Decreased affinity for oxygen of cytochrome-c oxidase in Leigh syndrome caused by SURF1 mutations

Petr Pecina,1 Erich Gnaiger,2 Jirí Zeman,3 Ewa Pronicka,4 and Josef Houstek1

1Institute of Physiology and Center for Integrated Genomics, Academy of Sciences of the Czech Republic, 142 20 Prague; 3Department of Pediatrics, 1st Medical Faculty, Charles University, 110 00 Prague, Czech Republic; 2Department of Transplant Surgery, D. Swarovski Research Laboratory, University Hospital Innsbruck, A-6020 Innsbruck, Austria; and 4Department of Metabolic Diseases, Children's Memorial Health Hospital, 04-730 Warsaw, Poland

Submitted 16 June 2004 ; accepted in final form 13 July 2004


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Mutations in the gene SURF1 prevent synthesis of cytochrome-c oxidase (COX)-specific assembly protein and result in a fatal neurological disorder, Leigh syndrome. Because this severe COX deficiency presents with barely detectable changes of cellular respiratory rates under normoxic conditions, we analyzed the respiratory response to low oxygen in cultured fibroblasts harboring SURF1 mutations with high-resolution respirometry. The oxygen kinetics was quantified by the partial pressure of oxygen (PO2) at half-maximal respiration rate (P50) in intact coupled cells and in digitonin-permeabilized uncoupled cells. In both cases, the P50 in patients was elevated 2.1- and 3.3-fold, respectively, indicating decreased affinity of COX for oxygen. These results suggest that at physiologically low intracellular PO2, the depressed oxygen affinity may lead in vivo to limitations of respiration, resulting in impaired energy provision in Leigh syndrome patients.

oxygen kinetics; mitochondrial disease


OXYGEN IS SUPPLIED TO TISSUES through the respiratory cascade. The transport of O2 through the respiratory tract and the cardiovascular system is characterized by a drop of the oxygen partial pressure (PO2) from 20 kPa in the inspired air to low intracellular PO2 (22). Under physiological conditions, therefore, mitochondria operate at PO2 as low as 0.3 kPa in some tissues (9). Nevertheless, mitochondrial and cellular respiration in health and disease is rarely studied under physiological low-oxygen conditions, and the oxygen affinity of cytochrome-c oxidase (COX) is frequently assumed to prevent any oxygen limitations of respiration under normoxia.

Mammalian COX is composed of 13 subunits: mitochondrial (mt)DNA genes encode the 3 largest subunits forming the catalytic core of the enzyme, and the other 10 subunits are encoded by the nuclear genome (27). In addition, numerous nucleus-encoded factors are required for efficient assembly and maintenance of the COX holoenzyme that are similar in yeast and humans (11, 18). Mutations in genes encoding these COX assembly factors are a frequent cause of COX deficiencies; in fact, they are much more common than mutations in genes encoding the COX subunits themselves (25). One of these assembly proteins is encoded by the SURF1 gene. Two groups identified mutations in this gene to be responsible for the autosomal recessive form of Leigh syndrome (LSCOX) (29, 33). Leigh syndrome, a subacute necrotizing encephalomyopathy, is a progressive neurodegenerative disease. The severe symptoms usually have onset within the first year of life and are characterized by general psychomotor retardation and bilaterally symmetrical lesions in the basal ganglia region. An increased level of lactate in both blood and cerebrospinal fluid is observed. The disease is fatal in the vast majority of cases; the patients usually die before 5 years of age (21). Leigh syndrome is the most common form of COX disorders and one of the most frequently occurring respiratory chain defects in infancy and childhood (23). To date, more than 30 different pathogenic mutations have been described in SURF1 (16). Most of these are nonsense mutations inducing the formation of a premature stop codon; missense and splicing site mutations are less common. It is generally assumed that the severe, isolated COX defect in patients harboring SURF1 mutations results from impaired assembly of the complex (3, 28). In a previous study (15), researchers at our laboratory further demonstrated that there exist several functional forms of COX in mitochondria of LSCOX cells that differ in subunit composition, electron transport, and proton pumping properties.

Because all previous studies with LSCOX cells were performed at much higher PO2 than is actually present in cells in vivo, we focused on low oxygen levels in our respirometric investigation of the functional impact of SURF1 mutations. We analyzed the oxygen kinetics of COX in fibroblasts from five Leigh syndrome patients by performing high-resolution respirometry. Although respiration at high oxygen levels was indistinguishable from control cells in these patients' cells, we observed a highly significant decrease of COX oxygen affinity in fibroblasts from all patients studied. These findings are discussed in the context of cell energetics in these pathological states.


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Patients. Fibroblast cultures from Leigh syndrome patients with isolated COX deficiency caused by SURF1 mutations were provided by the Department of Pediatrics, Charles University (patients 1 and 2), and the Department of Metabolic Diseases, Children's Memorial Health Hospital (patients 3–5). The SURF1 mutations of fibroblast cultures from five patients are presented in Table 1; all patients harbored premature stop codons, which prevent synthesis of the Surf1 protein. Five different fibroblast cultures without mitochondrial disorder served as controls. The study was carried out in accordance with the Declaration of Helsinki of the World Medical Association and was approved by the Committee of Medical Ethics. Informed consent was obtained from the parents of all patients.


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Table 1. SURF1 mutations in fibroblast cultures of patients 1–5

 
Cell culture. Skin fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 20 mM HEPES, pH 7.5, 0.2% NaHCO3, and gentamicin (2 mg/100 ml) at 37°C and 5% CO2 in air. Near-confluent cultures were harvested with 0.05% trypsin and 0.02% EDTA. Detached cells were diluted in ice-cold culture medium, sedimented by centrifugation (600 g), and washed twice in ice-cold phosphate-buffered saline (in mM: 140 NaCl, 5.4 KCl, 8 Na2HPO4·12H2O, and 1.4 KH2PO4, pH 7.2, at 25°C).

High-resolution respirometry and oxygen kinetics. The Oxygraph-2k (Oroboros) was used for measurements of oxygen consumption. This instrument provides sufficient sensitivity and time resolution for analysis of the oxygen kinetics of mitochondrial and cellular respiration (5). Before all measurements, the chambers were sterilized with 70% ethanol for 20 min. For calibration of the oxygen sensor, a constant signal was obtained in the opened chamber with medium in equilibrium with the air in the gas phase. Internal zero-oxygen calibration was performed with Datlab software (Oroboros) as described previously (10). All measurements were performed at 30°C in a 2-ml chamber volume.

For measurements with intact fibroblasts, an extracellular buffer (32) was used that contained (in mM) 137 NaCl, 5 KCl, 0.7 sodium phosphate, and 25 Tris·HCl (pH 7.4). A mitochondrial KCl medium (13) containing (in mM) 80 KCl, 10 Tris·HCl, 3 MgCl2, 1 EDTA, and 5 potassium phosphate (pH 7.4) was used for experiments when the cell membrane of the fibroblasts was permeabilized with digitonin (0.1 g/g protein). In this case, 10 mM succinate was added as a substrate and 1 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used to uncouple and maximally stimulate respiration (state 3u). The volume-specific rate of oxygen consumption (oxygen flux) was calculated as the negative slope of oxygen concentration recorded at 2-s time intervals. The signal was deconvoluted with the exponential time constant of the oxygen sensor (3–5 s). Cellular oxygen flux (J02) was corrected for instrumental background, which is a linear function of experimental PO2 and results from oxygen consumption by the sensor and oxygen back-diffusion from low-capacity oxygen reservoirs. P50 is the PO2 at which the cellular respiratory rate is half-maximal. This parameter was obtained from the hyperbolic function J02 = (Jmax·PO2)/(P50 + PO2) fitted over the low-oxygen range of 0–1.1 kPa, where Jmax is maximal flux. All calculations were performed with routine functions of the Datlab software (10).

Protein determination. Protein content was measured in 10-µl and 20-µl aliquots withdrawn from the Oxygraph chamber by the method of Bradford (2) with bovine serum albumin as a standard. Samples were sonicated for 20 s before protein determination.

Statistics. Data are presented as means ± SD from several measurements of each individual cell culture. The statistical significance between control and patient cells was evaluated with standard t-test and Kruskal-Wallis one-way ANOVA on ranks and by Tukey-Kramer multiple-comparison test with a freeware version of NCSS software.


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P50 was evaluated in two experimental settings: respiration of intact coupled cells with endogenous substrates (control subjects vs. patients 1, 2, 3, and 4) and unrestricted oxidation of exogenous succinate in digitonin-permeabilized cells after FCCP uncoupling (control subjects vs. patients 1, 3, 4, and 5). All patients presented with the typical biochemical phenotype of low COX activity (15, 17).

Typical experimental records of the simultaneous decrease of oxygen concentration and flux are shown in Fig. 1, A and B, for a control and a patient (patient 1) fibroblast culture, respectively. Oxygen flux and the corresponding hyperbolic fits are plotted as a function of oxygen concentration in Fig. 1C. In intact cells the P50 of control fibroblasts ranged from 0.03 to 0.05 kPa (0.039 ± 0.010 kPa), whereas in patient cells it increased 2.1-fold to a range of 0.08–0.10 kPa (0.082 ± 0.015 kPa) (Fig. 2A). No statistically significant differences were found between the average P50 of fibroblast cultures of individual patients (not shown). The P50 of cells for each patient (patients 1–4), however, was significantly higher compared with control subjects (Fig. 2A).



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Fig. 1. Aerobic-anoxic transition of fibroblast respiration. Figure shows a typical record of simultaneous decrease of oxygen partial pressure (PO2) (right y-axis; solid line) and volume-specific oxygen flux (left y-axis; {circ}) during the aerobic-anoxic transition in intact control (A) and patient (B) fibroblasts respiring on endogenous substrates. The parameters of the hyperbolic fit [maximal oxygen flux (Jmax) and PO2 at half-maximal respiration (P50)] are indicated. C: oxygen flux (individual data points) and corresponding hyperbolic fits calculated by DatLab2 (full lines) are plotted as a function of PO2 with data from A and B. {circ}, Control; {triangleup}, patient.

 


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Fig. 2. Mean P50 of respiration in intact cells (A) and of respiration at state 3u (B). Data in A are displayed for all control subjects (C; n = 14), for all patients (P; n = 17), and for individual patients 1 (n = 3), 2 (n = 2), 3 (n = 7), and 4 (n = 5). Data in B are displayed for all controls (C; n = 17), for all patients (P; n = 23), and for individual patients 1 (n = 8), 3 (n = 7), 4 (n = 7), and 5 (n = 1). Mean values are shown inside columns; error bars indicate ±SD. *P < 0.01, {dagger}P < 0.05.

 
Under the conditions of uncoupled state 3 respiration with succinate the P50 of control fibroblasts ranged from 0.020 to 0.042 kPa (0.032 ± 0.010 kPa), whereas in patient cells it increased 3.3-fold vs. control cells to a range from 0.09 to 0.17 kPa (0.104 ± 0.033 kPa) (Fig. 2B). As in intact cells, no statistically significant differences were found between the average P50 of fibroblast cultures of individual patients (not shown). The P50 of cells from each patient (patients 1, 3, and 4), however, was significantly higher compared with controls (Fig. 2B).

In contrast to P50, no statistical differences were found in respiratory rates at kinetic oxygen saturation normalized on protein content (Jmax) between control and patient cells, in agreement with a previous study (15). In intact control cells, Jmax ranged from 42.8 to 99.6 pmol O2·s–1·mg–1 (mean 60.5 ± 17.9 pmol O2·s–1·mg–1); in patient fibroblasts it ranged from 55.0 to 118.6 pmol O2·s–1·mg–1 (mean 78.8 ± 25.4 pmol O2·s–1·mg–1). In state 3u, Jmax increased to a range of 60.2–215.5 pmol O2·s–1·mg–1 (mean 108.1 ± 44.5 pmol O2·s–1·mg–1) in control fibroblasts compared with 68.1–146.0 pmol O2·s–1·mg–1 (mean 95.0 ± 24.6 pmol O2·s–1·mg–1) in patient fibroblasts.


    DISCUSSION
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The present study was aimed at resolving the apparent paradox that fatal SURF1 mutations associated with isolated COX deficiency present with barely detectable changes of cellular respiratory rates. We analyzed fibroblasts of five patients harboring mutations of the SURF1 gene, which prevent synthesis of the Surf1 protein and are responsible for a failure to assemble individual COX subunits into a fully functional enzyme complex in the inner mitochondrial membrane. Leigh syndrome caused by the dysfunction of Surf1 protein is the most common clinical presentation of COX deficiency in childhood (24). Defect in COX activity appears to be the only mitochondrial abnormality in these patients, and it is expressed in all tissues including skin fibroblasts. Because both the subunit composition of partially assembled complexes (3, 15) and the relative COX capacity are changed in patient cells, one might suspect that the oxygen affinity of such COX complexes might also be altered. A decrease of oxygen affinity could then have negative consequences on the oxygen availability for mitochondrial respiration.

We analyzed the COX oxygen kinetics by high-resolution respirometry (10). This method yields the P50 parameter, a measure of the sensitivity of cellular respiration to oxygen availability. Such a "macroscopic" approach is advantageous for several reasons: the method is relatively simple compared with, e.g., the flow-flash kinetic approach, which can yield microscopic oxygen binding constants (31). The P50 can be used to assess the oxygen dependence of respiration in mitochondria and even in small cells with relatively low aerobic activity such as fibroblasts (12) and human umbilical vein endothelial cells (26), where intracellular oxygen gradients are small and the P50 of cellular respiration is close to the P50 of isolated mitochondria. Possible changes of intracellular diffusion gradients to mitochondria in pathological cells, therefore, can be ignored in these cells but might be considerable in more active and larger cells (6). This is supported by our present study, in which P50 values of control fibroblasts are similar to P50 of mitochondria isolated from rat heart (7). The P50, therefore, can be conveniently analyzed in whole cells under conditions of intact mitochondrial membranes, where COX function is integrated in the respiratory chain. Results of such analyses might thus be more relevant in the context of mitochondrial and cellular physiology.

Our results clearly show that P50 is increased in patient cells compared with control cells. Basically, two reasons for the P50 increase can be considered: the P50 increase may be due to a decrease of the COX excess capacity or an increased apparent Michaelis-Menten constant (K'm) of COX for oxygen. A lower excess capacity of COX relative to the flux through the respiratory chain yields a corresponding increase of COX turnover, which then causes the P50 of oxygen flux through the respiratory chain to increase (8). To find out whether the observed increase of P50 in patient cells is due to a decrease of COX capacity, we plotted the P50 values versus the specific oxygen flux normalized on protein content (Fig. 3). P50 increased as a function of oxygen consumption of intact cells respiring on endogenous substrates (i.e., as COX turnover increased). In patient cells, P50 values increased without a corresponding increase of oxygen flux, indicating that the difference in P50 cannot be explained by the change of protein-specific flux observed in control and patient cells (Fig. 3A). Although the dependence of P50 on oxygen flux is more complex under uncoupled conditions (26), an analogous picture appeared when we analyzed P50 measurements in FCCP-treated cells (Fig. 3B). These data, therefore, provide strong evidence that the P50 increase in patient fibroblasts was due to changes in COX affinity for oxygen.



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Fig. 3. The relation of P50 and protein-specific oxygen flux. P50 values from individual measurements were plotted as a function of protein-specific oxygen flux (Jmax). A: endogenous respiration. {blacktriangleup}, Control fibroblasts; {bullet}, patient 1; {blacklozenge}, patient 2; {blacksquare}, patient 3; *, patient 4. B: uncoupled respiration. {blacktriangleup}, Control fibroblasts; {bullet}, patient 1; {blacksquare}, patient 3; *, patient 4; {blacklozenge}, patient 5.

 
The most important aspects of the present study are the consequences of a decreased affinity for oxygen on the energetics of the patient cells in vivo. Our previous study (15) indicated that the presence of incomplete COX assemblies with upregulated electron transport properties might serve as a kind of compensatory mechanism for the lower content of COX subunit I, allowing for near-normal oxygen flux through the respiratory chain under conditions of high PO2. The present data suggest that the situation might be completely different in vivo at low physiological oxygen levels, when oxygen flux would be significantly depressed in patient tissues. Although the SURF1 genetic defect is present in all cells, our results indicate that it would more pronouncedly manifest in tissues where energy demands are high relative to the cells' capacity to generate energy. This applies to the central nervous system (CNS), the principal site of pathology in Leigh syndrome. The CNS has very small energy reserves (for review see Ref. 1), and oxygen tensions well below 1 kPa were reported in the brain of experimental animals. The rate of oxygen utilization, in particular in neurons, is high, and it is expected that rapid consumption by the mitochondria creates around them a "well" in which PO2 is lower than at other cellular sites (4). This indicates that COX in LSCOX patient nerve cells would not be kinetically saturated by oxygen. On the basis of our measurements, for example, at PO2 of 0.3 kPa the mitochondrial respiration in patients would be limited to 70% compared with 90% saturation in control subjects. Such oxygen limitation of mitochondrial respiration in patient cells would cause impairment of oxidative energy production under normoxic conditions and especially under hypoxia. This hypothesis is supported by the fact that clinical symptoms in patients with Leigh syndrome significantly worsen during respiratory infections (19, 20), especially when the oxygen supply to tissues is decreased (30).

In addition, a recent study shows that the decrease of COX affinity for oxygen caused by nitric oxide results in decreased levels of hypoxia-induced factor 1-{alpha} and thus lower expression of "hypoxic genes" (14). If an analogous situation occurs when COX oxygen affinity is decreased because of SURF1 mutations, the patients not only would have impaired oxidative energy production due to oxygen limitation but would also suffer from impairment of glycolytic energy production due to lower expression of hypoxic genes.

The observed phenomenon of increased P50 in LSCOX fibroblasts could be one of the key mechanisms that translate the COX assembly defect triggered by SURF1 mutation to deleterious changes of cell energetics that finally result in the fatal symptoms of Leigh syndrome. In general, experimental and clinical investigations of cells and isolated mitochondria should be extended into the low physiological oxygen range, including evaluation of COX oxygen kinetics, to study the etiopathogenic mechanisms of COX deficiencies.


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This work was supported by the Grant Agency of the Czech Republic (303/03/0749), the Ministry of Education, Youth and Sports of the Czech Republic (institutional project VZ1110003 and Czech-Austrian grant Kontakt-Aktion 2004/S), and by the Academy of Sciences of the Czech Republic (institutional project AVOZ5011922).


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Houstek, Dept. of Bioenergetics, Institute of Physiology of the Czech Academy of Sciences, Vídenská 1083, 142 20 Prague, Czech Republic (E-mail: houstek{at}biomed.cas.cz)

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.


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