectively. This separation of the two effects of NO is related to electron turnover of the enzyme, because the addition of electron donors resulted in inhibition of respiration at progressively higher [O2], and to their eventual convergence. Our results indicate that partial inhibition of CcO by NO leads to an accumulation of reduced cytochrome c and, consequently, to an increase in electron flux through the enzyme population not inhibited by NO. Thus, respiration is maintained without compromising the bioenergetic status of the cell. We suggest that this is a physiological mechanism re...
Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010
We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O 2) conditions. The system measures the concentrations of O 2 and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O 2 concentration and electron turnover of the enzyme. At a high O 2 concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of L-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O 2. At low O 2 (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O 2 consumption. At both high and low O 2 concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.
Archives of Biochemistry and Biophysics, 1996
Nitric oxide (rNO) released by S-nitrosoglutathione peroxide anion; hydrogen peroxide. (GSNO) inhibited enzymatic activities of rat heart mitochondrial membranes. Cytochrome oxidase activity was inhibited to one-half at an effective rNO concentration of 0.1 mM, while succinate-and NADH-cyto-Nitric oxide (rNO) 2 is an endogenous free radical chrome-c reductase activities were half-maximally informed in a variety of cell types by NO-synthase by hibited at 0.3 mM rNO. Submitochondrial particles oxidation of L-arginine to L-citrulline (1). A function of treated with rNO (either from GSNO or from a pure rNO as a signal-transducing molecule has been desolution) showed increased O 0 2 and H 2 O 2 production scribed in endothelium and brain (2). In addition, rNO when supplemented with succinate alone, at rates that cytotoxic properties are considered as part of the nonwere comparable to those of control particles with specific immune response of activated macrophages added succinate and antimycin. Rat heart mitochonand neutrophils (3). dria treated with rNO also showed increased H 2 O 2 pro-Toxic effects of rNO, particularly by interfering with duction. Cytochrome spectra and decreased enzymatic activities in the presence of rNO are consistent with oxidative metabolism (4) or iron homeostasis , have a multiple inhibition of mitochondrial electron trans-been reported. Beckman (6) indicated that endothelial fer at cytochrome oxidase and at the ubiquinone-cytoinjury may arise from formation of peroxynitrite anion chrome b region of the respiratory chain, the latter (ONOO 0 ), the product of the diffusion-controlled reacleading to the increased O 0 2 production. Electrochemition between two molecules with unpaired electrons, cal detection showed that the buildup of a rNO concensuperoxide anion (O 0 2 ) and rNO. Peroxynitrite anion tration from GSNO was interrupted by submitochonhas been reported to produce membrane lipoperoxidadrial particles supplemented with succinate and antition (7) and sulfhydryl oxidation (8). Moreover, Radi et mycin and was restored by addition of superoxide al. (9) have reported oxidative damage to the electron dismutase. The inhibitory effect of rNO on cytochrome transport chain and the ATPase activity of rat heart oxidase was also prevented under the same conditions. mitochondria at biologically relevant ONOO 0 concen-Apparently, mitochondrial O 0 2 reacts with rNO to form trations (9) and that ONOO 0 reacts with isolated pig peroxynitrite and, by removing rNO, reactivates the aconitase, resulting in an irreversible loss of enzymatic previously inhibited cytochrome oxidase. It is sugactivity (10). gested that, at physiological concentrations of rNO, On the other hand, Cleeter et al. (11) reported that inhibition of electron transfer, rNO-induced O 0 2 pronitric oxide generated by S-nitrosoglutathione (GSNO) duction, and ONOO 0 formation participate in the reguproduces a reversible inhibition of respiration and cytolatory control of mitochondrial oxygen uptake. ᭧ 1996 Academic Press, Inc. 2 Abbreviations used: rNO, nitric oxide; ONOO 0 , peroxynitrite 1 To whom correspondence and reprint requests should be ad-anion; O 0 2
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.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
Background: The reactions between Complex IV (cytochrome c oxidase, CcOX) and nitric oxide (NO) were described in the early 60's. The perception, however, that NO could be responsible for physiological or pathological effects, including those on mitochondria, lags behind the 80's, when the identity of the endothelial derived relaxing factor (EDRF) and NO synthesis by the NO synthases were discovered. NO controls mitochondrial respiration, and cytotoxic as well as cytoprotective effects have been described. The depression of OXPHOS ATP synthesis has been observed, attributed to the inhibition of mitochondrial Complex I and IV particularly, found responsible of major effects. Scope of review: The review is focused on CcOX and NO with some hints about pathophysiological implications. The reactions of interest are reviewed, with special attention to the molecular mechanisms underlying the effects of NO observed on cytochrome c oxidase, particularly during turnover with oxygen and reductants. Major conclusions and general significance: The NO inhibition of CcOX is rapid and reversible and may occur in competition with oxygen. Inhibition takes place following two pathways leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) of the enzyme reduced, or a more labile nitrite-derivative (CcOX-NO 2 − ) of the enzyme oxidized, and during turnover. The pathway that prevails depends on the turnover conditions and concentration of NO and physiological substrates, cytochrome c and O 2 . All evidence suggests that these parameters are crucial in determining the CcOX vs NO reaction pathway prevailing in vivo, with interesting physiological and pathological consequences for cells. This article is part of a Special Issue entitled: Respiratory Oxidases.
Proceedings of The National Academy of Sciences, 2006
NO reversibly inhibits mitochondrial respiration via binding to cytochrome c oxidase (CCO). This inhibition has been proposed to be a physiological control mechanism and/or to contribute to pathophysiology. Oxygen reacts with CCO at a heme iron:copper binuclear center (a3/CuB). Reports have variously suggested that during inhibition NO can interact with the binuclear center containing zero (fully oxidized), one (singly reduced), and two (fully reduced) additional electrons. It has also been suggested that two NO molecules can interact with the enzyme simultaneously. We used steady-state and kinetic modeling techniques to reevaluate NO inhibition of CCO. At high flux and low oxygen tensions NO interacts predominantly with the fully reduced (ferrous/cuprous) center in competition with oxygen. However, as the oxygen tension is raised (or the consumption rate is decreased) the reaction with the oxidized enzyme becomes increasingly important. There is no requirement for NO to bind to the singly reduced binuclear center. NO interacts with either ferrous heme iron or oxidized copper, but not both simultaneously. The affinity (KD) of NO for the oxygen-binding ferrous heme site is 0.2 nM. The noncompetitive interaction with oxidized copper results in oxidation of NO to nitrite and behaves kinetically as if it had an apparent affinity of 28 nM; at low levels of NO, significant binding to copper can occur without appreciable enzyme inhibition. The combination of competitive (heme) and noncompetitive (copper) modes of binding enables NO to interact with mitochondria across the full in vivo dynamic range of oxygen tension and consumption rates. bioenergetics | mitochondria | signaling
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 ...
Regulation of Mitochondrial Respiration by Oxygen and Nitric Oxide
Annals of the New York Academy of Sciences, 2006
Although the regulation of mitochondrial respiration and energy production in mammalian tissues has been exhaustively studied and extensively reviewed, a clear understanding of the regulation of cellular respiration has not yet been achieved. In particular, the role of tissue pO2 as a factor regulating cellular respiration remains controversial. The concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers has evolved in the last few years and is still being researched. A recent concept that regulation of cellular respiration is regulated by ADP, O2 and NO preserves the notion that energy demands drive respiration but places the kinetic control of both respiration and energy supply in the availability of ADP to F1-ATPase and of O2 and NO to cytochrome oxidase. In addition, recent research indicates that NO participates in redox reactions in the mitochondrial matrix that regulate the intramitochondrial steady state concentration of NO itself and other reactive species such as superoxide radical (O2-) and peroxynitrite (ONOO-). In this way, NO acquires an essential role as a mitochondrial regulatory metabolite. No exhibits a rich biochemistry and a high reactivity and plays an important role as intercellular messenger in diverse physiological processes, such as regulation of blood flow, neurotransmission, platelet aggregation and immune cytotoxic response.
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.