Interaction of Peroxynitrite with Mitochondrial Cytochrome Oxidase
1998; Elsevier BV; Volume: 273; Issue: 47 Linguagem: Inglês
10.1074/jbc.273.47.30961
ISSN1083-351X
AutoresMartyn A. Sharpe, Chris E. Cooper,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoPurified mitochondrial cytochrome coxidase catalyzes the conversion of peroxynitrite to nitric oxide (NO). This reaction is cyanide-sensitive, indicating that the binuclear hemea 3/CuB center is the catalytic site. NO production causes a reversible inhibition of turnover, characterized by formation of the cytochrome a 3nitrosyl complex. In addition, peroxynitrite causes irreversible inhibition of cytochrome oxidase, characterized by a decreasedV max and a raised K m for oxygen. Under these conditions, the redox state of cytochromea is elevated, indicating inhibition of electron transfer and/or oxygen reduction reactions subsequent to this center. The lipid bilayer is no barrier to these peroxynitrite effects, as NO production and irreversible enzyme inhibition were also observed in cytochrome oxidase proteoliposomes. Addition of 50 μm peroxynitrite to 10 μm fully oxidized enzyme induced spectral changes characteristic of the formation of ferryl cytochromea 3, partial reduction of cytochromea, and irreversible damage to the CuA site. Higher concentrations of peroxynitrite (250 μm) cause heme degradation. In the fully reduced enzyme, peroxynitrite causes a red shift in the optical spectrum of both cytochromes a and a 3, resulting in a symmetrical peak in the visible region. Therefore, peroxynitrite can both modify and degrade the metal centers of cytochrome oxidase. Purified mitochondrial cytochrome coxidase catalyzes the conversion of peroxynitrite to nitric oxide (NO). This reaction is cyanide-sensitive, indicating that the binuclear hemea 3/CuB center is the catalytic site. NO production causes a reversible inhibition of turnover, characterized by formation of the cytochrome a 3nitrosyl complex. In addition, peroxynitrite causes irreversible inhibition of cytochrome oxidase, characterized by a decreasedV max and a raised K m for oxygen. Under these conditions, the redox state of cytochromea is elevated, indicating inhibition of electron transfer and/or oxygen reduction reactions subsequent to this center. The lipid bilayer is no barrier to these peroxynitrite effects, as NO production and irreversible enzyme inhibition were also observed in cytochrome oxidase proteoliposomes. Addition of 50 μm peroxynitrite to 10 μm fully oxidized enzyme induced spectral changes characteristic of the formation of ferryl cytochromea 3, partial reduction of cytochromea, and irreversible damage to the CuA site. Higher concentrations of peroxynitrite (250 μm) cause heme degradation. In the fully reduced enzyme, peroxynitrite causes a red shift in the optical spectrum of both cytochromes a and a 3, resulting in a symmetrical peak in the visible region. Therefore, peroxynitrite can both modify and degrade the metal centers of cytochrome oxidase. peroxynitrite diethylenetriaminepentaacetic acid N,N,N′,N′-tetremethyl-p-phenylenediamine hydrochloride mitochondrial permeability transition cytochrome c oxidase vesicle. The inhibition of cytochrome c oxidase by nitric oxide (NO) may play a normal role in controlling mitochondrial O2consumption (1Brown G.C. FEBS Lett. 1995; 369: 136-139Crossref PubMed Scopus (502) Google Scholar). Inhibition of cytochrome oxidase by NO could, for example, explain the observation that the apparentK m for O2 of whole cells is greater than that of isolated mitochondria (2Brown G.C. Cooper C.E. FEBS Lett. 1994; 356: 295-298Crossref PubMed Scopus (935) Google Scholar). The inhibition of cytochrome oxidase by elevated levels of NO, although readily reversible, can have profound consequence for the cell in pathophysiological disease states (3Cleeter M.W.J. Cooper J.M. Darley-Usmar V.M. Moncada S. Schapira A.H.V. FEBS Lett. 1994; 345: 50-54Crossref PubMed Scopus (1145) Google Scholar). Inhibition of the mitochondrial transport chain at the cytochrome oxidase level can produce superoxide (O2−) from O2. Peroxynitrite (ONOO−)1 can be formed by the reaction of NO and O2−. Thus, inhibition of mitochondrial cytochrome oxidase by NO can lead to the formation of both NO and O2− and thereby lead to the formation of ONOO− (4Packer M.A. Porteous C.M. Murphy M.P. Biochem. Mol. Biol. Int. 1996; 40: 527-534PubMed Google Scholar). In addition to this mechanism, ONOO− can also arise via the reaction of nitroxyl anion (NO−) and O2 (5Hogg N. Singh R.J. Kalyanaraman B. FEBS Lett. 1996; 382: 223-228Crossref PubMed Scopus (246) Google Scholar, 6Murphy M.E. Sies H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10860-10864Crossref PubMed Scopus (288) Google Scholar). We have recently demonstrated that both NO− and ONOO−are produced during the aerobic incubation of mitochondrial ferrocytochrome c and NO (7Sharpe M. Cooper C. Biochem. J. 1998; 332: 9-19Crossref PubMed Scopus (188) Google Scholar).In contrast to NO, ONOO− can irreversibly damage many of the mitochondrial enzymes and complexes of oxidative phosphorylation, including aconitase, NADH/co-Q reductase, quinol/cytochromec reductase, succinate dehydrogenase, and the ATP synthetase (8Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar, 9Radi R. Rodriguez M. Castro L. Telleri R. Arch. Biochem. Biophys. 1994; 308: 89-95Crossref PubMed Scopus (657) Google Scholar). The consequent collapse of the mitochondrial membrane potential (ΔΨ) can trigger the mitochondrial permeability transition (MPT). This causes the release of cytochrome c from the intra-mitochondrial space into the cytoplasm (10Liu X.S. Kim C.N. Yang J. Jemmerson R. Wang X.D. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4435) Google Scholar, 11Yang J. Liu X.S. Bhalla K. Kim C.N. Ibrado A.M. Cai J.Y. Peng T.I. Jones D.P. Wang X.D. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4384) Google Scholar, 12Kluck R.M. BossyWetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4256) Google Scholar). The movement of cytochrome c into the cytoplasm consequently triggers apoptotic cell death (13Kroemer G. Zanzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar). As ONOO− has been shown to induce the MPT (14Packer M.A. Murphy M.P. FEBS Lett. 1994; 345: 237-240Crossref PubMed Scopus (144) Google Scholar, 15Schweizer M. Richter C. Biochemistry. 1996; 35: 4524-4528Crossref PubMed Scopus (68) Google Scholar), the formation of ONOO− from NO may thus be responsible for a pro-apoptotic role of NO.Reversible inhibition of cytochrome oxidase by NO may therefore initiate a cascade of events that results in cell death. However, there is conflicting evidence as to whether ONOO− directly inhibits cytochrome oxidase function in cells and mitochondria, with some papers suggesting that there is little or no damage (9Radi R. Rodriguez M. Castro L. Telleri R. Arch. Biochem. Biophys. 1994; 308: 89-95Crossref PubMed Scopus (657) Google Scholar, 16Cassina A. Radi R. Arch. Biochem. Biophys. 1996; 328: 309-316Crossref PubMed Scopus (584) Google Scholar) and others disagreeing (17Bolanos J.P. Heales S.J.R. Land J.M. Clark J.B. J. Neurochem. 1995; 64: 1965-1972Crossref PubMed Scopus (472) Google Scholar). Here we resolve this controversy by clearly demonstrating that purified cytochrome oxidase when solubilized or in proteoliposomal form is irreversibly damaged by ONOO−. Interestingly, we also observe that cytochrome oxidase catalyzes NO production from ONOO−; this novel chemistry, converting an irreversible inhibitor into a reversible one, may play a role in ONOO− detoxification by the mitochondria.EXPERIMENTAL PROCEDURESBovine heart cytochrome c oxidase was purified according to the method of Kuboyama et al. (18Kuboyama M. Yong F.C. King T.E. J. Biol. Chem. 1972; 247: 6375-6383Abstract Full Text PDF PubMed Google Scholar), substituting Tween 80 for Emasol in the latter stages of the purification. Peroxynitrite (ONOO−) was prepared from amyl nitrite and hydrogen peroxide using the method of Uppu and Pryor (19Uppu R.M. Pryor W.A. Methods Enzymol. 1996; 269: 322-329Crossref PubMed Google Scholar); 26 ml of amyl nitrite (washed three times with water) was added to 100 ml of 2 m H2O2, 2 mNaOH, and 2 mm diethylenetriaminepentaacetic acid (DTPA), 4 °C. This solution was stirred vigorously for 3 h at 4 °C. The lower aqueous layer was removed from the organic phase using a separating funnel and washed three times with an equal volume of ice-cold hexane. To remove residual H2O2, 10 g of manganese dioxide was gradually added to the ONOO− solution while it was being stirred, on ice, for approximately 1 h. The ONOO− solution was filtered and then stored at 77 K. After thawing, the ONOO−solutions were passed down a 6 × 1-cm MnO2 column equilibrated with 0.1 m NaOH to remove any traces of H2O2. The ONOO− was then diluted with 0.1 m NaOH to approximately 30 mmimmediately before use. The concentration of ONOO− was determined spectrophotometrically using the extinction coefficient of 1670 m−1 cm−1 at 302 nm. Nitrite contamination of the ONOO− solution was determined to be approximately one third of the ONOO− concentration, as in (19Uppu R.M. Pryor W.A. Methods Enzymol. 1996; 269: 322-329Crossref PubMed Google Scholar). The addition of catalase to decomposed peroxynitrite at pH 7 resulted in no oxygen evolution, consistent with an absence of any H2O2 contamination in the solution.Crude NO was prepared by the addition of 2 mH2SO4 to solid NaNO2 in a Kipps apparatus. The NO gas was purified by passage through four NaOH (20%) traps and then through a dry ice trap, to remove NO2. NO solutions were prepared by the addition of purified NO gas into buffer solutions that had undergone four vacuum/N2 deoxygenation cycles. The concentration of the NO solution varied between 1.2 and 2 mm. The nitrite concentration in the NO solution was determined to be between 300 and 500 μm.The concentrations of O2 and NO were determined polarographically. A Perspex, water-jacketed O2 electrode (Rank Brothers, Bottisham, Cambridge, U.K.) was modified to allow the insertion of a WPI ISO-200 NO electrode (World Precision Instruments, Sarasota, FL). The NO electrode was connected to an ISO-NO II NO meter. Data from the O2 and the NO electrodes were collected using MacLab data collection and analysis system (ADI Instruments, Castle Hill, New South Wales, Australia). Nitrite concentrations were determined by the addition of aliquots of the solution to 100 mm KI, 100 mm H2SO4followed by measurement of the NO produced using the electrode (this procedure yields a 1:1 nitrite:NO stoichiometry).Cytochrome c oxidase turnover was assayed by adding 5 nm cytochrome oxidase to 20 mm sodium ascorbate, 300 μm N,N,N′,N′-tetremethyl-p-phenylenediamine hydrochloride (TMPD), 60 μm cytochrome c(horse heart, Sigma type C-7752) in 5 ml of 100 mmK+-Hepes or 100 mm K+-phosphate, 20 μm DTPA, 0.015% lauryl maltoside, pH 7.0, 30 °C.In the derived plots (Fig. 4), the non-enzymatic autoxidation rate was subtracted from the overall O2 consumption rate to give the enzymatic rate. We found that the autoxidation rate of ascorbate was directly proportional to the O2 concentration. We calculated the autoxidation rate for ascorbate at all O2concentrations and subtracted this from the O2 consumption rate.Figure 4Comparison of peroxynitrite and NO on the turnover of cytochrome oxidase. The effect of O2concentration on the turnover of cytochrome oxidase is shown in A. From this, it can be seen that the K mfor O2 is below 2 μm. In B, NO was added to a final concentration of approximately 750 nm at an O2 concentration of 180 μm. Following the disappearance of the NO the cytochrome turnover almost returns to the level before the addition of NO. The K m for O2 is again <2 μm. C shows the addition of 100 μm ONOO− to cytochrome oxidase. This releases 750 nm NO. Following the decay of NO, cytochrome oxidase turnover drops by approximately 50% and theK m for O2 is increased to approximately 20 μm. Conditions were as in Fig. 1. D shows the irreversible effect of ONOO− concentration on theK m for O2, measured using a Oroboros® Oxygraph high resolution respirometer. The line is the best fit linear regression forced to cross thevertical axis at 0.9 μmO2 (the K m for O2 in the absence of ONOO−). Conditions are as described for Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Precise measurement of the oxidase K m for O2 of cytochrome oxidase require high resolution respirometry (20Gnaiger E. Steinlechner R. Mendez G. Eberl Y. Margeiter R. J. Bioenerg. Biomembr. 1995; 27: 583-596Crossref PubMed Scopus (247) Google Scholar). This was done using an Oroboros®Oxygraph (Oroboros®, Innsbruck, Austria.) The experimental conditions were as in Fig. 1, but with a smaller chamber volume, 3.4 ml.Figure 1Effect of 100 μmONOO− on the oxygen consumption of cytochrome oxidase and the NO concentration. A, effect of 100 μm ONOO− on the O2 consumption of cytochrome c oxidase and on NO concentration detected simultaneously by O2 and NO electrodes. Following the addition of ONOO−, approximately 750 nm NO was generated (bottom trace), causing inhibition of the oxidase. B, generation of NO from ONOO− by cytochrome c oxidase under anaerobic conditions. ONOO− was added to an anaerobic solution of cytochromec. Conditions: 5 ml, 100 mmK+-Hepes, 0.015% lauryl maltoside, 20 μmDTPA, pH 7.0, 30 °C, 300 μm TMPD, 60 μmcytochrome c, 20 mm ascorbate, and 5 nm oxidase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cytochrome c oxidase vesicles (COV) were prepared as described previously (21Sharpe M. Perin I. Wrigglesworth J. Nicholls P. Biochem. J. 1996; 320: 557-561Crossref PubMed Scopus (8) Google Scholar). 0.25 g of soybean phospholipid (Sigma type II-S "phosphatidyl choline") and 0.1 g of sodium cholate were dispersed in 5 ml of 100 mm K+-Hepes, pH 7.0, by vortex mixer. The suspension was then sonicated on ice under a N2 stream for 8 min using a 30% duty cycle. Cytochrome oxidase was added to a final concentration of 6 μm and then sonicated for 30 s to disperse the oxidase. The mixture was centrifuged for 10 min at 20,000 × g and 4 °C to remove undispersed lipid and titanium particles. The solution was then twice dialyzed against 100 volumes of buffer and once against 200 volumes of buffer, over 3 days at 4 °C. The COV were passed down a Sephadex G-25 column to remove lipid aggregates. The sidedness was determined by the percentage ascorbate-reducibility in the presence of externally added cytochrome c and cyanide (22Wrigglesworth J. Wooster M.S. Elsden J. Danneel H. Biochem. J. 1987; 246: 737-744Crossref PubMed Scopus (34) Google Scholar) and was found to be approximately 80% externally facing cytochrome oxidase.Cytochrome c oxidase spectra were monitored in 100 mm K+-Hepes, 20 μm DTPA, 0.1% lauryl maltoside, pH 7.0. The change in the broad near-infrared band due to oxidized CuA was monitored at 830 nm, using the 722 and 900 nm wavepair as references, i.e. ΔCuA = ΔA 830 nm − (ΔA 722 nm + ΔA 900 nm)/2. Cytochrome a was monitored using the 605 minus 630 nm wavepair and an extinction coefficient of 23.5 mm−1 cm−1. The concentration of compound F was calculated using an extinction coefficient of 5.3 mm−1cm−1 for the 580 minus 630 nm wavepair; the contribution of cytochrome a reduction at this wavepair was corrected by using the relationship for a 2+ of (ΔA 605–630 nm)/(ΔA 580–630 nm) = 4.4.Steady state spectra (Fig. 8) were obtained as follows; 20 mm ascorbate was added to 10 μm cytochrome oxidase and 30 μm cytochrome c in K+-Hepes buffer. Following anaerobiosis, 10 nmcatalase and then 2 mm H2O2 was added to reoxygenate the solution. When steady state turnover was observed, an aliquot of ONOO− was added. The solution was allowed to become anaerobic again, and a few grains of dithionite were added to the solution to fully reduce the cytochrome oxidase. Enzyme inhibition following the addition of ONOO− was calculated from the change in the cytochrome c redox state. This is proportional to enzyme turnover under these conditions (23Nicholls P. van Buuren K.J.H. van Gelder B.F. Biochim. Biophys. Acta. 1972; 275: 279-287Crossref PubMed Scopus (69) Google Scholar).Figure 8Effect of peroxynitrite on cytochrome coxidase in the steady state. 10 μm cytochromec oxidase was incubated in 100 mmK+-phosphate, 20 μm DTPA, 0.015% lauryl maltoside, 30 μm cytochrome c, 30 °C. 300 μm TMPD and 20 mm ascorbate were than added, and the solution was allowed to become anaerobic. After ≈5 min of anaerobiosis, 1 nm catalase and 5 mmH2O2 were added. Once the solution came to steady state, aliquots of ONOO− were added. The solution was again allowed to become anaerobic, and after a few minutes a few grains of dithionite were added to the cuvette. A, changes in the cytochrome c (550 minus 540), cytochromesa and a 3 (605 minus 630 nm) and CuA redox states (see "Experimental Procedures"). Ascorbate/TMPD caused an increase in the steady state redox levels of cytochromes a and c, and in CuA. Anaerobiosis resulted in all of these respiratory components becoming reduced. Addition of 1 nm catalase and 5 mmH2O2 reoxygenated the sample. When the sample came to steady state, 250 μm ONOO− was added. After approximately 2 min, the sample again became anaerobic. Dithionite was then added. B, effect of ONOO−or NO on the steady state spectra of cytochrome oxidase with respect to the fully oxidized enzyme. Spectra were collected 5 s after the addition of between 100 μm and 1 mmONOO− or in the presence of 10 μm NO. The contribution due to the presence of cytochrome c was subtracted (see "Experimental Procedures"). C, the same spectra of oxidase plus NO (a) and oxidase plus 500 μm ONOO− (b) after the contribution of cytochrome a has been subtracted. Here it can be seen that there is an increase in the cytochrome a32+ NO, which increases with ONOO− up to 1 mm. The lower points (c) show the subtraction of spectrum a(multiplied by 0.55) minus spectrum b. D, effect of ONOO− on the steady state turnover and the maximum change in the 605–630 nm wavepair following the addition of dithionite (see "Experimental Procedures").View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Effect of peroxynitrite on cytochrome coxidase in the steady state. 10 μm cytochromec oxidase was incubated in 100 mmK+-phosphate, 20 μm DTPA, 0.015% lauryl maltoside, 30 μm cytochrome c, 30 °C. 300 μm TMPD and 20 mm ascorbate were than added, and the solution was allowed to become anaerobic. After ≈5 min of anaerobiosis, 1 nm catalase and 5 mmH2O2 were added. Once the solution came to steady state, aliquots of ONOO− were added. The solution was again allowed to become anaerobic, and after a few minutes a few grains of dithionite were added to the cuvette. A, changes in the cytochrome c (550 minus 540), cytochromesa and a 3 (605 minus 630 nm) and CuA redox states (see "Experimental Procedures"). Ascorbate/TMPD caused an increase in the steady state redox levels of cytochromes a and c, and in CuA. Anaerobiosis resulted in all of these respiratory components becoming reduced. Addition of 1 nm catalase and 5 mmH2O2 reoxygenated the sample. When the sample came to steady state, 250 μm ONOO− was added. After approximately 2 min, the sample again became anaerobic. Dithionite was then added. B, effect of ONOO−or NO on the steady state spectra of cytochrome oxidase with respect to the fully oxidized enzyme. Spectra were collected 5 s after the addition of between 100 μm and 1 mmONOO− or in the presence of 10 μm NO. The contribution due to the presence of cytochrome c was subtracted (see "Experimental Procedures"). C, the same spectra of oxidase plus NO (a) and oxidase plus 500 μm ONOO− (b) after the contribution of cytochrome a has been subtracted. Here it can be seen that there is an increase in the cytochrome a32+ NO, which increases with ONOO− up to 1 mm. The lower points (c) show the subtraction of spectrum a(multiplied by 0.55) minus spectrum b. D, effect of ONOO− on the steady state turnover and the maximum change in the 605–630 nm wavepair following the addition of dithionite (see "Experimental Procedures").View Large Image Figure ViewerDownload Hi-res image Download (PPT)All optical spectra were collected using a Hewlett Packard HP 8453 diode array spectrophotometer.RESULTSIt has been suggested that ONOO− does not inhibit cytochrome oxidase activity (9Radi R. Rodriguez M. Castro L. Telleri R. Arch. Biochem. Biophys. 1994; 308: 89-95Crossref PubMed Scopus (657) Google Scholar, 16Cassina A. Radi R. Arch. Biochem. Biophys. 1996; 328: 309-316Crossref PubMed Scopus (584) Google Scholar). Fig. 1 A shows that this is not the case. Following the addition of 100 μm ONOO−to actively respiring cytochrome oxidase, a transient but complete inhibition of enzyme activity was observed. The onset of enzyme inhibition occurred simultaneously with the generation of NO. The addition of 100 μm ONOO− induced the generation of approximately 750 nm NO, a value far above the apparent K i for NO inhibition of cytochrome oxidase turnover at the O2 tension used. NO is metabolized by cytochrome oxidase (24Zhao X. Samphath V. Caughey W.S. Biochem. Biophys. Res. Commun. 1995; 212: 1054-1060Crossref PubMed Scopus (72) Google Scholar) and its substrates (7Sharpe M. Cooper C. Biochem. J. 1998; 332: 9-19Crossref PubMed Scopus (188) Google Scholar, 25Kelm M. Yoshida K Feelisch M. Stamler J.S. Methods in Nitric Oxide Research. John Wiley & Sons, New York1996: 557-561Google Scholar, 26Liao G.-L. Palmer G. Biochim. Biophys. Acta. 1996; 1274: 109-111Crossref PubMed Scopus (75) Google Scholar, 27Squadrito G.L. Jin X. Pryor W.A. Arch. Biochem. Biophys. 1995; 322: 53-59Crossref PubMed Scopus (121) Google Scholar), and as the NO was consumed the oxidase began to turn over again.However, unlike the inhibition of cytochrome oxidase by the addition of authentic NO (where enzyme turnover is fully restored following the decay of NO; Ref. 2Brown G.C. Cooper C.E. FEBS Lett. 1994; 356: 295-298Crossref PubMed Scopus (935) Google Scholar), cytochrome oxidase turnover following the addition of ONOO− was always less than the rate prior to its addition. This shows that, unlike the reversible inhibition of cytochrome oxidase by NO, ONOO− is an irreversible inhibitor (2Brown G.C. Cooper C.E. FEBS Lett. 1994; 356: 295-298Crossref PubMed Scopus (935) Google Scholar, 28Koivisto A. Matthias A. Bronnikov G. Nedergaard J. FEBS Lett. 1997; 417: 75-80Crossref PubMed Scopus (117) Google Scholar). The production of NO from ONOO− by the enzyme was only marginally O2-sensitive. 100 μm ONOO− added to anaerobic 5 nmcytochrome c oxidase, in the presence of reductant, still induced the formation of approximately 500 nm NO (Fig. 1 B).Fig. 2 shows the effect of ONOO− concentration on the final cytochrome oxidase turnover (when all the NO generated has decayed away). It can be seen that 100 μm ONOO− caused a 50% drop in the turnover of the purified enzyme. This was a much greater drop in cytochrome oxidase activity than previously observed in both intact or sonicated mitochondria, even though in the experiments with mitochondria ONOO− concentrations as high as 2 mm were used (9Radi R. Rodriguez M. Castro L. Telleri R. Arch. Biochem. Biophys. 1994; 308: 89-95Crossref PubMed Scopus (657) Google Scholar).Figure 2Cytochrome oxidase turnover as a function of peroxynitrite concentration. Maximum rates after the disappearance of NO are shown in experiments under the same conditions as in Fig. 1 A. Each point is the average of three experiments. The error bars represent ±1 standard deviation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3 quantifies the amount of NO production as a function of the added ONOO− concentration. Both in the presence and absence of O2, NO production was proportional to the added ONOO− concentration. NO was not produced from ONOO− that had been allowed to decompose at neutral pH prior to addition. The formation of NO from peroxynitrite was inhibited by cyanide (20 mm). Addition of azide (10 mm) gave very similar results to those observed with cyanide. This suggests that the binuclear hemea 3/CuB center is the site of NO formation in cytochrome oxidase.Figure 3Nitric oxide production by cytochrome oxidase as a function of peroxynitrite concentration. The effect of ONOO− concentration on NO produced: aerobically in the presence of 5 nm oxidase (♦), anaerobically in the presence of 5 nm oxidase (▪), aerobically in the absence of oxidase (■), and aerobically in the presence of oxidase and 20 mm NaCN (▵). Each point is the average of three experiments. The error bars represent ±1 standard deviation. Other conditions are as described for Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Under these conditions, a small rate of NO production was seen even in the absence of the enzyme; this appears to be a reaction between peroxynitrite and ascorbate, as it did not occur when ascorbate was left out of the solution, nor did it occur if nitrite were added instead of peroxynitrite. Under certain conditions, NO donors can be formed from the addition of ONOO− to primary alcohols (29Moro M.A. Darley-Usmar V.M. Lizasoain I. Su Y.L. Knowles R.G. Radomski M.W. Moncada S. Br. J. Pharmacol. 1995; 116: 1999-2004Crossref PubMed Scopus (183) Google Scholar,30Zhu L. Gunn C. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 452-457Crossref PubMed Scopus (414) Google Scholar) such as Hepes. However, we found that there was no difference in the amount of NO produced following the addition of ONOO−to cytochrome oxidase in phosphate buffer or in Hepes buffer, ruling this out as a significant mechanism of NO production in this system.NO is a reversible inhibitor of cytochrome oxidase (2Brown G.C. Cooper C.E. FEBS Lett. 1994; 356: 295-298Crossref PubMed Scopus (935) Google Scholar, 28Koivisto A. Matthias A. Bronnikov G. Nedergaard J. FEBS Lett. 1997; 417: 75-80Crossref PubMed Scopus (117) Google Scholar). However, following ONOO− treatment, we observed an irreversible inhibition of enzyme turnover by as much as 50% (Figs. 1 Aand 2). The difference between the long term effects of NO and ONOO− on cytochrome oxidase activity can be clearly seen in Fig. 4, where we compared treatment with the same concentration of "authentic NO" (750 nm) as that formed following the addition of 100 μmONOO− to the enzyme. In a control trace, in the absence of NO or ONOO−, we see that oxidase turnover is insensitive to O2 concentration until this drops below 10 μm (Fig. 4 A). Following the addition of 750 nm NO, added at 180 μm O2 (Fig. 4 B), the enzyme was strongly inhibited until the NO concentration dropped below 300 nm. Following the complete removal of NO from the solution, the enzyme returned to its pre-inhibited rate. Addition of 100 μm ONOO−to cytochrome oxidase under steady state turnover conditions caused the release of approximately 750 nm NO. The oxidase was inhibited until almost all the NO had left the solution (Fig. 4 C). However, after all the NO had gone, the final rate was still approximately half the initial rate and the enzymes sensitivity to O2 concentration was increased. ONOO−treatment decreased the V max and significantly raised the K m for O2. Independent measurements in a dedicated high resolution respirometer demonstrate that under these conditions the K m for O2 in the control was 0.9 μm. After the addition of 100 μm ONOO−, theK m had risen to 14 μm (Fig. 4 D).One possible explanation for the lowering of theV max and the raising of theK m for O2 was that we were observing an artifact due to a slow degradation of the cytochrome oxidase throughout the time course of the experiment. We tested this hypothesis by allowing an oxidase solution that had been treated with ONOO− to become anaerobic. Following re-aeration 10 min later, the cytochrome oxidase turnover was found to have the same (inhibited) V max and K m for O2 as before. This indicates that there is no continuous degradation of cytochrome oxidase following the addition of ONOO− during the time course of the experiment,i.e. the damage occurred immediately following ONOO− addition.The ability of ONOO− to decrease theV max of purified cytochrome oxidase is consistent with reports that ONOO− inhibits cytochrome oxidase activity in astrocyte cultures, macrophages (31Szabo C. Day B.J. Salzman A.L. FEBS Lett. 1996; 381: 82-86Crossref PubMed Scopus (213) Google Scholar) and in rat brain mitochondria (32Lizasoain I. Moro M.A. Knowles R.G. Darley-Usmar V.M. Moncada S. Biochem. J. 1996; 246: 737-744Google Scholar). However, Radi and co-workers (9Radi R. Rodriguez M. Castro L. Telleri R. Arch. Biochem. Biophys. 1994; 308: 89-95Crossref PubMed Scopus (657) Google Scholar, 33Cassina A. Radi R. Arch. Biochem. Biophys. 1996; 328: 309-316Crossref PubMed Scopus (606) Google Scholar) reported that ONOO− caused no inhibition of cytochrome oxidase in intac
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