Nitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell

Free Radical Biology & Medicine, Vol. 34, No. 5, pp. 509 –520, 2003 Copyright © 2003 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter

doi:10.1016/S0891-5849(02)01326-6

Serial Review: Nitric Oxide in Mitochondria Guest Editors: Christoph Richter and Matthias Schweizer NITRIC OXIDE AND CYTOCHROME OXIDASE: REACTION MECHANISMS FROM THE ENZYME TO THE CELL PAOLO SARTI, ALESSANDRO GIUFFRE` , MARIA CECILIA BARONE, ELENA FORTE, DANIELA MASTRONICOLA, and MAURIZIO BRUNORI Department of Biochemical Sciences and CNR Institute of Molecular Biology and Pathology, University of Rome “La Sapienza”, Rome, Italy (Received 13 June 2002; Accepted 6 November 2002)

Abstract—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 (ⱕ ␮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. © 2003 Elsevier Science Inc. Keywords—Nitric oxide, Free radical, Respiratory chain, Cytochrome oxidase, Reaction mechanism, Respiration

INTRODUCTION

particular interest, being rapid (micro-milliseconds time scale) and reversible [10,11] as it is for many functional modulators, whereas the reaction with the other complexes is slower (time scale of several minutes to hours) or occurs at higher, nonphysiological, NO concentrations (for some complexes in the mM range) [12–14]. A number of reviews on the biomedical relevance of the NO chemistry has been published in the last few years [15–19], including an extensive analysis of the interactions between NO and metalloproteins [20,21]. The aim of this work is to review the information available on the molecular mechanisms by which NO reacts with CcOX either isolated in detergent solution, or in respiring cells. Since a role for CcOX in NO degradation has been envisaged [22,23], this issue will be also reviewed focusing on both eukaryotic and prokaryotic oxidases. A great body of evidence has been accumulated showing that NO inhibits cell respiration [3,24 –26] by reacting with CcOX at all integration levels, from the purified enzyme in detergent solution [10,27] to mitochondria [2– 4], cells [28 –31] and tissues [32,33], up to in vivo [34,35]. The reaction mechanism(s) involved in the inhibition have been elucidated, although the extent to

In 1990 Carr and Ferguson showed that nitric oxide (NO) catalytically generated by nitrite reductase was able to inhibit the respiration of bovine heart submitochondrial particles [1]. Clear-cut evidence showing that NO controls cell respiration was published in 1994 [2– 4]. Subsequent work showed that NO can react with several respiratory chain complexes [5–9]. Among these, the interaction with Cytochrome c Oxidase (CcOX) is of Paolo Sarti and Maurizio Brunori received their M.D. degrees from the University of Rome in 1972 and 1961, respectively; they are full Professors of Chemistry and Biochemistry in the Faculty of Medicine Ist and IInd of the University of Rome “La Sapienza”. Alessandro Giuffr`e, graduated in Biology at the University of Rome “La Sapienza” in 1993, was awarded the Ph.D. in Biochemistry in 1997; he is Researcher at the Institute of Molecular Biology and Pathology of the Consiglio Nazionale delle Ricerche. Maria Cecilia Barone graduated in Chemistry in 1999, while Elena Forte and Daniela Mastronicola both graduated in Biology in 1992 and 1999, respectively; they are all Ph.D. students in Biochemistry. The study of the structure and function of mitochondrial complex IV represents a major interest of the Rome group, nowadays focused on the interactions with the free radical nitric oxide. Address correspondence to: Paolo Sarti, Dipartimento di Scienze Biochimiche “A. Rossi-Fanelli”, Universita` di Roma “La Sapienza”, Piazzale Aldo Moro 5, I-00185 Rome, Italy; Tel: ⫹39 (06) 445-0291; Fax: ⫹39 (06) 444-0062; E-Mail: [email protected]. 509

510

P. SARTI et al.

Fig. 1. The active site of cytochrome oxidase. Heme a3 and CuB are shown together with their ligands. The structure refers to the fully oxidized beef heart enzyme. Tyr244 is covalently linked to His240, one of the CuB ligands. From the Protein Data Bank coordinates deposited by Tsukihara et al. [116,117].

which each of these is effective at the different integration levels is still a matter of study. We shall focus on the following main topics: 1) The mechanisms underlying the control exerted by NO on mitochondrial CcOX. 2) The role of mitochondrial and prokaryotic oxidases in the degradation of NO. HEME-COPPER OXIDASES: INFORMATION RELEVANT TO THE NO CHEMISTRY

The structure Since 1995, the 3D crystallographic structure of several heme-copper oxidases has been solved. This set of structures includes one eukaryotic cytochrome c oxidase (the aa3 from beef heart, see Fig. 1 and [36]), three prokaryotic cytochrome c oxidases (the aa3 oxidases from Paracoccus denitrificans [37] and from Rhodobacter sphaeroides [38] and the ba3 from the Thermus thermophilus [39]), and a quinol oxidase (the bo3 from Escherichia coli [40]). The view emerging from the structural data is that heme-copper oxidases share a highly conserved bimetallic active site, constituted by a high-spin heme (called either a3, o3, or b3, depending on the chemical identity of the heme) and a copper ion (called CuB). The high-spin heme is coordinated by His376 (numbering all throughout according to

the sequence of the beef-heart enzyme), while CuB is coordinated by His240, His290, and His291. The structure of the active site is reported in Fig. 1. A highly conserved tyrosine residue, Tyr244, is covalently bound to His240; this tyrosine was postulated to have a radical character in one of the catalytic intermediates, possibly playing a crucial role in catalysis [41]. The binuclear center is the binding site of the physiological substrate O2, as well as other ligands such as CN⫺, CO, and NO [42]. O2-binding demands a prior reduction of the active site by the low-spin heme (called heme a in the mitochondrial enzyme), which in CcOX is in turn reduced by CuA, the (bimetallic) site whereby the electrons donated by cytochrome c enter the enzyme [42]. NO reacts with protein-bound metal centers [20,21] as well as with activated thiols and radical tyrosines to yield, respectively, the nitroso- and the nitro-derivative [43– 46]. CcOX contains a solvent-exposed cysteine residue (Cys115 in subunit III) [47] and the above-mentioned Tyr244, displaying therefore a number of additional potential reaction sites for NO. This notwithstanding, in the mitochondrial enzyme, and at physiological NO concentrations (ⱕ ␮M) the only documented target for NO are the metals in the active site (heme a3-CuB). Other reactions between CcOX and NO (here including thiol-nitrosation or tyrosine-nitration) have never been shown to occur, whereas it has been reported that other nitrogen oxides, e.g., peroxynitrite, if present in 100-fold excess over CcOX, may induce formation of nitrotyrosine in the enzyme moiety [48]. The function In the respiratory chain of aerobic organisms, the heme-copper oxidases form a ubiquitous super-family of enzymes that transfer electrons from reducing substrates, such as cytochrome c or quinols, to O2 [49]. The exoergonic redox reaction is coupled to a vectorial proton pumping across the inner mitochondrial membrane, which contributes to formation and maintenance of the proton electrochemical gradient ⌬␮H⫹ (⬃200 mV) used by the cell to drive ATP-synthesis [49]: ⫹ 4cyt.c2⫹ ⫹ O2 ⫹ 8Hin⫹ 3 4cyt.c3⫹ ⫹ 2H2O ⫹ 4Hout

Electrons enter the enzyme via CuA, in rapid equilibrium with cytochrome a, and are transferred intra-molecularly to the O2-binding site (see structure). The catalytic cycle The scheme reported in Fig. 2 depicts the intermediates populated at the level of the active site of CcOX. According to this consensus simplified scheme, the catalytic cycle can be divided into a reductive and an

Reactions of cytochrome oxidase with nitric oxide

511

Fig. 2. The catalytic cycle of cytochrome oxidase and the interplay with NO. For simplicity, the scheme is centerd on the redox (e⫺) and ligand-binding changes of the heme a3-CuB active site. In the “slow” reductive phase, the fully oxidized species O is fully reduced to R, by two single-electron donations, and via formation of the (half-reduced) intermediate E. In the “fast” oxidative phase, R reacts with O2 to form in sequence P (see footnote) and F, regenerating eventually O. The heme a32⫹-NO (nitrosyl-adduct) or the heme a33⫹CuB2⫹-NO2⫺ (nitrite-bound adduct) are formed during the catalytic cycle where indicated. Intermediates having CuB2⫹ (oxidized) are circled, whereas those with Fe2⫹ (reduced) are squared.

oxidative part [50–52]. In the reductive part, the oxidized active site O accepts two electrons sequentially from CuA via cytochrome a. This is an intra-molecular electron transfer that yields the fully reduced site R, preceded by the formation of the single electron reduced intermediate E. In the single-electron reduced intermediate E, the electron can reside either on heme a3 (species E1) or on CuB (species E2) [53]. The rate-determining step in the overall cycle is the reduction of the binuclear site [42,54] prior to the reaction with O2. The much faster oxidative part of the cycle (␮s vs. ms) restores the initial O state by formation of intermediates P and F. The chemical identity of intermediates P and F is still debated, though it is agreed that both are oxo-ferryl adducts [55]1 We wish to point out that the NO chemistry of CcOX can be accounted for by the interaction with either reduced heme a3 or oxidized CuB. Thus, heme a3 is reduced in species E1 and R, while CuB is oxidized in species O, E1, P, and F. As it will be discussed in the next paragraph, the occupancy of each intermediate during turnover is crucial to the understanding of the overall mechanism of the reaction between CcOX with NO. THE MECHANISMS OF INTERACTION OF NO WITH CcOX

In 1955, Wanio first reported that NO reacts with CcOX [56]. In the early 1960s Gibson and Greenwood described the reaction of NO with the reduced enzyme [57], and later 1 Two different P intermediates can be generated. By reacting with O2, the two-electron reduced cytochrome c oxidase (so called Mixedvalence-CO), forms PM, whereas the fully reduced enzyme (R) forms PR. The data reported in this review have been obtained with PM, therefore throughout the paper P stands for PM.

Chan and co-workers observed that NO also reacts with the oxidized enzyme [58]. The physiological relevance of these reactions, however, was unknown and like CO, NO was simply used as an efficient but dull electron trapping ligand in heme-proteins reactions [59,60]. More than 10 years later, it was proposed that NO controls the mitochondrial respiration via a fully reversible, transient inhibition of CcOX (see Introduction). Since then, ample evidence has been collected supporting this view, which is becoming more and more exciting in view of predictable bioenergetic, thus patho-physiological implications. The reaction between CcOX and NO occurs at the active site with either the heme iron or the copper, in different redox states. NO binds to reduced heme a3 very quickly and with extremely high affinity, the reaction yielding the typical, for heme-proteins, Fe2⫹-NO nitrosyl-adduct [10,27]: 2⫹ ⫹ 关a32⫹Cu⫹ B 兴 ⫹ NO ^ 关a3 CuB NO兴

In the mitochondrial enzyme this reaction is a simple ligand binding process, whereas in some prokaryotic oxidases it is followed by the reduction of NO to yield N2O [61– 63]. A different reaction was shown to occur between NO and oxidised CuB, whereby NO is oxidised to nitrite, presumably via the transient formation of a nitrosonium ion (NO⫹) [64]. 3⫹ ⫹ 3⫹ 2⫹ ⫺ ⫹ 关a33⫹Cu2⫹ B 兴 ⫹ NO 3 关a3 CuB NO 兴 3 关a3 CuB NO2 兴

For the sake of clarity, we discuss separately the reactions of NO with each CcOX catalytic intermediate.

512

P. SARTI et al.

The kinetic information will be discussed based on the recent acquisition that CcOX reacts with NO following the two independent pathways just mentioned. The reaction of NO with the fully reduced state R The reaction of NO with R has been known for a long time and is well characterized from a kinetic point of view. It is described as a fast bimolecular process (k ⫽ 0.4 –1.0 ⫻ 108 M⫺1 s⫺1 at 20°C, [57,65]), yielding an inhibited enzyme through formation of a tight heme a32⫹-NO adduct [10,27]. This inhibited adduct of CcOX recovers its activity by dissociation of NO from the site, a process that is accelerated by illumination [66] in the absence of free NO. In the dark, functional recovery of respiration occurs at k’ ⫽ 4 ⫻ 10⫺3 s⫺1 at 20°C, a rate perhaps higher then expected when compared to other heme-proteins [67]. It is worth noticing that the rate of NO dissociation rises to k’ ⫽ 0.01 s⫺1 at 37°C [66], a value compatible with a fairly quick reversal of inhibition of CcOX, and thus preservation of cell viability. Dissociation of NO from the nitrosylated site has two consequences: (i) CcOX recovers activity, and (ii) NO dissociates into the medium unmodified. With the mitochondrial enzyme, upon reaction of NO with R, a single NO molecule binds to heme a3 with no redox change [68]. The absence of binding to CuB⫹ up to ⬃20 ␮M NO (upper limit for the reported amperometric measurements [68]) suggests that this metal in the reduced state has a very low affinity for NO. The latter information might be relevant, when considering that the mitochondrial enzyme cannot catalyze the reductive degradation of NO to N2O, a reaction recently documented for some prokaryotic oxidases [61– 63] (see below). When NO is bound to reduced heme a3, the enzyme is inhibited [10,27]. Inhibition is set rapidly and in competition with O2, being characterized by a KI ⫽ 60 nM at [O2 ] ⫽ 30 ␮M [3], consistent with the increase of the apparent Km of CcOX for O2 reported in vivo [69]; inhibition is fully reverted upon NO dissociation [70,71]. However, given the similarity between the rate constants for O2 and NO binding, the kinetics of the reaction of NO with R may not be sufficient to explain the rapid onset of inhibition (within seconds or less). For this reason, Torres et al. [10] first proposed that, differently from O2, NO can also bind to a single-electron reduced binuclear site, E [11,27]. The reaction of NO with the single-electron reduced intermediate E Although invoked in the past [10,27], experimental evidence supporting the reaction of NO with the singleelectron reduced intermediate E has been obtained only

recently, investigating the reactions of a site-directed mutant of the P. denitrificans cytochrome c oxidase [72]. E is thought to be a mixture of two forms, E1 and E2, the former with the electron residing on heme a3 and the latter on CuB; this makes it difficult to assess the primary metal target of NO in this intermediate. As for R, the end product of the reaction with E is the nitrosyl heme a32⫹-NO adduct. Unlike the other intermediates, however, E is expected to be populated during turnover but has not been isolated in a stable form. Torres et al. [10] proposed that NO binds to CuB⫹, and then is transferred to the nearby heme. This hypothesis is difficult to reconcile with the amperometric data collected on the R species [68], showing that only one NO binds in the ␮M range of concentration, thus excluding a high affinity for CuB⫹. Thus, it seems likely that binding of NO to E involves complexation with reduced heme a3. In addition, based on theoretical considerations and kinetic modeling, Giuffre` et al. [27] presented evidence that the reaction of NO with E would account for the rapid onset of inhibition, its O2-dependence, and the observed low KI. Thus, the reaction of NO with either the partially or the fully reduced active site of CcOX yields the inhibited NO-bound form of the enzyme. This reaction was considered the only one accounting for CcOX inhibition, until the kinetics of the reaction of NO with the oxidized CuB was reconsidered. The reaction of NO with the fully oxidized species O According to Brudvig et al. [58] this reaction involves binding of NO to CuB, as shown by low-temperature EPR spectroscopy; the reaction occurred only at high NO concentration and took hours [58]. The mechanism and functional relevance of this reaction became evident in 1997 when Cooper et al. [64], using a so called fastpulsed preparation of CcOX devoid of ligands at the binuclear center, reported that NO could rapidly react with CuB in the oxidized enzyme, a reaction prevented by bound chloride [73]. In the reaction with O, NO is oxidized to nitrite, which binds to the binuclear site perturbing the spectrum of the heme a33⫹ (Fig. 3), [74,75] and yields an inhibited enzyme [23,76]. However, upon reduction of CcOX the enzyme recovers activity, likely because of the much lower affinity of the reduced active site for nitrite (Fig. 4) [23,75]. It is worth noticing that this may represent a pathway of oxidative degradation of NO into the less toxic nitrite. The reaction of NO with O occurs rather rapidly, with a bimolecular rate constant k ⫽ 2 ⫻ 105 M⫺1s⫺1 at 20°C [64,73,74]. Interestingly enough, although more than two orders of magnitude slower than the reaction with R, the reaction of NO with O may still

Reactions of cytochrome oxidase with nitric oxide

513

Fig. 3. Reaction of NO with O, P, and F species of cytochrome c oxidase. Difference spectra obtained by mixing CcOX (O, P, F) with stoichiometric NO. After subtraction of the optical contribution of reduced cytochrome a, all transitions are very similar (solid spectra) and superimposable to the O-NO2⫺ adduct produced statically (dashed spectrum) (modified from ref. [75]).

be sufficiently fast to be functionally relevant in the cell. This is because the overall occupancy of the oxidized CuB-containing intermediates increases when turnover is sustained by a slow electron supply to CcOX (see below) [75]. The reaction of NO with intermediates P and F Intermediates P and F can be prepared in a sufficiently stable form (several minutes) to allow their reactivity towards NO to be studied both by optical spectroscopy [74,75] and by NO amperometry [75]. The relevant information emerging from these studies is that intermediates P and F react with NO somewhat similarly to O (k ⬇ 104 ⫼ 105 M⫺1 s⫺1 at 20°C), yielding as a final adduct the inhibited nitrite-bound enzyme (O-NO2⫺). Relevant to cell physiology, the latter derivative will be promptly reactivated on reduction by the natural electron donor,

Fig. 4. Number of turnovers elicited and residual activity of CcOX nitrite-bound. The data show that the function of CcOX inhibited by nitrite bound to the active site, is fully recovered upon increasing the concentration of reduced cytochrome c, i.e., the number of turnovers performed (modified from ref. [75]).

cytochrome c (Fig. 4) [75]. Similarly to O, intermediates P and F contain oxidised CuB and on this basis Torres et al. [74] proposed a mechanism common to the three intermediates, involving the oxidation of NO to nitrite via formation of NO⫹ at the level of CuB. These results were extended by Giuffre` et al. [75], with a combination of amperometric and spectroscopic measurements, showing that the reaction of O, P, or F with stoichiometric NO invariably leads to formation of the O-nitrite adduct. The reaction of NO with CcOX in situ Before considering the reaction of NO with CcOX integrated in the respiratory chain, it is worth mentioning the spatial cell distribution of the NO sources. Endogenous NO is enzymatically produced by the NO synthase (NOS) [77]. Three distinct NOSs isoforms have been identified so far and named, respectively, neuronal (nNOS or type I-NOS), inducible (iNOS or type IINOS), and endothelial (eNOS or type III-NOS) [78]; four variants have been described for the neuronal NOS [79]. The existence of a mitochondrial NOS (mtNOS) was proposed in the mid-1990s based on immuno-cytochemical evidence [80 – 83]. In 1997 Ghafourifar and Richter further supported this proposal showing that this mitochondrion-bound NOS was functionally active and its activity was Ca⫹⫹-dependent [84], as it is for nNOS and eNOS. Consistently, mitochondria supplemented with L-arginine were able to produce significant amounts of NO [85]. Recently Kanai et al. [86] reported that cardiomyocytes from knockout mice for nNOS do not produce NO in the mitochondrion, contrary to wild-type mice; on this basis these authors deduced that the mtNOS is actually a nNOS. Despite the fairly large mass of data, the existence of a mitochondrial NOS isoform is still controversial and debated. Indeed, the bioenergetic role of NO would be strengthened by the unequivocal demonstration of the existence of a mtNOS; however, even in

514

P. SARTI et al.

the absence of a mitochondrial source of NO, the control on respiration may be understood because NO is freely permeable to biological membranes, due to its high solubility in lipid environments [87]. Cleeter et al. [2] first reported that the NO generator S-nitrosylglutathione can reversibly inhibit respiration of mitochondria isolated from skeletal muscle, and indicated cytochrome oxidase as the primary target. In agreement with this hypothesis, Brown and Cooper [3] showed that upon illuminating brain synaptosomes respiring in the presence of nitroprusside, the photoreleased NO was inhibiting O2 consumption. In the dark, respiration was promptly (several seconds) recovered, indicating that a constant flux of NO is necessary for inhibition. In 1995, Takehara et al. [70] reported that the respiration of phosphorilating mitochondria is reversibly depressed on exposure to NO, the degree of inhibition depending on both the NO and O2 concentration [70,71]. Results in line with these observations were obtained by several groups working with a wide variety of systems, from isolated mitochondria to cells (see Introduction). The susceptibility of cell respiration to NO has also been studied by taking advantage of the electrophoretic import into mitochondria of cationic fluorescent dyes, such as rhodamine123 [29,88] or JC-1 [4,89,90]. The mitochondrial fluorescence pattern analyzed by fluorescence microscopy, after cell treatment with exogenous NO, or with effectors rapidly stimulating the NOsynthase (like NMDA) allowed to confirm the ability of NO to affect the mitochondrial function of cultured cells (Fig. 5) [29]. Always, the onset of mitochondrial inhibition is fast, pointing to CcOX as the primary target. Thus, in a physiological concentration range (ⱕ ␮M) NO inhibits CcOX by forming either the Fenitrosyl [a32⫹NO] or the derivative with nitrite bound at the active site [a33⫹CuB2⫹NO2⫺]. In the former case, activity is recovered at the rate of the thermal dissociation of NO from the reduced site; following this path, NO is released in the bulk phase/cell-cytoplasm as such, i.e., as a reactive radical and needs to be further degraded and/or scavenged. On the contrary, in the latter case, toxic NO is degraded to harmless NO2⫺, and is thereafter displaced upon intramolecular reduction of the site, with prompt restoration of activity. Recently, Sarti et al. [66] designed an experimental protocol based on the well-known photosensitivity of the a32⫹NO complex [91], to discriminate between the accumulation of the two adducts that can be formed when CcOX in turnover is exposed to NO. Typically, a CcOX-containing system (from the purified enzyme to a cell suspension) is allowed to respire in an O2-electrode vessel until NO

Fig. 5. Single-cell fluorescence microscopy: effect of endogenous NO on mitochondrial membrane potential. Top panel: HaCat keratinocytes whose mitochondria have imported electrophoretically the fluorescent dye rhodamine123. Bottom panel: image analysis of cells treated with the NOS inhibitor 7-nitroindazole (7-N) or with the NOS stimulate N-methyl-D-aspartate (NMDA), either alone or in combination. One may observe an increase of fluorescence due to inhibition of NOS, and a decrease that follows its stimulation by NMDA. Specimens treated with nigericin (converting the trans-membrane ⌬pH into ⌬⌿) are taken as control of the 100% fluorescence (⌬␮H⫹ ⬇ 200 mV), while those treated with valinomycin (collapsing the membrane potential, ⌬⌿) represent background (see also ref. [29]).

is added and respiration is inhibited. Once inhibition is set, the rapid removal of free NO by addition of oxy-Hb, used as a scavenger, is allowed to follow the time course of recovery of respiration. Using the purified enzyme, it was shown (Fig. 6) that recovery is different depending on the concentration of the reductants (ferrocytochrome c) sustaining respiration [66]. At high [cytochrome c2⫹], the recovery in the dark occurs at a rate compatible with the off-rate of NO from reduced cytochrome a3, and consistently is accelerated by illuminating the sample, as the a32⫹NO

Reactions of cytochrome oxidase with nitric oxide

515

distinguish two extreme electron flux regimes (high and low) through the respiratory chain (Fig. 7). High electron flux Under this regime, the “nitrosylation” mechanism prevails, because the occupancy of R and E1 is higher [75]. At high electron flux inhibition appears to depend on the rapid and tight binding of NO to reduced heme a3 and O2-competition is observed, showing that binding of both ligands occurs with a reduced site. By extrapolation from the data obtained using the K354M mutant oxidase from Paracoccus denitrificans, Giuffre` et al. [72] concluded that NO binds also to the single electron-reduced binuclear site, thus confirming the kinetic advantage of NO over O2 that justifies the rapid onset of inhibition. The presence of O2 reacting with free NO in the bulk phase drives the (light-sensitive) NO dissociation from reduced cytochrome a3 and accounts for the time-dependent reversal of inhibition. Low electron flux

Fig. 6. Light-induced changes of activity of NO-inhibited CcOX. Schematic representation of typical O2-electrode measurements. Turnover of CcOX is sustained by reductants; NO inhibition is set, and at a given time Hb is added to scavenge free NO. Top panel (high electron flux, i.e., higher amount of reductants): the time course of recovery is different in the dark and light pointing to the presence of the nitrosyl a32⫹-NO derivative. Bottom panel (low electron flux, i.e., lower amount of reductants): recovery is fast (not resolved) and the time course is light insensitive, indicating the presence of the a33⫹ NO2⫺adduct. Notice the different time scale due to the different concentration of reductants.

adduct is light sensitive [91]. On the contrary, when turnover is sustained by a low concentration of cytochrome c2⫹, after free NO removal the recovery is almost immediate (not time resolved in the O2 electrode) and light insensitive, as expected for the nitriteadduct [66]. The existence of these two reaction pathways has been also proved spectroscopically using soluble CcOX [66].

ATTEMPTING TO DEPICT AN OVERALL PICTURE

All together, the evidence summarized above suggests that the electron transfer rate through the mitochondrial respiratory chain is an important parameter driving the NO-to-CcOX interaction towards accumulation of either the nitrosyl- or the nitrite-derivative of the enzyme. These adducts are both inhibited but follow different metabolic fates. To better clarify this point, we shall

Under these conditions the overall occupancy of the intermediates having CuB oxidized increases [75,92]. Oxidized CuB reacts with NO more slowly than (reduced) heme a32⫹, but still rapidly enough to account for the inhibition of CcOX during turnover. This pathway leads to a slow metabolization of NO. Species O, P, and F react with NO at the level of CuB2⫹, forming NO2⫺ in the active site, the nitrite being released into the bulk upon reduction of the site. In conclusion, evidence has been collected for two reaction mechanisms, both accounting for the reversible CcOX inhibition, but only one leading to oxidative degradation of NO. NO SCAVENGING IN THE CELL

Cells producing NO as a messenger, activator, or modulator are faced with its potential mitochondrial toxicity. It may be recalled that a consequence of the pathway leading to accumulation of the nitrosyl-adduct is that, once dissociated from the active site, NO would be released in the cell cytoplasm as such. NO free in the cell environment for a long enough time can ultimately induce apoptosis and cell death [18,93]. This is expected based on the finding that NO not only downregulates the respiratory chain, but also reacts with mitochondrial aconitase [94] and with the superoxide ion to form toxic peroxynitrite (ONOO⫺) [18,95]. The mitochondrial NO toxicity, in the absence of suitable control mechanisms would lead to irreversible opening of the permeability transition pore [96,97], release of cytochrome c and activation of pro-apoptotic factors [98,99]. It is therefore clear that mitochondrial toxicity would be attenuated if

516

P. SARTI et al.

Fig. 7. Schematic representation of the interaction between NO and CcOX. The metabolic electron flux through the respiratory chain is simply defined as high or low to allow us to distinguish between two well different pathways, whereby NO: (i) binds to heme a32⫹, inhibits and is thereafter released as such in the bulk phase, or (ii) binds to CuB2⫹, inhibits and is thereafter degraded to nitrite.

the reaction between NO and CcOX follows the nitrite pathway. In the red muscles, a reaction pathway ensuring the control of intracellular NO concentration was proposed independently by Brunori [100,101] and Flo¨ gel et al. [102]. The mechanism is based on oxymyoglobin (MbO2) playing the role of NO scavenger, the reaction yielding NO3⫺. In these tissues, rich both in mitochondria and Mb, the fast reaction of CcOX with NO [85,103] would depress respiration, while the reaction of oxy-Mb with free NO leads to its efficient degradation to the nontoxic nitrate. In this respect, it is worth mentioning that the interplay between NO and CcOX in the presence of all cellular components, particularly NO scavengers/buffers, still needs to be clarified.

The contribution of CcOX to NO degradation While the capacity of CcOX to oxidize NO to nitrite has never been controversial, doubts have been raised about the property of CcOX, particularly the mammalian, to metabolize NO to N2O via a reductive pathway. The existence of a marginal NO- and NO2⫺-reductase activity of mammalian CcOX was first proposed by Brudvig et al. [58] and later revisited by others [22,104,105], stimulated by the newly discovered implications of NO in the mitochondrial physiology. The capability of CcOX to catalyze the reduction of NO has been postulated not only because of the similarities between NO and O2, but also on the basis of the striking similarities between heme-copper oxidases and bacterial NO-reductases (NOR) [106,107]. On this basis of struc-

Table 1. NO Reductase Activity of Oxidases from Mammals and Bacteria Compared to Bona Fide NOR

Purified aa3, Mitos T.th ba3 Ps. st cbb3 NOR a

TN (mol NO/mol enzyme ⫻ min)

KM (␮M)

Oxidation by 125 ␮M NO k (min⫺1)

Inactive 3 ⫾ 0.7 100 ⫾ 9 300–6000

Inactive 40a 12 ⫾ 2.5 0.002–0.25

Inactive 2.4 120 n.d.

Dissociation constant (KD). Notice that the bovine CcOX, either purified (detergent-solubilized aa3) or in mitochondria (mitos), is unable to reduce NO, whereas both the Thermus thermophilus ba3 and the Pseudomonas stutzeri cbb3-type oxidases display a significant turnover activity that correlates with the kinetics of the enzyme oxidation by NO.

Reactions of cytochrome oxidase with nitric oxide

tural similarities, one may speculate that these two enzymes may use both O2 and NO as substrates, although with different specificity and efficiency. In line with this hypothesis, Fujiwara and Fukumori found that the bona fide NOR from Paracoccus denitrificans (ATCC 35512) displays a measurable O2 reductase activity [108]. The experimental evidence supporting the NO reductase activity of mammalian CcOX, however, has been controversial. Some initial proposals suggested that CcOX may reduce NO to N2O [22,58,104,105]. Stubauer et al. [68], however, demonstrated both optically and amperometrically that purified beef heart CcOX, as well as mitochondrial particles (same source), do not significantly contribute to a reductive degradation of NO. The reduction of NO to N2O by prokaryotic heme-copper oxidases In terms of NO reductase activity, heme-copper oxidases purified from Thermus thermophilus have been found to behave very differently from mammalian CcOX [61]. This microorganism expresses the ba3 cytochrome oxidase when grown under conditions of low O2 tension [109]. The CuB⫹ in this oxidase displays an unusually high affinity for CO, compared with mesophilic oxidases (Kaff ⫽ 103–104 M⫺1 vs. Kaff ⫽ 101–102 M⫺1) [110,111]. If extended to O2 and NO, the higher affinity of CuB⫹ for these ligands would have some important implications. First of all it may favor O2 binding, an important task for the microorganisms living in an oxygen-poor environment. Secondly, the binding of two NO molecules at the active site provides a reasonable molecular basis for formation of N2O. It is worth mentioning that this feature originally proposed for the bo3 oxidase from Escherichia coli [112] has been recently confirmed experimentally [63]. Whatever the mechanism, the ba3type oxidase can catalyze the reduction of NO to N2O at a significant rate (Table 1) [61]. This finding has been confirmed by amperometric and spectrophotometric experiments, and by detection of N2O production by gas chromatography. The same procedures failed to reveal a significant NO-reductase activity by mammalian CcOX, and consistently the affinity of CuB⫹ for CO (and presumably NO) is very much lower (Table 1). The cbb3-type oxidases are the most divergent members of the heme-copper oxidase family, closely related to NOR as suggested by sequence alignment, subunit composition, and type of heme in the binuclear active site (heme b). These enzymes are reported to have an O2 affinity definitely higher than aa3 oxidases [113]. Interestingly, the cbb3 oxidase expressed by Pseudomonas stutzeri under low O2 tension displays the highest NO reductase activity so far detected for oxidases [62]. Relevant from the biomedical viewpoint, the cbb3-type cy-

517

tochrome oxidases are expressed in many human indigenous or pathogenic microorganisms, which can either adapt to (Rhodobacter, Paracoccus spp.) or thrive under microaerophilic condition (Helicobacter and Campylobacter spp.) [114]. Their capacity to exploit both O2 and NO provides these microorganisms with an additional enzymatic tool to cope with the antimicrobial action of NO produced by the host macrophage [115]; this would further protect their respiratory capacity from NO, in an environment that contains both gases. Acknowledgements — Work supported by Ministero dell’Istruzione, dell’Universita` e della Ricerca (MIUR) of Italy (PRIN “Bioenergetica: aspetti genetici, biochimici e fisiopatologici” and Prg. Biotecnologie 5% - Neuroscienze). The research grant “Marker periferici e danno mitocondriale da nitrossido nelle demenze”, by the University of Rome “La Sapienza”, is also gratefully acknowledged. We wish to thank Victor Darley-Usmar (University of Alabama, USA) and Andrea Urbani (University of Chieti, Italy) for stimulating discussions.

REFERENCES [1] Carr, G. J.; Ferguson, S. J. Nitric oxide formed by nitrite reductase of Paracoccus denitrificans is sufficiently stable to inhibit cytochrome oxidase activity and is reduced by its reductase under aerobic conditions. Biochim. Biophys. Acta 1017:57– 62; 1990. [2] Cleeter, M. W. J.; Cooper, J. M.; Darley-Usmar, V. M.; Moncada, S.; Schapira, A. H. V. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. FEBS Lett. 345:50 –54; 1994. [3] Brown, G. C.; Cooper, C. E. Nanomolar concentration of nitric oxide reversibly inhibit synaptosomal cytochrome oxidase respiration, by competing with oxygen at cytochrome oxidase. FEBS Lett. 356:295–298; 1994. [4] Schweizer, M.; Richter, C. NO potently and reversibly deenergizes mitochondria at low oxygen tension. Biochem. Biophys. Res. Commun. 204:169 –175; 1994. [5] Hibbs, J. B. Jr.; Taintor, R. R.; Vavrin, Z.; Rachlin, E. M. NO: a cytotoxic-activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 157:87–94; 1988. [6] Riobo, N. A.; Clementi, E.; Melani, M.; Boveris, A.; Cadenas, E.; Moncada, S.; Poderoso, J. J. Nitric oxide inhibits mitochondrial NADH: ubiquinone reductase activity through peroxynitrite formation. Biochem. J. 359:139 –145; 2001. [7] Clementi, E.; Brown, G. C.; Feelisch, M.; Moncada, S. Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc. Natl. Acad. Sci. USA 95:7631–7636; 1998. [8] Poderoso, J. J.; Lisdero, C.; Schopfer, F.; Riobo, N.; Carreras, M. C.; Cadenas, E.; Boveris, A. The regulation of mitochondrial oxygen uptake by redox reactions involving nitric oxide and ubiquinol. J. Biol. Chem. 274:37709 –37716; 1999. [9] Welter, R.; Yu, C.; Yu, A. The effects of NO on electron transport complexes. Arch. Biochem. Biophys. 331:9 –14; 1996. [10] Torres, J.; Darley-Usmar, V. M.; Wilson, M. T. Inhibition of cytochrome c oxidase in turnover by nitric oxide: mechanism and implications for control of respiration. Biochem. J. 312: 169 –173; 1995. [11] Brunori, M.; Giuffr`e, A.; Sarti, P.; Stubauer, G.; Wilson, M. T. Nitric oxide and cellular respiration. Cell. Mol. Life Sci. 56:549 – 557; 1999. [12] Henry, Y.; Lepoivre, M.; Drapier, J. C.; Ducrocq, C.; Boucher, J. L.; Guissani, A. EPR characterization of molecular targets for NO in mammalian cells and organelles. FASEB J. 7:1124 –1134; 1993. [13] Pearce, L. L.; Epperly, M. W.; Greenberger, J. S.; Pitt, B. R.;

518

[14]

[15] [16] [17]

[18]

[19]

[20] [21] [22]

[23]

[24] [25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

P. SARTI et al. Peterson, J. Identification of respiratory complexes I and III as mitochondrial sites of damage following exposure to ionizing radiation and nitric oxide. Nitric Oxide 5:128 –136; 2001. Brooks, K. J.; Hargreaves, I. P.; Bates, T. E. Nitric-oxideinduced inhibition of mitochondrial complexes following aglycaemic hypoxia in neonatal cortical rat brain slices. Dev. Neurosci. 22:359 –365; 2000. Ignarro, L. J. Nitric oxide: a unique endogenous signaling molecule in vascular biology. Biosci. Rep. 19:51–71; 1999. Lane, P.; Gross, S. S. Cell signaling by nitric oxide. Semin. Nephrol. 19:215–229; 1999. Brennan, P. A.; Moncada, S. From pollutant gas to biological messenger: the diverse actions of nitric oxide in cancer. Ann. R. Coll. Surg. Engl. 84:75–78; 2002. Moncada, S.; Erusalimsky, J. D. Does nitric oxide modulate mitochondrial energy generation and apoptosis? Nat. Rev. Mol. Cell Biol. 3:214 –220; 2002. Ignarro, L. J.; Napoli, C.; Loscalzo, J. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview. Circ. Res. 90:21–28; 2002. Cooper, C. E. Nitric oxide and iron proteins. Biochim. Biophys. Acta 1411:290 –309; 1999. Torres, J.; Wilson, M. T. The reactions of copper proteins with nitric oxide. Biochim. Biophys. Acta 1411:310 –322; 1999. Zhao, X.-J.; Sampath, V.; Caughey, W. S. Cytochrome c oxidase catalysis of the reduction of nitric oxide to nitrous oxide. Biochem. Biophys. Res. Commun. 212:1054 –1060; 1995. Torres, J.; Sharpe, M. A.; Rosquist, A.; Cooper, C. E.; Wilson, M. T. Cytochrome c oxidase rapidly metabolises nitric oxide to nitrite. FEBS Lett. 475:263–266; 2000. Brown, G. C. Nitric oxide and mitochondrial respiration. Biochim. Biophys. Acta 1411:351–369; 1999. Heales, J. R.; Bolan˜ os, J. P.; Stewart, V. C.; Brookes, P. S.; Land, J. M.; Clark, J. B. NO, mitochondria and neurological disease. Biochim. Biophys. Acta 1410:215–228; 1999. Wolin, M. S.; Xie, Y. W.; Hintze, T. H. Nitric oxide as a regulator of tissue oxygen consumption. Curr. Opin. Nephrol. Hypertens. 8:97–103; 1999. Giuffr`e, A.; Sarti, P.; D’ Itri, E.; Buse, G.; Soulimane, T.; Brunori, M. On the mechanism of inhibition of cytochrome c oxidase by nitric oxide. J. Biol. Chem. 271:33404 –33408; 1996. Balligand, J. L.; Ungureanu-Longrois, D.; Kelly, R. A.; Kobzik, L.; Pimental, D.; Michel, T.; Smith, T. W. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J. Clin. Invest. 91:2314 –2319; 1993. Sarti, P.; Lendaro, E.; Ippoliti, R.; Bellelli, A.; Benedetti, P. A.; Brunori, M. Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy. FASEB J. 13:191–197; 1999. Stumpe, T.; Decking, U. K. M.; Schrader, J. Nitric oxide reduces energy supply by direct action on the respiratory chain in isolated cardiomyocytes. Am. J. Physiol Heart Circ. Physiol. 280: H2350 –H2356; 2001. Brady, A. J.; Warren, J. B.; Poole-Wilson, P. A.; Williams, T. J.; Harding, S. E. Nitric oxide attenuates cardiac myocyte contraction. Am. J. Physiol. 265:H176 –H182; 1993. Shen, W.; Hintze, T. H.; Wolin, M. S. Nitric oxide. An important signaling mechanism between vascular endothelium and parenchymal cells in the regulation of oxygen consumption. Circulation 92:3505–3512; 1995. Xie, Y. W.; Shen, W.; Zhao, G.; Xu, X.; Wolin, M. S.; Hintze, T. H. Role of endothelium-derived nitric oxide in the modulation of canine myocardial mitochondrial respiration in vitro. Implications for the development of heart failure. Circ. Res. 79:381– 387; 1996. Hare, J. M.; Keaney, J. F.; Balligand, J. L.; Loscalzo, J.; Smith, T. W.; Colucci, W. S. Role of nitric oxide in parasympathetic modulation of ␤-adrenergic myocardial contractility in normal dogs. J. Clin. Invest. 95:360 –366; 1995. Zhao, G.; Bernstein, T. H.; Hintze, T. H. Nitric oxide and

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43] [44]

[45]

[46] [47]

[48]

[49] [50]

[51]

[52]

[53]

[54]

[55]

[56] [57]

oxygen utilization: exercise, heart failure and diabetes. Coron. Artery Dis. 10:315–320; 1999. Yoshikawa, S.; Shinzawa-Itoh, K.; Tsukihara, T. X-ray structure and the reaction mechanism of bovine heart cytochrome c oxidase. J. Inorg. Biochem. 82:1–7; 2000. Iwata, S.; Ostermeier, C.; Ludwig, B.; Michel, H. Structure at 2.8 Å resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376:660 – 669; 1995. Svensson-Ek, M.; Abramson, J.; Larsson, G.; Tornroth, S.; Brzezinski, P.; Iwata, S. The X-ray crystal structures of wildtype and EQ(I-286) mutant cytochrome c oxidases from Rhodobacter sphaeroides. J. Mol. Biol. 321:329 –339; 2002. Soulimane, T.; Buse, G.; Bourenkov, G. P.; Bartunik, H. D.; Huber, R.; Than, M. E. Structure and mechanism of the aberrant ba(3)-cytochrome c oxidase from Thermus thermophilus. EMBO J. 19:1766 –1776; 2000. Abramson, J.; Riistama, S.; Larsson, G.; Jasaitis, A.; SvenssonEk, M.; Laakkonen, L.; Puustinen, A.; Iwata, S.; Wikstr¨om, M. The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site. Nat. Struct. Biol. 7:910 –917; 2000. Proshlyakov, D. A.; Pressler, M. A.; DeMaso, C.; Leykam, J. F.; DeWitt, D. L.; Babcock, G. T. Oxygen activation and reduction in respiration: involvement of redox-active tyrosine 244. Science 290:1588 –1591; 2000. Malatesta, F.; Antonini, G.; Sarti, P.; Brunori, M. Structure and function of a molecular machine: cytochrome c oxidase. Biophys. Chem. 54:1–33; 1995. Gaston, B. Nitric oxide and thiol groups. Biochim. Biophys. Acta 1411:323–333; 1999. Stubauer, G.; Giuffr`e, A.; Sarti, P. Mechanism of S-nitrosothiol formation and degradation mediated by copper ions. J. Biol. Chem. 274:28128 –28133; 1999. Patel, R. P.; McAndrew, J.; Sellak, H.; White, C. R.; Jo, H.; Freeman, B. A.; Darley-Usmar, V. M. Biological aspects of reactive nitrogen species. Biochim. Biophys. Acta 1411:385– 400; 1999. Hogg, N. Biological chemistry and clinical potential of S-nitrosothiols. Free Radic. Biol. Med. 28:1478 –1486; 2000. Malatesta, F.; Georgevich, G.; Capaldi, R. A. Structural studies on beef heart cytochrome c oxidase from which subunit II has been removed by chimotrypsin treatment. In: Quagliarello, E.; Palmieri, F., eds. Structure and function of membrane proteins. Amsterdam: Elsevier; 1983:223. Pearce, L. L.; Pitt, B. R.; Peterson, J. The peroxynitrite reductase activity of cytochrome c oxidase involves a two-electron redox reaction at the heme a3-CuB site. J. Biol. Chem. 274:35763– 35767; 1999. Ferguson-Miller, S.; Babcock, G. T. Heme-copper terminal oxidases. Chem. Rev. 96:2889 –2907; 1996. Babcock, G. T.; Wikstr¨om, M. Oxygen activation and the conservation of energy in cell respiration. Nature 356:301–309; 1992. Michel, H. Cytochrome c oxidase: catalytic cycle and mechanisms of proton pumping, a discussion. Biochemistry 38:15129 – 15140; 1999. Sucheta, A.; Szundi, I.; Einarsdo´ ttir, O. Intermediates in the reaction of fully reduced cytochrome c oxidase with dioxygen. Biochemistry 37:17905–17914; 1998. Zaslavsky, D.; Gennis, R. B. Proton pumping by cytochrome oxidase: progress, problems and postulates. Biochim. Biophys. Acta 1458:164 –179; 2000. Verkhovsky, M. I.; Morgan, J. E.; Wikstr¨om, M. Control of electron delivery to the oxygen reduction site of cytochrome c oxidase: a role for protons. Biochemistry 34:7483–7491; 1995. Fabian, M.; Palmer, G. Redox state of peroxy and ferryl intermediates in cytochrome c oxidase catalysis. Biochemistry 38: 6270 – 6275; 1999. Wanio, W. W. Reactions of cytochrome oxidase. J. Biol. Chem. 212:723–733; 1955. Gibson, Q. H.; Greenwood, C. Reactions of cytochrome oxidase

Reactions of cytochrome oxidase with nitric oxide

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67] [68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

with oxygen and carbon monoxide. Biochem. J. 86:541–554; 1963. Brudvig, G. W.; Stevens, T. H.; Chan, S. I. Reactions of nitric oxide with cytochrome c oxidase. Biochemistry 19:5275–5285; 1980. Malatesta, F.; Sarti, P.; Antonini, G.; Vallone, B.; Brunori, M. Electron transfer to the binuclear center in cytochrome oxidase: catalytic significance and evidence for an additional intermediate. Proc. Natl. Acad. Sci. USA 87:7410 –7413; 1990. Brunori, M.; Giuffr`e, A.; D’Itri, E.; Sarti, P. Internal electron transfer in Cu-heme oxidases. Thermodynamic or kinetic control? J. Biol. Chem. 272:19870 –19874; 1997. Giuffr`e, A.; Stubauer, G.; Sarti, P.; Brunori, M.; Zumft, W. G.; Buse, G.; Soulimane, T. The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: evolutionary implications. Proc. Natl. Acad. Sci. USA 96:14718 –14723; 1999. Forte, E.; Urbani, A.; Saraste, M.; Sarti, P.; Brunori, M.; Giuffr`e, A. The cytochrome cbb3 from Pseudomonas stutzeri displays nitric oxide reductase activity. Eur. J. Biochem. 268:6486 – 6491; 2001. Butler, C. S.; Forte, E.; Scandurra, F. M.; Arese, M.; Giuffr`e, A.; Greenwood, C.; Sarti, P. Cytochrome bo3 from Escherichia coli: the binding and turnover of nitric oxide. Biochem. Biophys. Res. Commun. 296:1272–1278; 2002. Cooper, C. E.; Torres, J.; Sharpe, M. A.; Wilson, M. T. Nitric oxide ejects electrons from the binuclear center of cytochrome c oxidase by reacting with oxidised copper: a general mechanism for the interaction of copper proteins with nitric oxide? FEBS Lett. 414:281–284; 1997. Blackmore, R. S.; Greenwood, C.; Gibson, Q. H. Studies of the primary oxygen intermediate in the reaction of fully reduced cytochrome oxidase. J. Biol. Chem. 266:19245–19249; 1991. Sarti, P.; Giuffr`e, A.; Forte, E.; Mastronicola, D.; Barone, M. C.; Brunori, M. Nitric oxide and cytochrome c oxidase: mechanisms of inhibition and NO degradation. Biochem. Biophys. Res. Commun. 274:183–187; 2000. Gibson, Q. H.; Roughton, F. J. W. Reaction of hemoglobin with nitric oxide. J. Physiol. 136:507–516; 1957. Stubauer, G.; Giuffr`e, A.; Brunori, M.; Sarti, P. Cytochrome c oxidase does not catalyze the anaerobic reduction of NO. Biochem. Biophys. Res. Commun. 245:459 – 465; 1998. Brown, G. C. Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase. FEBS Lett. 369:136 –139; 1995. Takehara, Y.; Kanno, T.; Yoshioka, T.; Inoue, M.; Utsumi, K. O2-dependent regulation of mitochondrial energy metabolism by nitric oxide. Arch. Biochem. Biophys. 323:27–32; 1995. Koivisto, A.; Matthias, A.; Bronnikov, G.; Nedergaard, J. Kinetics of the inhibition of mitochondrial respiration by NO. FEBS Lett. 417:75– 80; 1997. Giuffr`e, A.; Barone, M. C.; Brunori, M.; D’ Itri, E.; Ludwig, B.; Malatesta, F.; Mu¨ ller, H. W.; Sarti P. Nitric oxide reacts with the single-electron reduced active site of cytochrome c oxidase. J. Biol. Chem. 277:22402–22406; 2002. Giuffr`e, A.; Stubauer, G.; Brunori, M.; Sarti, P.; Torres, J.; Wilson, M. T. Chloride bound to oxidized cytochrome c oxidase controls the reaction with nitric oxide. J. Biol. Chem. 273: 32475–32478; 1998. Torres, J.; Cooper, C. E.; Wilson, M. T. A common mechanism for the interaction of nitric oxide with the oxidised binuclear center and oxygen intermediates of cytochrome c oxidase. J. Biol. Chem. 273:8756 – 8766; 1998. Giuffr`e, A.; Barone, M. C.; Mastronicola, D.; D’Itri, E.; Sarti, P.; Brunori, M. Reaction of nitric oxide with the turnover intermediates of cytochrome c oxidase: reaction pathway and functional effects. Biochemistry 39:15446 –15453; 2000. Paitian, N. A.; Markossian, K. A.; Nalbandyan, R. M. The effect of nitrite on cytochrome oxidase. Biochem. Biophys. Res. Commun. 133:1104 –1111; 1985. Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Nitric oxide

[78] [79]

[80]

[81]

[82]

[83]

[84] [85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

519

synthases: structure, function and inhibition. Biochem. J. 357: 593– 615; 2001. Stamler, J. S.; Meissner, G. Physiology of nitric oxide in skeletal muscle. Physiol. Rev. 81:209 –237; 2001. Huber, A.; Saur, D.; Kurjak, M.; Schusdziarra, V.; Allescher, H. D. Characterization and splice variants of neuronal nitric oxide synthase in rat small intestine. Am. J. Physiol. 275: G1146 –G1156; 1998. Kobzik, L.; Stringer, B.; Balligand, J. L.; Reid, M. B.; Stamler, J. S. Endothelial type nitric oxide synthase in skeletal muscle fibers: mitochondrial relationships. Biochem. Biophys. Res. Commun. 211:375–381; 1995. Bates, T. E.; Loesch, A.; Burnstock, G.; Clark, J. B. Mitochondrial nitric oxide synthase: a ubiquitous regulator of oxidative phosphorylation? Biochem. Biophys. Res. Commun. 218:40 – 44; 1996. Bates, T. E.; Loesch, A.; Burnstock, G.; Clark, J. B. Immunocytochemical evidence for a mitochondrially located nitric oxide synthase in brain and liver. Biochem. Biophys. Res. Commun. 213:896 –900; 1995. Frandsen, U.; Lopez-Figueroa, M.; Hellsten, Y. Localization of nitric oxide synthase in human skeletal muscle. Biochem. Biophys. Res. Commun. 227:88 –93; 1996. Ghafourifar, P.; Richter, C. Nitric oxide synthase activity in mitochondria. FEBS Lett. 418:291–296; 1997. Tatoyan, A.; Giulivi, C. Purification and characterization of a nitric-oxide synthase from rat liver mitochondria. J. Biol. Chem. 273:11044 –11048; 1998. Kanai, A. J.; Pearce, L. L.; Clemens, P. R.; Birder, L. A.; VanBibber, M. M.; Choi, S. Y.; de Groat, W. C.; Peterson, J. Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection. Proc. Natl. Acad. Sci. USA 98:14126 –14131; 2001. Rubbo, H.; Radi, R.; Anselmi, D.; Kirk, M.; Barnes, S.; Butler, J.; Eiserich, J. P.; Freeman, B. A. Nitric oxide reaction with lipid peroxyl radicals spares alpha-tocopherol during lipid peroxidation. Greater oxidant protection from the pair nitric oxide/alphatocopherol than alpha-tocopherol/ascorbate. J. Biol. Chem. 275: 10812–10818; 2000. Johnson, L. V.; Walsh, M. L.; Chen, L. B. Localization of mitochondria in living cells with rhodamine 123. Proc. Natl. Acad. Sci. USA 77:990 –994; 1980. Richter, C.; Gogvadze, V.; Schlapbach, R.; Schweizer, M.; Schlegel, J. Nitric oxide kills hepatocytes by mobilizing mitochondrial calcium. Biochem. Biophys. Res. Commun. 205:1143– 1150; 1994. Laffranchi, R.; Gogvadze, V.; Richter, C.; Spinas, G. A. Nitric oxide (nitrogen monoxide, NO) stimulates insulin secretion by inducing calcium release from mitochondria. Biochem. Biophys. Res. Commun. 217:584 –591; 1995. Boelens, R.; Wever, R.; Van Gelder, B. F.; Rademaker, H. An EPR study of the photodissociation reactions of oxidised cytochrome c oxidase-nitric oxide complexes. Biochim. Biophys. Acta 724:176 –183; 1983. Moody, A. J. Ligation and electronation states of cytochrome c oxidase in relation to other oxidases and peroxidases. Biochem. Soc. Trans. 19:617– 621; 1991. Ghafourifar, P.; Schenk, U.; Klein, S. D.; Richter, C. Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation. J. Biol. Chem. 274:31185–31188; 1999. Stadler, J.; Billiar, T. R.; Curran, R. D. Z.; Stuehr, D. J.; Ochoa, J. B.; Simmons, R. L. Effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes. Am. J. Physiol. 260:C910 –C916; 1991. Sharpe, M. A.; Cooper, C. E. Interaction of peroxynitrite with mitochondrial cytochrome oxidase. Catalytic production of nitric oxide and irreversible inhibition of enzyme activity. J. Biol. Chem. 273:30961–30972; 1998. Halestrap, A. P.; Woodfield, K. Y.; Connern, C. P. Oxidative

520

[97]

[98]

[99] [100] [101] [102]

[103]

[104]

[105]

[106]

[107]

[108]

P. SARTI et al. stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase. J. Biol. Chem. 272:3346 –3354; 1997. Horn, T. F.; Wolf, G.; Duffy, S.; Weiss, S.; Keilhoff, G.; MacVicar, B. A. Nitric oxide promotes intracellular calcium release from mitochondria in striatal neurons. FASEB J. 16: 1611–1622; 2002. Brookes, P.; Levonen, A.; Shiva, S.; Sarti, P.; Darley-Usmar, V. Mitochondria: regulators of signal transduction by reactive oxygen and nitrogen species. Free Radic. Biol. Med. 33:755; 2002. Brown, G. C.; Borutaite´ , V. Nitric oxide, cytochrome c and mitochondria. Biochem. Soc. Symp. 66:17–25; 1999. Brunori, M. Nitric oxide, cytochrome c oxidase and myoglobin. Trends Biochem. Sci. 26:21–23; 2001. Brunori, M. Nitric oxide moves myoglobin center stage. Trends Biochem. Sci. 26:209 –210; 2001. Flogel, U.; Merx, M. W.; Godecke, A.; Decking, U. K.; Schrader, J. Myoglobin: a scavenger of bioactive NO. Proc. Natl. Acad. Sci. USA 98:735–740; 2001. Frandsen, U.; Lopez-Figueroa, M.; Hellsten, Y. Localization of nitric oxide synthase in human skeletal muscle. Biochem. Biophys. Res. Commun. 227:88 –93; 1996. Borutait´e, V.; Brown, G. C. Rapid reduction of nitric oxide by mitochondria, and reversible inhibition of mitochondrial respiration by nitric oxide. Biochem. J. 315:295–299; 1996. Clarkson, R. B.; Norby, S. W.; Smirnov, A.; Boyer, S.; Vahidi, N.; Nims, R. W.; Wink, D. A. Direct measurement of the accumulation and mitochondrial conversion of nitric oxide within Chinese hamster ovary cells using an intracellular electron paramagnetic resonance technique. Biochim. Biophys. Acta 1243:496 –502; 1995. Saraste, M.; Castresana, J. Cytochrome oxidase evolved by tinkering with denitrification enzymes. FEBS Lett. 341:1– 4; 1994. Van der Oost, J.; De Boer, A. P. N.; De Gier, J. W. L.; Zumft, W. G.; Stouthamer, A. H.; Van Spanning, R. J. M. The hemecopper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase. FEMS Microbiol. Lett. 121:1–10; 1994. Fujiwara, T.; Fukumori, Y. Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512. J. Bacteriol. 178:1866 –1871; 1996.

[109] Keightley, J. A.; Zimmermann, B. H.; Mather, M. W.; Springer, P.; Pastuszyn, A.; Lawrence, D. M.; Fee, J. A. Molecular genetic and protein chemical characterization of the cytochrome ba3 from Thermus thermophilus HB8. J. Biol. Chem. 270:20345– 20358; 1995. [110] Einarsdo´ ttir, O.; Killough, P. M.; Fee, J. A.; Woodruff, W. H. An infrared study of the binding and photodissociation of carbon monoxide in cytochrome ba3 from Thermus thermophilus. J. Biol. Chem. 264:2405–2408; 1989. [111] Giuffr`e, A.; Forte, E.; Antonini, G.; D’Itri, E.; Brunori, M.; Soulimane, T.; Buse, G. Kinetic properties of ba3 oxidase from Thermus thermophilus: effect of temperature. Biochemistry 38: 1057–1065; 1999. [112] Butler, C. S.; Seward, H. E.; Greenwood, C.; Thomson, A. J. Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB. Biochemistry 36:16259 –16266; 1997. [113] Preisig, O.; Zufferey, R.; Thony-Meyer, L.; Appleby, C. A.; Hennecke, H. A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum. J. Bacteriol. 178:1532–1538; 1996. [114] Myllykallio, H.; Liebl, U. Dual role for cytochrome cbb3 oxidase in clinically relevant proteobacteria? Trends Microbiol. 8:542– 543; 2000. [115] Clark, I. A.; Rockett, K. A. Nitric oxide and parasitic disease. Adv. Parasitol. 37:1–56; 1996. [116] Tsukihara, T.; Aoyama, H.; Yamashita, E.; Tomizaki, T.; Yamaguchi, H.; Shinzawa-Itoh, K.; Nakashima, R.; Yaono, R.; Yoshikawa, S. Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 Å. Science 269:1069 –1074; 1995. [117] Tsukihara, T.; Aoyama, H.; Yamashita, E.; Tomizaki, T.; Yamaguchi, H.; Shinzawa-Itoh, K.; Nakashima, R.; Yaono, R.; Yoshikawa, S. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å. Science 272:1136 – 1144; 1996.

ABBREVIATIONS

CcOX— cytochrome c oxidase 7-N—7-nitro-indazole NMDA—N-methyl-D-aspartate NOR— bacterial NO reductase NOS—NO synthase