Indirect inhibition of mitochondrial dihydroorotate dehydrogenase activity by nitric oxide

Free Radical Biology & Medicine, Vol. 28, No. 8, pp. 1206 –1213, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(00)00239-2

Original Contribution INDIRECT INHIBITION OF MITOCHONDRIAL DIHYDROOROTATE DEHYDROGENASE ACTIVITY BY NITRIC OXIDE CLAIRE BEUNEU,* RODOLPHE AUGER,* MONIKA L¨OFFLER,† ANNIE GUISSANI,‡ GENEVIE` VE LEMAIRE,* MICHEL LEPOIVRE*

and

*Unite´ 8619, Centre National de la Recherche Scientifique, Universite´ Paris-Sud, Orsay, France; †Institute for Physiological Chemistry, School of Medicine, Philips University, Marburg, Germany; and ‡Unite´ 350, Institut National de la Sante´ et de la Recherche Me´dicale, Institut Curie, Orsay, France (Received 16 December 1999; Revised 29 February 2000; Accepted 29 February 2000)

Abstract—Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate in the pyrimidine biosynthesis pathway. It is functionally connected to the respiratory chain, delivering electrons to ubiquinone. We report here that inhibition of cytochrome c oxidase by nitric oxide (NO) indirectly inhibits DHODH activity. In digitonin-permeabilized cells, DEA/NO, a chemical NO donor, induced a dramatic decrease in DHO-dependent O2 consumption. The inhibition was reversible and more pronounced at low O2 concentration; it was correlated with a decrease in orotate synthesis. Since orotate is the precursor of all pyrimidine nucleotides, indirect inhibition of DHODH by NO may significantly contribute to NO-dependent cytotoxicity. © 2000 Elsevier Science Inc. Keywords—Dihydroorotate dehydrogenase, Nitric oxide, Mitochondria, Cytochrome c oxidase, Orotate, Respiration, Free radicals

INTRODUCTION

plexes I, II, IV, and ATP-synthase (for review see Brown [7]). Irreversible inhibition of complexes I and II has been proposed to be mediated by peroxynitrite, a product of the reaction of NO with O2•⫺ [8,9]. Cytochrome c oxidase (complex IV) is strongly inhibited by NO, but in a reversible manner [9 –13]. This inhibition was shown to be competitive with oxygen [10]. Two molecules of NO compete with one molecule of O2 for the same binding site [14], the cyt a3-CuB binuclear center [15–17]. Whether NO binds first to the CuB center or to the cyt a3 is still controversial [18,19]. Cytochrome c oxidase is the final complex of the mitochondrial respiratory chain and is thus located downstream of DHODH. Since the latter links mitochondrial respiration to pyrimidine biosynthesis, we investigated whether inhibition of cytochrome c oxidase by NO could indirectly inhibit DHODH activity, therefore impairing orotate production required for pyrimidine nucleotide de novo biosynthesis.

Dihydororotate dehydrogenase (DHODH, EC 1.3.9911) catalyzes the oxidation of DHO to orotate, i.e., the fourth step of de novo pyrimidine biosynthesis. In higher eukaryotes, the enzyme is located in the inner mitochondrial membrane and is a component of the respiratory chain (for review see Jones [1]). Electrons resulting from DHO oxidation are accepted by ubiquinone and finally by cytochrome c oxidase (Fig. 1). It has been suggested by Lo¨ffler et al. that any dysfunction of the respiratory chain downstream of DHODH (i.e., at complexes III or IV) could cause an impairment of DHODH activity, thereby decreasing pyrimidine nucleotide pools [2]. Nitric oxide (NO) is an important signaling molecule in mammals that also supports cytotoxic functions in phagocytes (for review see MacMicking [3]). It has recently been suggested that NO could be a physiological regulator of mitochondrial respiration [4 – 6]. Exposure of mitochondria to NO or NO-derived nitrogen oxides results in inhibition of mitochondrial respiration at different sites, including com-

MATERIALS AND METHODS

Material Address correspondence to: Claire Beuneu, UMR 8619 CNRS, Baˆt. 430, UPS Orsay, F-91405 Orsay Cedex, France; Tel: ⫹33 1 6915-7972; Fax: ⫹33 1 6985-3715; E-Mail: [email protected].

Ascorbic acid and digitonin were from Merck (Darmstadt, Germany). Digitonin was purified following the 1206

Indirect inhibition of DHODH by NO

Fig. 1. Schematic location of DHODH in the inner mitochondrial membrane with respect to respiratory chain and pyrimidine biosynthesis. I: NADH-coenzyme Q reductase, II: Succinate-coenzyme Q reductase, III: Coenzyme Q-cytochrome c reductase, IV: Cytochrome c oxidase. Inhibition sites by cyanide (CN–), antimycin A, A771726, and nitrogen oxides (NOx) are also indicated.

1207

solution with argon gas and/or allowing cells to consume oxygen in the oxygraph cuvette before starting the experiment. A basal slope of O2 disappearance, in the absence of any cells, ranging between 0.3 and 0.8 ␮M/ min was consistently observed. This value was subtracted from any other rate, prior to any calculations. In several experiments, NO was measured simultaneously with oxygen using a Clark-modified NO electrode (WPI, Stevenage, England) introduced into the oxygraph cuvette. This electrode was monitored by the DUO-18 software v.1.1 from WPI. Calibration of the NO electrode using sodium nitrite and potassium iodide in sulfuric acid, as recommended by the manufacturers, gave inconsistent results when this acidic medium was replaced by the respiration buffer. Thus, the calibration was done using DEA/NO and aliquots of known concentrations of hemoglobin to trap NO. Orotate assay

method of Kun et al. [20]. DEA/NO was purchased from Cayman (Ann Arbor, MI, USA) and solutions were prepared in KOH 10 mM. The concentration was measured from the absorbance at 246 nm using ⑀M ⫽ 6500 M⫺1 cm⫺1 [21]. A771726 was obtained from Hoechst (Frankfurt, Germany). All other reagents were obtained from Sigma (L’isle d’Abeau Chesnes, France). Cell culture Cell lines were cultured at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 0.2 ␮g/ml streptomycin, 0.2 U/ml penicillin, and 10% (L1210, CEM-SS, RAW 264.7, and K-562), 7.5% (U-937), or 5% (HL-60) (v/v) heat-inactivated fetal calf serum. Respiration measurement Respiration medium containing 250 mM sucrose, 20 mM HEPES, 10 mM MgCl2, 2 mM KH2PO4, and 1 mM EGTA was adjusted to pH 7.4 with KOH. Before the assay, 0.7% fatty acid free bovine serum albumin was added to the respiration medium. Cells were permeabilized by exposure to 0.005% digitonin in respiration medium, followed by two washes at 4°C. Permeabilization was checked by trypan blue staining. Each sample contained 15.106 cells. The oxygen concentration was measured in a final volume of 1 ml using a Clark-type O2 electrode from Hansatech (Norfolk, England). The oxygraph was monitored by the Oxygraph System software v.1.10 from Hansatech. Substrates and inhibitors were added with a Hamilton microsyringe (see figure legends for concentrations). When required, the oxygen concentration in the solution was decreased by flushing the

Fifty microliters of HClO4 0.5 M were added to a 50 ␮l cell sample withdrawn from the oxygraph cuvette. The sample was shaken for 10 min at room temperature and centrifuged for 30 min at 17,000 ⫻ g. The supernatant was analyzed for orotate content by HPLC on an ion-exchange Partisil 10 SAX column. Gradient elution was from potassium phosphate 10 mM pH 3.5 to potassium phosphate 1 M pH 3.5 over 7 min at 1 ml/min. Under these conditions, orotate was detected by UV absorption at 280 nm, with a typical retention time of 8.0 min. Determination of theoretical DEA/NO breakdown Simulation of the theoretical kinetics of NO production from DEA/NO at 160 ␮M O2, 37°C, and pH 7.4 was performed by the Runge-Kutta integration method considering the following parameters: DEA/NO half-life for NO release in these conditions is 2.1 min [21] and the kinetic constant of NO autoxidation in aqueous solution is 8.106 M⫺2 s⫺1 [22]. The oxygen concentration was considered constant. Enzyme assay Recombinant human and rat DHODH were obtained from purification protocols and were assayed as described previously [23]. The oxidation of the substrate DHO with the quinone cosubstrate was coupled to the reduction of the chromogen 2,6-dichlorophenol-indophenol. The reaction mixture of 1 ml contained 1 mM DHO, 0.1 mM decylubiquinone, 0.12 mM dichlorophenol-indophenol, 0.1% Triton X-100 in 50 mM Tris/HCl buffer,

C. BEUNEU et al.

1208

rectly block DHODH activity. As expected, O2 consumption was also inhibited by antimycin A and cyanide (Fig. 2B,C), which are known inhibitors of complex III and complex IV respectively, thus acting downstream of DHODH in the mitochondrial respiratory chain. These results show that DHODH can be inhibited indirectly through respiration inhibition, as it has been suggested by Lo¨ffler et al. [2]. Inhibition of DHO-dependent respiration by DEA/NO

Fig. 2. Inhibition of DHODH activity by various respiratory chain inhibitors. L1210 cells (15.106) were permeabilized with 0.005% digitonin and incubated at 37°C with 4 mM DHO as a substrate. Inhibitors (A: 10 ␮M A771726; B: 2 ␮M antimycin A; C: 1 mM KCN) were added after a few minutes of DHO-dependent respiration. Rates of O2 consumption are indicated in parentheses as ␮M/min.

150 mM KCl, pH 7.5 or pH 8.0. For evaluating the effect of NO, the compound DEA/NO (from a freshly prepared stock solution of 0.5 mM in 10 mM KOH) was added to give a final concentration of 25 ␮M. Control assays contained the same amount of KOH. The reaction was started by addition of the enzyme. The loss of absorbance of the blue dye was monitored at 600 nm, (⑀M ⫽ 18,800 M⫺1 cm⫺1) for 8 min. RESULTS

DHO-dependent respiration in L1210 cells L1210 cells were permeabilized with digitonin and incubated at 37°C with DHO as a substrate in an oxygraph cuvette. Under these conditions, cell respiration was ADP independent, ranging between 1.8 and 2.9 ␮M/min for 15.106 cells. A771726, the active metabolite of leflunomide and a potent inhibitor of DHODH activity [23], completely inhibited O2 consumption, therefore demonstrating that respiration with the DHO substrate was entirely dependent on DHODH activity (Fig. 2A). Since electrons resulting from DHODH activity are transferred to ubiquinone, complex III, and complex IV successively, inhibition of these complexes should indi-

In further experiments, an NO electrode was added in the oxygraph cuvette in order to measure simultaneously NO and O2 concentrations. NO was generated from DEA/NO, a chemical NO precursor with a short half-life of 2.1 min. Introduction of 10 ␮M DEA/NO into the cuvette resulted in a transient production of NO peaking at 2.8 ␮M after 2.9 min (Fig. 3A, lower trace). NO was then consumed by autoxidation, so that after approximately 15 min, it had almost completely disappeared from the solution. DHO-dependent O2 consumption was linear until pO2 had decreased to very low levels, i.e., under 40 ␮M (data not shown). The first addition of DEA/NO in a sample containing 160 ␮M O2 caused a decrease in O2 consumption rate, from 3.37 to 1.88 ␮M/min (Fig. 3A, upper trace), i.e., 58% after correction of a basal, DHO-independent rate of O2 disappearance of 0.8 ␮M/min (see Material and Methods). Maximal inhibition coincided exactly with the NO production peak (compare Fig. 3A upper trace and lower trace). It was rapidly and spontaneously reversible, but only 80% of the initial respiration rate was recovered after NO had decreased to negligible levels (2.66 vs. 3.37 ␮M/min). Respiration inhibition by DEA/NO was also reversed by hemoglobin, which binds NO (Fig. 3B). Again, reversion was partial with a recovery of about 80% even when adding up to 25 ␮M hemoglobin. The DHO-dependent O2 consumption of the human or murine cell lines CEM-SS, RAW 264.7, HL60, U937, and K562 was similarly inhibited by micromolar concentrations of DEA/NO (data not shown). When a second addition of 10␮M DEA/NO was performed at a lower O2 concentration of 100 ␮M (Fig. 3A), a greater inhibition of respiration was noted. This phenomenon was consistently observed between 0.5 and 10 ␮M of DEA/NO (Fig. 4). At 160 ␮M O2, maximal inhibition of DHO-dependent respiration did not exceed 65%, whereas it reached 90% at 80 ␮M O2. IC50 for DEA/NO were respectively 2.5 ␮M and 0.25 ␮M at 160␮M and 80 ␮M O2. These results are in agreement with a competition of O2 and NO binding to complex IV. When DEA/NO was tested on purified human or rat

Indirect inhibition of DHODH by NO

1209

Fig. 4. Concentration-dependent inhibition of DHODH activity by DEA/NO. L1210 cells were treated as described in Fig. 2. DEA/NO was added at 80 ␮M (●) and 160 ␮M (Œ) O2 level. Rates after DEA/NO addition were measured at the maximum of the inhibition. They are expressed as the percentage of O2 consumption prior to the addition of DEA/NO. Data are the means ⫾ SEM of three independent experiments.

Comparison of NO production from DEA/NO at different oxygen tensions

Fig. 3. Kinetics of inhibition of DHODH activity by DEA/NO. L1210 cells were treated as described in Fig. 2. Respiration was induced by DHO addition. Oxygen (upper trace) and NO (lower trace) were measured simultaneously in the incubation vessel using a Clark-type O2 electrode and an NO electrode. (A) 10 ␮M DEA/NO was added twice, at 160 ␮M O2 and at 100 ␮M O2. (B) 5 ␮M DEA/NO was added to DHO-respiring cells at 80 ␮M O2, then 0.5, 0.75, and 10 ␮M HbO2 were added. Rates of O2 consumption are indicated in parentheses as ␮M/min. Those indicated just after DEA/NO addition correspond to maximal inhibition.

DHODH activity, no inhibition was observed in the presence of 25 ␮M DEA/NO (data not shown). Complete inhibition of the purified enzymes has been previously observed with 10 ␮M A771726, under similar experimental conditions [23]. Therefore, a direct effect of DEA/NO on DHODH can be excluded.

In an aqueous medium, NO is consumed by autoxidation. The rate of this reaction is first order with respect to O2 concentration. Thus, at 100 ␮M O2 NO autoxidation is decreased and a higher accumulation of NO resulting from DEA/NO breakdown is expected. This was actually observed (Figs. 3A and 5). However, the difference remained slight. Only a 29% increase in NO concentration at peak was measured using 10 ␮M DEA/NO at 100 ␮M O2, compared to the value obtained at 160 ␮M O2. At 5 ␮M DEA/NO, the difference reached 34% (2.1 vs. 2.8 ␮M NO at peak). Considering the concentration-dependent curves shown in Fig. 4, it is evident that this moderate increase in NO concentration cannot solely explain the strong increase in DEA/NO efficiency at lower O2 tensions. This is particularly apparent above 7 ␮M DEA/NO, where DHODH inhibition leveled out (i.e., became independent of DEA/NO concentration). The observed production of NO generated from 10 ␮M DEA/NO at 160 ␮M O2 fit well with the theoretical kinetics calculated for the same conditions, when taking into account NO autoxidation (Fig. 5). It has been proposed that mitochondria could catalyze NO breakdown [12]. In our model, NO production from DEA/NO was not significantly different in cell-free and cell-containing samples (Fig. 5).

1210

C. BEUNEU et al.

Fig. 5. Time-course of NO production from DEA/NO in different conditions. Nitric oxide release was monitored at 37°C using an NO electrode in the presence and absence of 15.106 permeabilized L1210 cells, and at different O2 concentrations. The dashed line represents theoretical NO production from 10 ␮M DEA/NO. Solid lines represent experimental values. Upper trace: 10 ␮M DEA/NO ⫹ L1210 cells, 100 ␮M O2; middle trace: 10 ␮M DEA/NO without cells, 160 ␮M O2; lower trace: 10 ␮M DEA/NO ⫹ L1210 cells, 160 ␮M O2.

Inhibition of orotate production by DEA/NO To confirm DHODH inhibition by NO, we also measured orotate production, which directly reflects DHODH activity. This was done simultaneously with cell respiration measurements, by withdrawing cell aliquots from the oxygraph cuvette before and after DEA/NO addition. Orotate production was linear with time in DHO-respiring cells (Fig. 6). As expected, addition of 10 ␮M A771726 totally blocked orotate production. Addition of 10 ␮M DEA/NO inhibited orotate synthesis only for a few minutes. Then, orotate production spontaneously resumed. After 15–20 min, the initial rate of orotate synthesis was almost recovered (Fig. 6). The discrete determinations of orotate concentrations at 3– 4 time points were not accurate enough to establish whether recovery was partial or complete. It has been suggested that NO could accept electrons from cytochrome c oxidase or from cytochrome c [24], therefore allowing electron transfer without O2 consumption. Therefore, there could be an extra orotate production using NO as a final acceptor instead of O2. To test this possibility, we tested the effect of DEA/NO addition on cyanide-inhibited orotate synthesis. DHODH-dependent respiration was completely inhibited by 1 mM cyanide in the presence or absence of DEA/NO. Orotate production was also inhibited by cyanide and did not

Fig. 6. Kinetics of inhibition of orotate production by DEA/NO. L1210 cells were treated as described in Fig. 2. Orotate production by the DHO-respiring cells was measured at different times. Time of addition of A771726 and DEA/NO are indicated by an open and a closed arrow, respectively. Cells without inhibitors (E, solid line), with 10 ␮M A771726 (■, dotted line), or with 10 ␮M DEA/NO (●, dashed line).

increase upon addition of DEA/NO (data not shown). It seems, therefore, that NO in our model is not an efficient electron acceptor from cytochrome c or from complex IV. Inhibition of orotate production increased as a function of DEA/NO concentration, as shown in Table 1. Maximal inhibition was approximately 60% at 160 ␮M O2, very similar to the inhibition level noted for O2 consumption (Fig. 4), whereas the inhibitory effects of the lowest concentrations of DEA/NO on orotate synthesis seem less pronounced than those on O2 consumption (Fig. 4); however, the former values were obtained over a 10 min interval including a significant period where orotate production resumed to almost the control rate. Conversely, data for Fig. 3 were calculated at the maxTable 1. Concentration-dependent Inhibition of Orotate Production by DEA/NO DEA/NO 1 ␮M 4 ␮M 7 ␮M 10 ␮M 10 ␮M decomposed

% of control (mean ⫾ SEM) 95.2 ⫾ 4.5 66.1 ⫾ 8.4 40.0 ⫾ 10.6 37.9 ⫾ 26.8 98

Rates of orotate synthesis in the presence of DEA/NO were determined over approximately a 10 min interval, in experiments similar to the one shown in Fig. 6. Rates are expressed as the percentage of orotate production before DEA/NO addition (control). Values are the means ⫾ SEM of at least two experiments, except for decomposed DEA/NO (n ⫽ 1).

Indirect inhibition of DHODH by NO

imum of the inhibition of O2 consumption by NO. A previously decomposed NO donor did not inhibit orotate production (Table 1). Two electrons are produced by reduction of one DHO molecule, and four are necessary to reduce O2 into H2O. Therefore, the theoretical ratio between orotate production and O2 consumption is 2:1. In the experiments described in Fig. 6 and Table 1, the mean calculated ratio between these two parameters was 1.7:1, in good agreement with the theory. DISCUSSION

In higher eukaryotes, pyrimidine biosynthesis is linked to the mitochondrial respiratory chain and ATP production at the level of the DHODH enzyme. In the present study we demonstrate for the first time that NO generated from DEA/NO inhibits DHO-dependent respiration. This effect does not result from a direct inhibition of DHODH, since DEA/NO did not decrease human or rat DHODH activities. We propose that impairment of DHODH activity might be a consequence of cytochrome c oxidase inhibition by NO. The observation that NOdependent inhibition of DHODH activity was much stronger at a low O2 concentration suggested competition between NO and O2 for the same binding site. This was consistent with an inhibition of cytochrome c oxidase, which is the O2 binding complex in the respiratory chain. IC50 values determined for DHODH inhibition were 2.5 and 0.25 ␮M DEA/NO at 160 and 80 ␮M O2, respectively. Assuming a t1/2 of 2.1 min [21] and a monoexponential decay, we calculated a concentration of NO at peaks of 0.9 and 0.12 ␮M NO, respectively. These values are in agreement with the IC50 for cytochrome c oxidase inhibition already reported by other groups: 60 nM NO at 30 ␮M O2 and 270 nM NO at 145 ␮M O2 in synaptosomes [10], 69 nM at 72 ␮M O2 and 364 nM NO at 180 ␮M O2 in isolated mitochondria from brown adipose tissue [14], and up to 2 ␮M NO in submitochondrial particles [25]. According to Borutaite and Brown, the sensitivity of the inhibition also depends on the respiratory state (phosphorylating or nonphosphorylating) and on the substrate type [12]. Since we used DHO for the first time as a substrate of the respiratory chain in this kind of inhibitory experiments, comparison with previous work using other substrates has to be made with caution. In most previous studies, cytochrome c oxidase inhibition by NO was found to be completely reversible, but in some cases a partial irreversibility was noted [26,27]. We observed that DHODH inhibition by NO was essentially reversible, either spontaneously or in the presence of hemoglobin, but the initial respiration rate was never totally recovered. There is some evidence that irreversible inhi-

1211

bition of cytochrome c oxidase and complex III can be caused by peroxynitrite [28,29], a product of the reaction of NO with O2•⫺. The superoxide anion radical can be produced by reduced complex III when electron transfer is blocked downstream in the respiratory chain (i.e., during inhibition of cytochrome c oxidase) [11,13]. Moreover, production of O2•⫺ by DHODH has also been described previously [30 –32]. There is thus some probability that peroxynitrite could be formed under our experimental conditions, and might be responsible for the minor irreversible component of DHODH inhibition by DEA/NO; however, we have not tested the effect of peroxynitrite itself on DHO-dependent respiration. We do not have any convincing arguments in favor of a production of peroxynitrite in our system and we cannot rule out other mechanisms, for instance, modification of thiol groups as was proposed for complex I by Clementi et al. [33]. Inhibition of DHO-dependent respiration by NO is accompanied by a consistent decrease in orotate production, which is also reversible and exhibits a similar dose dependency with respect to DEA/NO concentration. Since there is no direct inhibition of DHODH activity by DEA/NO, the impairment of orotate synthesis by NO is again best explained by inhibition of cytochrome c oxidase. It has been suggested that NO could accept electrons from cytochrome c oxidase or from cytochrome c [24]. It has also been proposed that mitochondria could catalyze NO breakdown [12]. In our model, NO decay was not significantly different in cell-free and cell-containing samples at 160 ␮M O2. Interestingly, there is a substantial decrease in NO breakdown by mitochondria, and especially by cytochrome c oxidase, when O2 tension increases from 0 to 52– 65 ␮M [12]. We suppose that at 160 ␮M O2, if NO was consumed by mitochondria, this activity was too low to be detected. This assumption is reinforced by the observation that orotate production is completely blocked by cyanide, showing that NO cannot efficiently accept electrons from a segment of the respiratory chain between DHODH and cytochrome c oxidase (e.g., complex III or cytochrome c). Finally, in most experiments the stoichiometry between orotate production vs. O2 consumption is slightly less than the theoretical value of 2. Therefore, it seems that no significant orotate synthesis occurs independently of O2 reduction. We conclude that NO reduction does not support DHODH activity. Under our experimental conditions, NO is not a functional electron acceptor from cytochrome c or cytochrome c oxidase. Considering that cytochrome c oxidase is efficiently inhibited by physiological concentrations of NO, it has been proposed that control or inhibition of cytochrome c oxidase by NO may occur in (patho-)physiological conditions [4,34]. Consequently, indirect inhibition of

1212

C. BEUNEU et al.

DHODH activity by NO may also happen in vivo. DHODH belongs to the metabolic pathway leading to de novo synthesis of pyrimidine nucleotides, which are required for RNA and DNA synthesis, as well as production of UDP sugars and CDP lipids. Inhibitors of pyrimidine synthesis are potent chemotherapeutic agents [35, 36]. Despite the fact that DHODH is not the rate-limiting enzyme in de novo pyrimidine biosynthesis [1], and despite the existence of a salvage pathway for pyrimidine nucleotide synthesis, DHODH inhibitors display marked immunosuppressive and antitumour effects, in vitro and in vivo [36 –38]. It is therefore important now to determine to what extent indirect inhibition of DHODH activity might contribute to NO-mediated immunosuppression and NO-induced toxicity against tumors and microbes. Acknowledgements — We thank Jean-Claude Drapier for lending us the NO electrode, Be´atrice Wolfersberger for excellent technical assistance, and Helen Withrow and Sarah Dolling for proofreading the manuscript. This work was supported by l’Association pour la Recherche contre le Cancer (grant 9426).

REFERENCES [1] Jones, M. E. Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP biosynthesis. Annu. Rev. Biochem. 49:253–279; 1980. [2] Lo¨ffler, M.; Jockel, J.; Schuster, G.; Becker, C. Dihydroorotateubiquinone oxidoreductase links mitochondria in the biosynthesis of pyrimidine nucleotides. Mol. Cell Biochem. 174:125–129; 1997. [3] MacMicking, J.; Xie, Q. W.; Nathan, C. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15:323–350; 1997. [4] Brown, G. C. Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase. FEBS Lett. 369: 136 –139; 1995. [5] 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. [6] Torres, J.; Darley-Usmar, V.; 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. [7] Brown, G. C. Nitric oxide and mitochondrial respiration. Biochim. Biophys. Acta 1411:351–369; 1999. [8] Radi, R.; Rodriguez, M.; Castro, L.; Telleri, R. Inhibition of mitochondrial electron transport by peroxynitrite. Arch. Biochem. Biophys. 308:89 –95; 1994. [9] Cassina, A.; Radi, R. Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport. Arch. Biochem. Biophys. 328:309 –316; 1996. [10] Brown, G. C.; Cooper, C. E. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. 356:295–298; 1994. [11] Cleeter, M. W.; Cooper, J. M.; Darley-Usmar, V. M.; Moncada, S.; Schapira, A. H. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett. 345:50 –54; 1994. [12] Borutaite, 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.

[13] Poderoso, J. J.; Carreras, M. C.; Lisdero, C.; Riobo, N.; Schopfer, F.; Boveris, A. Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. Arch. Biochem. Biophys. 328:85–92; 1996. [14] Koivisto, A.; Matthias, A.; Bronnikov, G.; Nedergaard, J. Kinetics of the inhibition of mitochondrial respiration by NO. FEBS Lett. 417:75– 80; 1997. [15] Brudvig, G. W.; Stevens, T. H.; Chan, S. I. Reactions of nitric oxide with cytochrome c oxidase. Biochemistry 19:5275–5285; 1980. [16] 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. [17] Stevens, T. H.; Brudvig, G. W.; Bocian, D. F.; Chan, S. I. Structure of cytochrome a3-Cua3 couple in cytochrome c oxidase as revealed by nitric oxide binding studies. Proc. Natl. Acad. Sci. USA 76:3320 –3324; 1979. [18] Giuffre, 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. [19] Cooper, C. E.; Torres, J.; Sharpe, M. A.; Wilson, M. T. Nitric oxide ejects electrons from the binuclear centre 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. [20] Kun, E.; Kirsten, E.; Piper, W. N. Stabilization of mitochondrial functions with digitonin. Methods Enzymol. 55:115–118; 1979. [21] Maragos, C. M.; Morley, D.; Wink, D. A.; Dunams, T. M.; Saavedra, J. E.; Hoffman, A.; Bove, A. A.; Isaac, L.; Hrabie, J. A.; Keefer, L. K. Complexes of NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects. J. Med. Chem. 34:3242–3247; 1991. [22] Wink, D. A.; Beckman, J. S.; Ford, P. C. Kinetics of nitric oxide reaction in liquid and gas phase. In: Feelisch, M.; Stamler, J. S., eds. Methods in Nitric Oxide Research. Chichester, England: Wiley Ltd.; 1996: 29 –37. [23] Bader, B.; Knecht, W.; Fries, M.; Lo¨ffler, M. Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase. Protein Expr. Purif. 13:414 – 422; 1998. [24] Sharpe, M. A.; Cooper, C. E. Reactions of nitric oxide with mitochondrial cytochrome c: a novel mechanism for the formation of nitroxyl anion and peroxynitrite. Biochem. J. 332:9 –19; 1998. [25] Lizasoain, I.; Moro, M. A.; Knowles, R. G.; Darley-Usmar, V.; Moncada, S. Nitric oxide and peroxynitrite exert distinct effects on mitochondrial respiration which are differentially blocked by glutathione or glucose. Biochem. J. 314:877– 880; 1996. [26] Hurst, R. D.; Chowdhury, R.; Clark, J. B. Investigations into the mechanism of action of a novel nitric oxide generator on cellular respiration. J. Neurochem. 67:1200 –1207; 1996. [27] Brown, G. C.; Bolanos, J. P.; Heales, S. J.; Clark, J. B. Nitric oxide produced by activated astrocytes rapidly and reversibly inhibits cellular respiration. Neurosci. Lett. 193:201–204; 1995. [28] 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. [29] Bolanos, J. P.; Heales, S. J.; Land, J. M.; Clark, J. B. Effect of peroxynitrite on the mitochondrial respiratory chain: differential susceptibility of neurones and astrocytes in primary culture. J. Neurochem. 64:1965–1972; 1995. [30] Forman, H. J.; Kennedy, J. Superoxide production and electron transport in mitochondrial oxidation of dihydroorotic acid. J. Biol. Chem. 250:4322– 4326; 1975. [31] Forman, H. J.; Kennedy, J. Mammalian dihydroorotate dehydrogenase: physical and catalytic properties of the primary enzyme. Arch. Biochem. Biophys. 191:23–31; 1978. [32] Lakaschus, G.; Kruger, H.; Heese, D.; Lo¨ffler, M. Evidence from

Indirect inhibition of DHODH by NO

[33]

[34]

[35]

[36]

in vitro studies that dihydroorotate dehydrogenase may be a source of toxic oxygen species. Adv. Exp. Med. Biol. 309A:361– 364; 1991. Clementi, E.; Brown, G. C.; Feelisch, M.; Moncada, S. Persistent inhibition of cell respiration by nitric oxide: crucial role of Snitrosylation of mitochondrial complex I and protective action of glutathione. Proc. Natl. Acad. Sci. USA 95:7631–7636; 1998. Loke, K. E.; McConnell, P. I.; Tuzman, J. M.; Shesely, E. G.; Smith, C. J.; Stackpole, C. J.; Thompson, C. I.; Kaley, G.; Wolin, M. S.; Hintze, T. H. Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption. Circ. Res. 84:840 – 845; 1999. Brazelton, T. R.; Morris, R. E. Molecular mechanisms of action of new xenobiotic immunosuppressive drugs: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide. Curr. Opin. Immunol. 8:710 –720; 1996. Silva Jr., H. T.; Morris, R. E. Leflunomide and malononitrilamides. Am. J. Med. Sci. 313:289 –301; 1997.

1213

[37] Chen, S.-F.; Ruben, R. L.; Dexter, D. L. Mechanism of action of the anticancer agent 6-fluoro-2-(2⬘-fluoro-1, 1⬘-biphenyl-4-yl)-3methyl-4-quinolinecarboxylic acid sodium salt (NSC 368390): inhibition of de novo pyrimidine nucleotide biosynthesis. Cancer Res. 46:5014 –5019; 1986. [38] Pizzorno, G.; Wiegand, R. A.; Lentz, S. K.; Handschumacher, R. E. Brequinar potentiates 5-fluorouracil antitumor activity in a murine model colon 38 tumor by tissue-specific modulation of uridine nucleotide pools. Cancer Res. 52:1660 –1665; 1992.

ABBREVIATIONS

DEA/NO—1,1-diethyl-2-hydroxy-2-nitroso-hydrazine, sodium salt DHODH— dihydroorotate dehydrogenase