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Frataxin Acts as an Iron Chaperone Protein to Modulate Mitochondrial Aconitase Activity

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Science  09 Jul 2004:
Vol. 305, Issue 5681, pp. 242-245
DOI: 10.1126/science.1098991

Abstract

Numerous degenerative disorders are associated with elevated levels of prooxidants and declines in mitochondrial aconitase activity. Deficiency in the mitochondrial iron-binding protein frataxin results in diminished activity of various mitochondrial iron-sulfur proteins including aconitase. We found that aconitase can undergo reversible citrate-dependent modulation in activity in response to pro-oxidants. Frataxin interacted with aconitase in a citrate-dependent fashion, reduced the level of oxidant-induced inactivation, and converted inactive [3Fe-4S]1+ enzyme to the active [4Fe-4S]2+ form of the protein. Thus, frataxin is an iron chaperone protein that protects the aconitase [4Fe-4S]2+ cluster from disassembly and promotes enzyme reactivation.

Aconitase, a Krebs-cycle enzyme that converts citrate to isocitrate, belongs to the family of iron-sulfur–containing dehydratases whose activities depend on an intact cubane [4Fe-4S]2+ cluster (1, 2). The purified enzyme is highly susceptible to oxidant-induced inactivation due to release of the solvent-exposed Fe-α and formation of a [3Fe-4S]1+ cluster (3, 4). Loss of mitochondrial aconitase activity is an intracellular indicator of superoxide generation and of oxidative damage in a variety of degenerative diseases and aging (5, 6). Nevertheless, aconitase is rapidly inactivated and subsequently reactivated when isolated rat cardiac mitochondria are treated with H2O2, suggesting that aconitase may be an intramitochondrial sensor of redox status (7). The presence of the enzyme's substrate citrate diminishes Fe-α release, cluster disassembly, and enzyme inactivation, and it is required for enzyme reactivation (7). However, the physiological mechanisms responsible for preventing full cluster disassembly and for reduction of the [3Fe-4S]1+ center and reinsertion of Fe(II) are unclear (1).

The mitochondrial matrix protein frataxin and its yeast homolog Yfh1p are thought to play a role in the storage of iron within mitochondria (812) and to promote Fe(II) availability (10, 13, 14) as one of the components involved in the maturation of cellular iron-sulfur–containing and heme-containing proteins (1319). Friedreich's ataxia, a neurodegenerative and cardiac disorder, is characterized by a deficiency in frataxin, an accumulation of iron in the mitochondria, and diminished activity of various mitochondrial iron-sulfur proteins, including aconitase (2022). The crystal structure of human frataxin reveals a conserved, primarily hydrophobic region on the surface of the protein that may interact with other proteins (23). We sought evidence for a role of frataxin distinct from the assembly of protein iron-sulfur clusters. It is possible that frataxin could act as an iron chaperone protein to protect aconitase from cluster disassembly and serve as a iron donor to the [3Fe-4S]1+ cluster during pro-oxidant–induced modulation of aconitase activity.

We treated isolated rat cardiac mitochondria with 100 μM H2O2 in the presence or absence of 2.0 mM citrate, then immunoprecipitated aconitase. This resulted in the copurification of frataxin eluting at a molecular weight (17 kD) consistent with the mature form of the protein (Fig. 1A). No interaction between aconitase and frataxin was detected in the absence of citrate (Fig. 1A). Citrate prevents aconitase cluster disassembly and is required for enzyme reactivation in rat cardiac mitochondria treated with H2O2 (7, 24). Thus, the requirement for citrate for interaction of frataxin and aconitase supports the contention that frataxin can stabilize the [4Fe-4S]2+ cluster and facilitate enzyme reactivation.

Fig. 1.

Citrate-dependent interaction between aconitase and frataxin. (A) Isolated rat cardiac mitochondria (0.5 mg/ml) were incubated with 100 μMH2O2 in the presence or absence of 2.0 mM citrate at 25°C for 2.0 min. Mitochondria were then solubilized (0.05% Triton X-100), followed by immunoprecipitation with antiserum raised against rat aconitase. Western blot analysis was performed on immunoprecipitated protein with polyclonal antibodies to aconitase or frataxin as indicated (31). Blots are representative of five separate experiments. (B) Mitochondria were isolated from YC-FRDA or pG3-FRDA strains of yeast. Western blot analysis (40 μg of total mitochondrial protein) was performed with polyclonal antibodies to human frataxin and yeast aconitase. (C) Isolated mitochondria (0.5 mg/ml) from YC-FRDA and pG3-FRDA yeast were incubated with 100 μM H2O2 in the presence or absence of 2.0 mM citrate at 25°C for 2.0 min. Immunoprecipitation was then performed with antiserum raised against yeast aconitase, followed by Western blot analysis with polyclonal antibodies to yeast aconitase or human frataxin as indicated.

Mitochondria were isolated from YC-FRDA or pG3-FRDA strains of yeast lacking the yeast homolog of frataxin, Yfh1p, but complemented with human frataxin expressed from a low-copy or high-copy plasmid, respectively (25, 26). Frataxin polyclonal antibody recognized two bands representing the intermediate (21 kD) and mature (17 kD) forms of frataxin (Fig. 1B) (26, 27). The level of human frataxin in pG3-FRDA cells was fourfold higher than in YC-FRDA cells. Nevertheless, no appreciable differences in mitochondrial respiratory rates were observed (Table 1). In addition, the level of aconitase was not altered between strains (Fig. 1B). However, aconitase activity in pG3-FRDA cells was ∼1.6-fold higher than in YC-FRDA cells (Table 1), indicating that frataxin is probably involved in aconitase [4Fe-4S]2+ cluster assembly and/or prevention of disassembly. Human frataxin interacted with yeast aconitase in a citrate-dependent fashion in both YC-FRDA and pG3-FRDA strains, and the ratio of frataxin to aconitase increased in accordance with overall levels of frataxin (Fig. 1C). The yeast strain YC-FH1, lacking Yfh1p (28), exhibited levels of frataxin, aconitase activity, and respiratory rates similar to those of the YC-FRDA cells (Table 1) (25).

Table 1.

Tabulated representation of data from Fig. 2A, respiratory rates, and mitochondrial frataxin and iron content for indicated yeast strains (31).

Yeast strain YC-YFH1 YC-FRDA pG3-FRDA
Relative frataxin levels 100 130 520
Aconitase activity (nmol/min/mg) 100 ± 10 115 ± 8 181 ± 18
State 3 respiration (nmol O/min/mg) 181 186 217
State 4 respiration (nmol O/min/mg) 75 75 80
% Inhibition of activity 81 52 27
% Recovery of activity 107 21 76
Iron content (nmol/mg protein) 10.2 ± 2.1 11.8 ± 1.8 9.9 ± 0.6

Treatment of mitochondria isolated from yeast strains containing different levels of frataxin with H2O2 in the presence of citrate resulted in maximal inhibition of aconitase within 5.0 min (Fig. 2A). The level of inhibition relative to untreated samples paralleled the level of frataxin (Table 1). Mitochondria from each strain of yeast exhibited similar rates of H2O2 removal (Fig. 2, C and D). In the absence of citrate, H2O2 treatment led to a greater than 80% loss of activity, with no subsequent recovery (Fig. 2B). When citrate was included, recovery of aconitase activity was evident (Fig. 2A). The degree of reactivation depended on both the level of frataxin and the origin of the protein (Table 1) (12, 29, 30). Iron-catalyzed formation of free radicals and subsequent oxidative inactivation of aconitase may also limit aconitase reactivation. Total iron present in mitochondria isolated from the three yeast strains was not significantly different (Table 1). This does not, however, preclude the possibility that higher concentrations of free iron were present in mitochondria from YC-FRDA yeast and may have contributed to irreversible inactivation of aconitase. Thus, frataxin plays a critical role in protecting aconitase from pro-oxidant–induced inactivation and in facilitating enzyme reactivation.

Fig. 2.

Role of frataxin in the inactivation and reactivation of aconitase in intact yeast mitochondria treated with H2O2 Mitochondria isolated from YC-FRDA (⚫), pG3-FRDA (◯), or YC-YFH1 (▢) cells were incubated with 100 μM H2O2 in the presence (A and C) or absence (B and D) of 2.0 mM citrate (31). [(A) and (B)] At indicated times, mitochondria were disrupted and aconitase activity was determined as previously described (7). [(C) and (D)] The concentration of H2O2 was in the incubation mixture determined by p-hydroxyphenylacetate fluorescence on addition of horseradish peroxidase (31). Data are represented as mean ± standard error from five experiments with independent cell culture and mitochondrial preparations.

The purified inactive [3Fe-4S]1+ form of bovine mitochondrial aconitase was used to test the ability of frataxin to donate iron and convert inactive aconitase to the active [4Fe-4S]2+ form of the enzyme. The protein had a residual activity of 0.5 nmol/min/mg that could be reconstituted under anaerobic conditions to an activity of 2.4 nmol/min/mg (24, 31). To assess whether the presence of frataxin could facilitate enzyme reactivation under more physiologically relevant conditions, purified human frataxin in assembled form (3.0 μM) was preincubated for 10 min in the presence of Fe(II) (30 μM) under aerobic conditions at 30°C (9). Under these conditions, ∼19 μM Fe(II) was in a form available to bipyridine as compared to less than 7 μM in buffer without frataxin (32). Thus, frataxin maintained a substantial level of iron in a bioavailable state. Aconitase (3.0 μM) and dithiothreitol (DTT) (1.0 mM), required for reduction of the [3Fe-4S]1+ cluster (24), were then incubated with iron-loaded frataxin for 5.0 min. After the addition of citrate (0.5 mM), electron paramagnetic resonance (EPR) analysis indicated that the [3Fe-4S]1+ cluster of aconitase (2) was converted to the EPR-silent [4Fe-4S]2+ cluster within 30 min (Fig. 3A). This resulted in the time-dependent activation of aconitase that was most efficient in the presence of citrate (Fig. 3B). Thus, citrate facilitated the interaction between frataxin and aconitase (Fig. 1, A and C) and promoted activation of aconitase (Fig. 3B).

Fig. 3.

Transfer of iron to, and reactivation of purified [3Fe-4S]1+ aconitase by, frataxin. (A) Purified assembled frataxin (3.0 μM) was incubated with 30 μM Fe(II) for 10 min in 10 mM Hepes, pH 7.3, at 30°C. Purified [3Fe-4S]1+ aconitase (3.0 μM) and DTT (1.0 mM) were then incubated with Fe(II)-loaded frataxin for 5 min. On addition of citrate (0.5 mM), the incubation was allowed to proceed for 30 min before samples were frozen for EPR analysis (9.45 GHz with 10 gauss field modulation at 100 kHz at 10 K) (dashed line). EPR analysis was also performed on purified [3Fe-4S]1+ aconitase (3.0 μM) (solid line). g, Lande's g factor. (B) Aconitase activity was measured after incubation for indicated periods of time under the conditions described in (A) with the following alterations: ◼, no alterations; ▢, in the absence of citrate; ⚫, in the absence of frataxin; and △, in the absence of frataxin and citrate. (C) Aconitase activity was measured after incubation for indicated periods of time under the conditions described in (A) with the following alterations: ◼, no alterations; ▲, citrate (0.5 mM) was incubated for 5.0 min with Fe(II)-loaded frataxin before addition of purified [3Fe-4S]1+ aconitase (3.0 μM) and DT T (1.0 mM); and ♦, frataxin was replaced with BSA (3.0 μM). Data are represented as mean ± standard error from three independent experiments.

Incubation of aconitase and DTT with Fe(II) in the absence of frataxin restored 10% of total recoverable activity (Fig. 3B). Inclusion of citrate under these conditions resulted in a loss in residual activity (Fig. 3B). This is likely the result of citrate-catalyzed conversion of Fe(II) to Fe(III) and oxidative inactivation of aconitase. The inability of 30 μM Fe(II) to cause substantial enzyme reactivation suggests that frataxin transfers Fe(II) directly to aconitase or supports efficient recycling of Fe(III) to Fe(II) in the presence of DTT. Direct transfer is supported by data indicating that when citrate (0.5 mM) was added to Fe(II)-loaded frataxin (5 min) before aconitase and DTT, recovery of aconitase activity did not occur (Fig. 3C). Thus, citrate can effectively compete for Fe(II) in the absence of aconitase. An interaction between frataxin and aconitase likely shields the Fe(II) and allows for effective reinsertion of iron-sulfur into the enzyme's [3Fe-4S]1+ cluster. Finally, bovine serum albumin (BSA) was loaded with Fe(II). Incubation of aconitase with Fe(II)-loaded BSA, under the same conditions used for frataxin, resulted in no reactivation of aconitase (Fig. 3C). Thus, the reactivation observed in the presence of frataxin did not reflect nonspecific protein stabilization.

Frataxin can play a dynamic role in the regulation of aconitase during oxidative stress. Four important roles for frataxin include (i) maintenance of iron in a bioavailable state; (ii) prevention of the production of potentially deleterious free radicals; (iii) protection of the [4Fe-4S]2+ cluster from disassembly; and (iv) facilitation of Fe(II) transfer to the [3Fe-4S]1+ cluster of aconitase, resulting in reactivation of the enzyme. The labile iron released from the enzyme's [4Fe-4S]2+ cluster is involved in binding of citrate to the enzyme's active site (2). The role of citrate in stabilizing the frataxin-aconitase interaction and promoting enzyme reactivation may be to act as a bridge between the two proteins and as a co-chaperone, orienting iron for appropriate reinsertion and stabilization of protein structure. The identification of frataxin as an iron chaperone protein that is required for reversible modulation of aconitase activity expands on what is likely to be a complex and highly integrated set of molecular events that participate in redox regulation.

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