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J. Biol. Chem., Vol. 277, Issue 16, 13556-13562, April 19, 2002

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The Catabolic Fate of Nitric Oxide

THE NITRIC OXIDE OXIDASE AND PEROXYNITRITE REDUCTASE ACTIVITIES OF CYTOCHROME OXIDASE^*

Linda L. Pearce^§, Anthony J. Kanai^¶, Lori A. Birder^¶, Bruce R. Pitt, and Jim Peterson^§

From the  Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, Pittsburgh, Pennsylvania 15261, ^¶ Department of Medicine/Renal Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and  Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 and Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Received for publication, October 11, 2001, and in revised form, January 31, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stimulation of cardiomyocytes to endogenously evolve nitric oxide is shown by microsensor measurements on single cells to lead to transient nitric oxide concentrations of a few hundred nanomolar. At these submicromolar concentrations, no evidence could be found for the expected reaction between nitric oxide generated and the oxymyoglobin present in the cells: nitric oxide + oxymyoglobin  nitrate + metmyoglobin. No metmyoglobin formation was detected by electron paramagnetic resonance spectroscopy, and microsensor measurements revealed near quantitative conversion of the nitric oxide to nitrite rather than nitrate ion. Moreover, the rate of nitrite formation is shown to be too rapid to be accounted for by non-enzymatic means. The essentially quantitative and rapid catabolism of nitric oxide to nitrite ion can plausibly be explained on the basis of a cycle of reactions catalyzed by cytochrome c oxidase. It is demonstrated with the purified hemoproteins in vitro that the terminal oxidase can outcompete oxymyoglobin for available nitric oxide. It is proposed that under normal physiological and most pathological (non-inflammatory) conditions, reaction with cytochrome c oxidase is the major route by which NO is removed from mitochondria-rich cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It has been widely accepted that the reactions of nitric oxide (NO) with either oxyhemoglobin or oxymyoglobin to form nitrate ion, and respectively, methemoglobin or metmyoglobin constitute the major catabolic pathways by which excess NO is removed from tissues in vivo. The in vitro reactions are facile with second-order rate constants at pH 7.0 and 20-25 °C of about 4 × 10⁷ M¹ s¹ (1, 2). Consequently, it is reasonable to suppose that the relevant rate constants governing the same reactions at pH 7.4 and 37 °C are around 10⁸ M¹ s¹. The physiological significance of these processes is of considerable importance, exerting a fundamental influence on our understanding of cardiovascular NO levels, and thus, is an important consideration in the design of various NO delivery therapies. Recently, the conventional belief that the hemoglobin reaction is the primary catabolic process responsible for NO removal from the vasculature has been questioned (3, 4). Until now, the physiological significance of the myoglobin reaction has not been the subject of much debate. Indeed, at relatively high levels of NO (as might be encountered, for example, during inflammation), it has been conclusively demonstrated that metmyoglobin is slowly formed in Langendorff-perfused mouse hearts (5). Here, however, we demonstrate that at NO levels prevalent under more typically encountered physiological conditions, there is no measurable increase in metmyoglobin content of cardiomyocytes. Furthermore, rather than nitrate ion, the major product of cellular catabolism is nitrite ion (NO), probably formed in a reaction involving cytochrome c oxidase. It is proposed that the reaction catalyzed by cytochrome c oxidase (and not the stoichiometric one with oxymyoglobin) constitutes the major pathway for NO catabolism in mitochondria-rich cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Analyses, Enzymes, and Reagents-- Cytochrome c oxidase (mitochondrial respiratory complex IV) was prepared from beef heart as described previously (method 2) (6). Crystalline, bovine cytochrome c (type III), and equine myoglobin were obtained from Sigma and used without further purification. Lauryl maltoside was obtained from Anatrace. Sodium dithionite was obtained from EM Science and used under anaerobic conditions to make up solutions volumetrically, employing the manufacturer's assay (93%) to calculate dithionite ion concentration. Other reagents were ACS-certified and supplied by Aldrich, Sigma, or Fisher. Stable, hydrogen peroxide-free, alkaline (pH ~12) solutions of sodium peroxynitrite were prepared following the recommendations of Beckman et al. (7). Concentrations of cytochrome c oxidase, ferrocytochrome c, myoglobin, and peroxynitrite were determined using, respectively, the extinction coefficients ₆₀₄ = 24 mM¹ cm¹ (reduced minus oxidized, total heme) (8), ₅₅₀ = 28 mM¹ cm¹ (9), ₅₅₇ = 34.5 mM¹ cm¹ (pyridine hemochromogen) (10), and ₃₀₂ = 1.67 mM¹ cm¹ (11). Determinations of nitrite ion concentration were performed by a modification of the method of Greiss as described by Granger et al. (12), and also, in some cases, electrochemically (see below). First, protein was precipitated by heating aqueous solutions to near boiling and removed by centrifugation. Next, 100-µl aliquots of the supernatant were mixed with 100 µl of the Greiss reagent (0.1% N-(1-napthyl)ethylenediamine hydrochloride freshly mixed with an equal volume of 1.0% sulfanilamide in 5% phosphoric acid). Nitrite concentrations were then determined colorimetrically at 550 nm by comparison with a sodium nitrite standard curve. Nitrate ion concentrations were determined by the same method following reduction of nitrate to nitrite ion (12), the difference in nitrite level before and after the reduction step being used to calculate nitrate concentration in samples. Ferrocytochrome c:O₂ oxidoreductase activity was determined employing the high ionic strength conditions of Sinjorgo et al. (13).

Cardiomyocyte Preparations-- Noncontracting, Ca²⁺-tolerant cardiomyocytes were prepared from 8-12-week-old wild-type or mdx mice (14). Briefly, after inducing deep anesthesia with sodium pentobarbital (50 mg/kg), the thoracic cavity was opened, and the heart was rapidly excised, cannulated at the aorta, and then retrogradely perfused (37 °C, 120 mm Hg) with a modified Langendorff preparation. The heart was first perfused for 5 min with a solution of NaCl (144 mM), KCl (5.4 mM), NaH₂PO₄ (0.4 mM), HEPES (10 mM), MgCl₂ (1 mM), and CaCl₂ (1.8 mM), pH 7.4; next perfused for 5 min with a similar solution to the first but containing no CaCl₂ and containing additionally EGTA (0.1 mM), creatine (5 mM), and taurine (10 mM); finally perfused for 15 min with a similar solution to the second but containing no EGTA and containing in addition 1.5 mg/ml collagenase, 0.5 mg/ml protease, and 0.01 mg/ml elastase. After perfusion, the atria and right ventricle were removed and then discarded. The remaining left ventricle was minced and incubated with stirring in the collected enzyme mix, additionally containing bovine serum albumin (10 mg/ml). Aliquots (10 ml) of the stirred mixture of enzymes and suspended, dissociated cells were withdrawn and centrifuged (1,000 × g, 5 °C) for 5 min. The supernatant was returned to the stirring mixture, and the pellet was resuspended in medium 199 (Sigma) at 25 °C, under which conditions the cardiomyocytes can be reliably maintained for 4-6 h.

For spectroscopic experiments, the heart was excised as above, and then, in order to remove the blood, perfused with the first solution only. Next, the whole heart was minced in two passes at right angles to each other, using a McIlwain motorized tissue chopper set to chop at 150-µm intervals. As cardiomyocytes can be considered roughly cylindrical with a length of ~75 µm and a diameter of ~15 µm, this treatment ensured that a high proportion of intact cardiomyocytes remained in samples. The minced tissue was suspended in 0.2 ml of buffer (first perfusion solution) and then introduced to a 5-mm outer diameter quartz EPR tube. Following the addition of any further reagents (if required by a particular experimental procedure), the sample was quickly frozen (~8 s) by immersion in liquid nitrogen and then stored at 77 K. Subsequently, samples were transferred to the EPR cryostat for measurement without thawing.

Microsensor Methods-- Porphyrinic microsensors (0.1-15-µm tip diameter, 1 nM NO detection limit, 1-ms response time) were prepared from carbon strands (1-5 fibers, 5-µm diameter each, AMOCO) as described previously (15). Single-fiber microsensors to be used for intracellular nitrite measurements were thermally sharpened to a 0.1-µm tip diameter. Monomeric tetrakis(3-methoxy-4-hydroxyphenyl)nickel(II)-porphyrin (Frontier Scientific) was dissolved in 0.1 M NaOH and deposited as a polymeric film on the carbon fiber by cyclic voltammetry (0.2 to +1.0 volts, 20 cycles, EG&G 283 Potentiostat). Nafion (Sigma) was then applied to NO microsensors by dipping in a 1% ethanolic solution. Measurements were performed using a three-electrode system consisting of a working microsensor, a saturated calomel reference, and a platinum counter electrode. Microsensors were characterized by differential pulse voltammetry to determine the effective redox potentials of NO and nitrite. Quantitations by chronoamperometry were performed at a constant overpotential of 50 mV to determine sensitivities and detection limits. High purity (>99.99%) NO and nitrite standards were prepared for accurate calibration as described previously (16). The microsensor was mounted on an ultra-micromanipulator (0.2-µm resolution), enabling the tip to be placed on the cell surface for NO measurements or to gently pierce the membrane for nitrite determinations. The currents generated by oxidation of the analyte at the porphyrinic interface were amplified, converted to voltages, and then digitized for real-time viewing and storage.

Spectroscopic Measurements-- Electronic absorption spectra in the range 200-900 nm were recorded, and colorimetric quantitations were performed employing a Shimadzu UV-2501PC dual-beam dispersive (double monochromator) spectrophotometer. X-band electron paramagnetic resonance spectra were obtained using an IBM ESP 300 spectrometer equipped with a Bruker B-E 25 electromagnet and Bruker ER4116DM resonant cavity. Cryogenic temperatures were achieved by means of an Oxford Instruments ESR 910 liquid helium flow cyrostat in conjunction with a VC30 controller. Signal frequency and intensity calibrations were verified daily using, respectively, diphenylpicrylhydrazyl and ethylenediaminetetraacetocuprate(II) standard solutions.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Quantitative Production ofNO by Cardiomyocytes-- The response of single mdx mouse cardiomyocytes to electrical stimulation was a transient production of NO, typically reaching a maximum concentration of ~130 nM (Fig. 1A). The mdx mouse lacks the gene coding for the protein dystrophin, which tethers endothelial nitric oxide synthase to the cell membrane. It is an experimentally useful attribute of the mdx cell that NO production at readily measurable levels can be electrically stimulated in this highly reproducible manner (14). Following the NO pulse, a buildup of nitrite ion (NO) could routinely be detected, reaching a final concentration of ~115 nM (Fig. 1A). Allowing for some loss of NO by diffusion away, detection of NO at 85% the level of NO initially produced effectively represents quantitative conversion of the NO remaining in the cell to NO.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Microelectrode traces of NO and NO production at 22 °C, pH 7.4. A, single cultured mouse (mdx) cardiomyocyte stimulated to release NO by electrical pulse. B, section of perfused (blood-free) mouse aorta stimulated to release NO by flow-induced (peristaltic pump) shear stress.

There are known mechanisms by which NO may be converted to NO in vitro (17, 18). These processes are, however, quite slow at the low NO concentrations in question. To demonstrate this, a length of rat aorta was perfused with buffered saline solution to apply shear stress to the endothelial cells and evoke NO production (Fig. 1B). In this case, simultaneous measurement of the NO produced showed that only ~12% of the NO generated was converted to NO (Fig. 1B). Moreover, noting the different time scales of the experimental traces shown in Fig. 1, A and B, it is clear that the rate of NO production in Fig. 1B lagged well behind that observed in Fig. 1A. In short, the rapid and near quantitative conversion of NO to NO observed in the cardiomyocytes must have required the participation of one or more essential cellular components.

Low Reactivity of Cytoplasmic Oxymyoglobin toward NO-- It has been previously shown that in vitro, oxymyoglobin reacts rapidly with NO to produce metmyoglobin and nitrate ion (NO) (1, 2). Monitoring the course of this reaction during a titration by visible region electronic absorption spectroscopy results in well maintained isosbestic points being observed (Fig. 2A). Thus, the reaction involves the straightforward conversion of oxymyoglobin to another single derivative. Signals appearing at ~1,150 G (g = 6) in the cryogenic electron paramagnetic resonance spectra of oxymyoglobin samples following the addition of NO (Fig. 2B) confirm that the product in question is metmyoglobin, oxymyoglobin itself being EPR silent. Analyses of the solutions following the recording of these particular spectra (data not shown) were fully consistent with NO being the majority reaction product, which is in keeping with the findings of the earlier authors (1, 2).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Anaerobic reaction of bovine oxymyoglobin with NO at 22 °C, pH 7.4. A, visible region electronic absorption spectra with 1.00-cm path length, 20 µM in total heme. The sample was initially prepared as oxymyoglobin (solid line) and then titrated with aliquots of NO until fully converted to metmyoglobin (dotted line). The arrows indicate the directions of the observed spectral changes during the course of the titration with NO. B, X-band EPR spectra at 15 K, 100 µM in total heme. Samples were prepared at ambient temperature and frozen within ~8 s for storage prior to recording data. The sample was initially prepared as oxymyoglobin (featureless spectrum) and then titrated with aliquots of NO until fully converted to metmyoglobin (derivative-shaped g = 6 signal).

Fortunately, one does not require optically transparent samples for EPR measurements, and so it is quite possible to record useful data on less highly purified materials. Preparations of minced heart tissue from both wild-type and mdx mice in which most of the cardiocytes remain intact and viable prior to rapid freezing (see "Experimental Procedures") were found to exhibit small EPR signals at g = 6 due to the presence of metmyoglobin at a low concentration (~1%) relative to total myoglobin (Fig. 3A, left panel). Note that after dispersing the minced tissue in a small volume of buffered saline, the total myoglobin concentrations in Figs. 2B and 3 were about the same. Unexpectedly, when the minced heart of a wild-type mouse was first stimulated to release NO by application of norepinephrine (14) before quickly freezing, there was no apparent evidence of metmyoglobin production (Fig. 3B, left panel). Even more surprising, when the minced tissue was flooded with excess NO prior to freezing, there was still no significant increase in the metmyoglobin content of samples (Fig. 3C, left panel). There were, however, new signals around 3,400 G (g = 2) characteristic of nitrosomyoglobin present in the spectrum (Fig. 3C, right panel) corresponding to <10% of the total myoglobin present. Nitrosomyoglobin is rapidly formed by the combination of deoxymyoglobin with NO, and the appearance of signals at g = 2 confirms the presence of NO in the experiment. Moreover, the sample manipulations were performed aerobically, the proportion of the total myoglobin in the oxy form was almost certainly higher than in vivo, and this would have been the majority species present. When the minced heart tissue of an mdx mouse was flooded with excess NO prior to freezing, an analogous spectrum to that of Fig. 3C was obtained (not shown). Significantly, at least two other groups have detected the presence of nitrosomyoglobin in cardiac tissue. (19, 20)

View larger version (19K): [in this window] [in a new window]   Fig. 3.   X-band EPR spectra at 15 K of minced whole mouse heart. Left panel, g = 6 region showing signals arising from metmyoglobin. Right panel, g = 2 region showing signals arising from nitrosomyoglobin. A, as isolated (control), minced tissue dispersed in an equal volume of buffer prior to freezing in an EPR tube. The total myoglobin concentration was approximately the same as in Fig. 2B. B, as in panel A plus the addition of norepinephrine to 10 µM. C, as in panel A plus the addition of NO to a instantaneous concentration of around 20 µM.

To summarize, in the tissue at physiologically relevant concentrations, no evidence for significant reaction of NO with myoglobin species was found (Fig. 3B). When NO was added to (potentially overwhelming) excess, it appeared to preferentially react with deoxymyoglobin to form nitrosomyoglobin (Fig. 3C) and did not undergo the conventionally expected reaction with oxymyoglobin to form metmyoglobin and NO. This was observed to be the case irrespective of the particular cardiomyocyte cell line employed. Moreover, the near quantitative conversion of NO to NO (Fig. 1) rather than NO independently confirms the lack of reaction between NO and oxymyoglobin.

The Reaction of NO with Cytochrome c Oxidase-- During turnover, cytochrome c oxidase has been reported to bind NO with an affinity more than an order of magnitude larger than its affinity for oxygen (21). Therefore, even in the presence of excess oxygen, significant (but incomplete) inhibition of the oxidase by NO is expected. At physiological pH and temperature, we found that significant inhibition of the cytochrome c oxidase-catalyzed reduction of molecular oxygen by ferrocytochrome c was observed at oxygen/NO ratios of around 100 (Fig. 4). There was a very narrow range of conditions over which the system appeared to follow Michaelis-Menten kinetics, rendering extrapolation of the plots to find the intercept unreliable. Nevertheless, a result indicative of mixed, or non-competitive, inhibition of electron transfer from ferrocytochrome c to its oxidase was reproducibly obtained at relatively low NO levels. This suggests that binding of NO at the heme a₃-Cu_B pair of cytochrome c oxidase affects the rate of electron transfer through the complex. Knowing something of the affinity of NO toward cytochrome c oxidase naturally led us to hypothesize that this enzyme, rather than one or more myoglobin species, might be the primary target for NO in cardiomyocytes. Consequently, we investigated the competition between these two hemoproteins for NO under aerobic conditions at physiological pH and temperature, where the oxidase was turning over and all the myoglobin was initially present as oxymyoglobin (Fig. 5). Cytochrome c oxidase (variable amounts) and oxymyoglobin (20 µM) were preincubated aerobically (20 mM sodium phosphate buffer, 200 µM in oxygen at 37 °C) in a 1-ml reaction vessel. Enzyme turnover was initialized by addition of ferrocytochrome c (to 100 µM), and then the completely filled vessel was rapidly sealed with a septum through which gaseous NO was immediately injected (to 20 µM) using a gas-tight microsyringe. After 5 min of reaction time, the septum was removed, and the sample was frozen for subsequent NO and NO analyses as described under "Experimental Procedures." Clearly, when the oxidase was absent, added NO was free to react with oxymyoglobin and was almost quantitatively converted to NO. However, if the ratio of total oxidase:myoglobin was 0.5:1 or greater, essentially all added NO was converted to NO, demonstrating the marked preference of NO toward one or more cytochrome c oxidase turnover species as compared with oxymyoglobin. In vivo, there must be additional (presently unknown) factors that either increase the rate of reaction between NO and cytochrome c oxidase or decrease the rate of reaction between NO and oxymyoglobin as the oxidase:myoglobin ratio is certainly much less than 0.5:1.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Lineweaver-Burk plots showing inhibition of cytochrome c oxidase activity by NO at 37 °C, pH 7.4. Total cytochrome c oxidase and initial oxygen concentrations were 50 nM and around 200 µM respectively, for each individual assay. Reactions were monitored spectrophotometrically by following the disappearance of the ferrocytochrome c band at 550 nm. The non-linear data obtained at 5 µM NO are included to illustrate the very limited range over which the system appeared to follow Michaelis-Menten kinetics.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Competition between oxymyoglobin and cytochrome c oxidase during turnover for NO at 37 °C, pH 7.4. In all individual experiments, initial concentrations of oxymoglobin, NO, ferrocytochrome c, and oxygen were 20, 20, 100, and 200 µM, respectively. Turnover of cytochrome c oxidase was initiated by addition of ferrocytochrome c followed immediately by NO introduction to the reaction mixture. Nitrite and nitrate determinations were performed by standard procedures as described under "Experimental Procedures."

The Question of Peroxynitrite Reductase Activity-- We have previously shown that oxidation of the centers in cytochrome c oxidase by peroxynitrite (peroxynitrite reductase activity) is reasonably fast and leads to the generation of NO (22). If peroxynitrite forms in vivo, this may very well be in circumstances in which NO levels are elevated. Thus, the reaction of peroxynitrite with the NO-inhibited oxidase is of some interest. The electronic absorption spectrum of the reduced enzyme in the presence of a 2-fold excess of NO exhibited a pronounced shoulder at about 440 nm on the low energy side of a broad Soret band (Fig. 6, solid trace). This is indicative, as expected, of an incompletely formed NO adduct, a fraction of the substrate-binding sites (i.e. heme a₃) remaining uncomplexed. Anaerobic addition of a 3-fold excess of sodium peroxynitrite to the sample led to the appearance of an absorption spectrum, which, not unreasonably, was suggestive of a partially reduced, NO-bound derivative (broken trace). It was necessary to finally add a 12-fold excess of peroxynitrite over the enzyme before a 424-nm Soret maximum (dotted trace) demonstrated that the fully oxidized (pulsed) derivative had been formed. This result suggests that the rate of oxidation of NO-bound oxidase by peroxynitrite is slower than the isomerization of peroxynitrite to nitrate. These findings are in contrast to the behavior of the uninhibited cytochrome c oxidase, which only requires a 2-fold excess of peroxynitrite over the reduced enzyme for a quantitative reaction to be observed (22). So in vitro, whereas the NO-free enzyme is a competent peroxynitrite reductase, NO-inhibited cytochrome c oxidase is not.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Electronic absorption spectra showing the reaction of fully re- duced NO-inhibited cytochrome c oxidase (solid traces) with peroxynitrite, 3-fold excess (broken traces), and 12-fold excess (dotted traces) at 22 °C, pH 7.4. Spectra shown have 1.00-cm path lengths with ~7 µM enzyme concentration for each trace.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NO Catabolism under Normal Physiological Circumstances-- It has recently and quite reasonably been proposed that oxymyoglobin might constitute a scavenging system in red muscle, which prevents inhibition of the mitochondrial respiratory chain by NO (23). Unfortunately, the present data clearly argue against this suggestion being correct under normal physiological (and most pathological) circumstances. Monitoring the formation of metmyoglobin by ¹H NMR spectroscopy, it has been shown that the reaction of oxymyoglobin with NO is just measurable in Langendorff-perfused hearts from wild-type mice when 1-2 µM NO is delivered continuously for 15 min (5). Such NO levels are seemingly achievable by continuous perfusion with either authentic NO or bradykinin (modeling inflammation), but our own experience with a variety of cell lines (and where we do not mimic inflammatory conditions) is that endogenously generated NO reaches at most a few hundred nanomolar in concentration (14) (e.g. Fig. 1).

Failure to detect any metmyoglobin formation at submicromolar NO concentrations in the earlier work was ascribed to the cytochrome b₅-dependent reductase system rapidly reconverting the oxidized form to deoxymyoglobin (5). However, in our opinion, there are two major problems associated with this interpretation. First, the existing literature (24, 25) does not support the idea that the cytochrome b₅-dependent reduction of metmyoglobin to deoxymyoglobin is necessarily fast in vivo. In free solution, with the membrane-anchoring section of polypeptide removed, the second-order rate constant governing reduction of metmyoglobin by NADH/cytochrome b₅ is ~10³ M¹ s¹ (25). This rate constant is more than 4 orders of magnitude slower than the rate of reaction between oxymyoglobin and NO in vitro (1, 2). In other words, for the reductase reaction to reconvert metmyoglobin formed by reaction with NO to deoxymyoglobin within about 1 s, cytochrome b₅ would have to be present at >10⁴ times the NO concentration, i.e. >1 mM in the cell. Furthermore, in a cardiomyocyte, myoglobin is almost certainly not present as an unrestricted molecule in aqueous solution: the medium is more like a gel (26), and at ~0.2 mM concentration in red muscle (27), the protein is close to being saturated (i.e. in aqueous solution at pH 7.4). Consequently, it is difficult to envisage the cellular myoglobin being able to rapidly diffuse to the sarcoplasmic reticulum where cytochrome b₅ resides (28), and the effective electron transfer rate between these two molecules can be expected to be even slower than the in vitro rate constants might otherwise suggest. Second and most importantly, of course, the present data of Fig. 1A show essentially quantitative conversion of NO to nitrite, proving there was no significant reaction of NO with oxymyoglobin. In addition, the EPR samples of Fig. 3, in which only 8-12 s of reaction time were allowed prior to freezing, show no evidence for reaction of NO with oxymyoglobin to form metmyoglobin (and nitrate). Flogel et al. (5) report observing changes in their spectra following the cessation of NO delivery at >1-2 µM, which was described as rapid, but this was on a time scale of several minutes, and perhaps this (slow by our standards) recovery can indeed be ascribed to the cytochrome b₅-dependent reductase system. As lower levels of NO (i.e. submicromolar) were used in the present experiments and the time scales were much shorter (i.e. 1-10 s), there is absolutely no conflict between the two data sets.

The concentration of myoglobin in red muscle is typically ~0.2 mM (27), much higher than in other cell types. So if at normal physiological NO concentrations the reaction between NO and oxymyoglobin does not occur to any great extent in cardiomyocytes (Figs. 1 and 3), it almost certainly is of very little consequence in other cell lines. Therefore, the present findings lead us to suspect that, in addition to straightforward loss by diffusion, the reaction with cytochrome c oxidase may well turn out to represent the major biochemical pathway for NO removal from all eukaryotic cells (except erythrocytes, perhaps) under normal physiological (and non-inflammatory, pathological) circumstances. It follows that the low mitochondrial content of NO-producing endothelial cells (29) may be necessary to ensure that most of the NO generated diffuses into the vasculature and is not consumed before it has a chance to activate guanylate cyclase or interact with other targets. It should be noted that the data of Fig. 1B in comparison with the data of Fig. 1A support this suggestion.

Modulation of Cellular Respiration by NO-- The ability of NO to inhibit cytochrome c oxidase was known long before the extensive biological activity of NO emerged. Consequently, the notion that NO might modulate mitochondrial activity can hardly be considered a revolutionary idea. On the contrary, given the extraordinarily high affinity of the oxidase for NO (21) and the low NO/O₂ ratio at which inhibition is effective (Fig. 4), arguing against a respiratory control function for NO appears to be a much less tenable position. Certainly, the uptake of O₂ by cardiac mitochondria has been observed to be affected by NO (30, 31), cardiac function is measurably inhibited at submicromolar NO levels (5), and NO/O₂ ratios have been implicated in regulating a diverse array of bioenergetic processes, such as O₂ consumption by endothelial cells (32), apoptosis in human T cells (33), and firefly flashing (34).

The question of whether NO is a significant modulator of cytochrome c oxidase activity in vivo and the mechanism(s) by which this might be achieved are clearly interrelated issues. If in vitro experiments can identify the intermediates involved and establish their rates of interconversion, then some reasonable assessment of the possibility that such processes can occur in vivo can be made. The finding that cytochrome c oxidase catalyzes the conversion of NO to NO is not controversial (35, 36). However, there is some disagreement regarding the likely mechanism(s) of the reaction(s) involved when NO is consumed while the enzyme is turning over in the presence of added reductant and oxygen. While acknowledging that much work remains to be undertaken before the mechanism(s) in question may be satisfactorily delineated, Giuffrè et al. (36) have postulated a complicated reaction scheme involving the reaction of NO with one or more oxyferryl intermediates in the normal reaction cycle. We presently have no data that contradict this suggestion but would like to point out that there is another plausible mechanism that might be operative. This scheme is presented in Fig. 7 and is based in part on the recent observation that the peroxynitrite reductase activity of cytochrome c oxidase leads to the quantitative production of NO and sustains electron transport from ferrocytochrome c (22). Also, as the reactions of oxyhemoglobin and oxymyoglobin with NO seemingly involve peroxynitrite intermediates (37), the proposed scheme is broadly related to other known hemoprotein chemistry. Initially, NO may bind to one of the metal ions at the active site (i.e. the binuclear pair). Next, O₂ could enter the active site, which is known to be able to accommodate at least two exogenous diatomic ligands (38), where it reacts with the other metal ion. Following reduction of O₂ to superoxide ion (O), NO can then combine with O to form ONO in a reaction that has been shown to be very rapid (39). The subsequent conversion of ONO to NO in a two-electron reduction at the active site is also a known facile reaction (22). Following the transfer of electrons from the other components of the electron transport chain, the cycle of reactions may then be repeated. If the proposed NO-driven process (a net three-electron reduction) is slower than the familiar four-electron reduction of O₂ to H₂O, then it follows that the reaction of NO at the enzyme active site is inhibitory, which is the case. Note that the "NO oxidase" scheme proposed in Fig. 7 represents transient inhibition of the enzyme; it is not reversible in the strict sense.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Plausible scheme (working hypothesis) for the reaction of NO-inhibited cytochrome c oxidase with oxygen (NO oxidase activity). Fig. 7 shows the proposed two reaction cycle, normal (fast) and inhibitory (slow). The precise nature of the intermediates, number of discrete steps, and possible importance of alternative competing mechanisms are currently under investigation.

Suppression of Peroxynitrite Formation in Mitochondria-- The rapid non-enzymatic reaction of NO with O and the cytotoxicity of the ONO produced have been implicated in numerous pathological situations (18, 40). It is clearly of importance as to whether mitochondria are a major source of ONO under any of these circumstances. Here, we have shown that cytochrome c oxidase has good NO oxidase activity, and previously, we have demonstrated the same enzyme to have reasonable peroxynitrite reductase activity (22). Consequently, the actively respiring mitochondrial electron transport chain appears to function (in part) to suppress ONO formation but also appears to have some capacity to detoxify any that might be formed in small amounts. Respiring mitochondria are, therefore, only likely to produce ONO in miniscule quantities. The same is clearly not correct if the respiratory chain ceases to maintain electron transport to cytochrome c oxidase. Non-functional mitochondria, where the terminal oxidase is no longer turning over, generate large fluxes of O (41). Moreover, additional substrate (O₂) will be available to NO synthases (42, 43), resulting in elevated NO concentrations. A sustained production of ONO is, therefore, probably unavoidable once the mitochondrion has been compromised and may explain much of the evidence for ONO formation in dead tissues (18, 40, 44). From this point of view, it follows that mitochondrially generated ONO could either simply represent collateral damage or be an amplification step in an etiological chain of events signaling other organelles and/or neighboring cells.

	FOOTNOTES

^* This work was supported by Research Grants HL61411 (to J. P. and B. R. P.) and HL57985 (to A. J. K.) from the NHLBI National Institutes of Health and by Grant 9950029N from the American Heart Association (to A. J. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Mellon Institute, 4400 Fifth Ave., Pittsburgh, PA 15213. Tel.: 412-268-5670; E-mail: lip10@pitt.edu or jamesp{at}cmu.edu.

Published, JBC Papers in Press, February 1, 2002, DOI 10.1074/jbc.M109838200

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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