Inhibitory effect of palmitate on the mitochondrial NADH:ubiquinone oxidoreductase (complex I) as related to the active-de-active enzyme transition

Abstract

Palmitate rapidly and reversibly inhibits the uncoupled NADH oxidase activity catalysed by activated complex I in inside-out bovine heart submitochondrial particles (IC50 extrapolated to zero enzyme concentration is equal to 9 microM at 25 degrees C, pH 8.0). The NADH:hexa-ammineruthenium reductase activity of complex I is insensitive to palmitate. Partial (approximately 50%) inhibition of the NADH:external quinone reductase activity is seen at saturating palmitate concentration and the residual activity is fully sensitive to piericidin. The uncoupled succinate oxidase activity is considerably less sensitive to palmitate. Only a slight stimulation of tightly coupled respiration with NADH as the substrate is seen at optimal palmitate concentrations, whereas complete relief of the respiratory control is observed with succinate as the substrate. Palmitate prevents the turnover-induced activation of the de-activated complex I (IC50 extrapolated to zero enzyme concentration is equal to 3 microM at 25 degrees C, pH 8.0). The mode of action of palmitate on the NADH oxidase is qualitatively temperature-dependent. Rapid and reversible inhibition of the complex I catalytic activity and its de-active to active state transition are seen at 25 degrees C, whereas the time-dependent irreversible inactivation of the NADH oxidase proceeds at 37 degrees C. Palmitate drastically increases the rate of spontaneous de-activation of complex I in the absence of NADH. Taken together, these results suggest that free fatty acids act as specific complex I-directed inhibitors; at a physiologically relevant temperature (37 degrees C), their inhibitory effects on mitochondrial NADH oxidation is due to perturbation of the pseudo-reversible active-de-active complex I transition.

Figure 1. Effect of palmitate on the catalytic activities of SMP

(A) Actual tracing of the NADH oxidase activity catalysed by pre-activated SMP (SMP_A, 10 μg/ml) at 25 °C. Gramicidin D (0.05 μg/ml) was present in the standard assay mixture containing 0.5 mM NADH. Palmitate (15 μM) and BSA (2 mg/ml) were added where indicated. (B) Inhibition of the NADH oxidase (curve 1, ●), NADH:hexa-ammineruthenium reductase (curve 2, ■), NADH:Q₁ reductase (curve 3, ○) and succinate oxidase (curve 4, □) activities by palmitate. Gramicidin (0.05 μg/ml) was present in all the assays. Specific activity of 100% corresponds to 1.0, 1.1, 0.33 and 0.65 μmol of the substrate oxidized·min⁻¹·(mg of protein)⁻¹ for NADH oxidase, NADH:hexa-ammineruthenium reductase, NADH:Q₁ reductase and succinate oxidase respectively. The residual, palmitate-insensitive NADH:Q₁ reductase activity [0.16 μmol·min⁻¹·(mg of protein)⁻¹] was decreased down to <5% by piericidin (10 nmol/mg of protein). Coupled (●) and uncoupled (○, 0.05 μg/ml gramicidin was present) NADH (C) and succinate oxidase activities (D) were measured.

Figure 2. Effect of palmitate on the de-activated NADH oxidase at 25 °C

(A) Time course of NADH oxidation catalysed by de-activated and activated particles [SMP_D (curve 1) and SMP_A (curve 2) respectively]. The reaction was started by the addition of SMP (10 μg/ml) to the standard mixture containing 0.1 mM NADH and gramicidin D (0.05 μg/ml). BSA did not influence the lag-phase in NADH oxidation catalysed by SMP_D (results not shown). (B) Effect of palmitate on time course of NADH oxidation catalysed by de-activated particles. The experimental conditions were the same as in (A) except for a different time-sensitivity resolution. Palmitate (15 μM) was present (curves 2 and 3) and BSA (2 mg/ml) was added (curve 3) where indicated.

Figure 3. Effect of palmitate on the turnover-dependent activation of NADH oxidase

The time course of NADH oxidation catalysed by de-activated particles was followed as shown in Figure 2 in the presence of different palmitate concentrations. The apparent first-order rate constants for the time-dependent increase of the catalytic activity traced at better time resolution were calculated from the linear log[v_t→∞/(v_t→∞−v_t)]−t dependence, where v_t and v_t→∞ are the reaction rates measured at time t and those measured for SMP_A in the presence of a given palmitate concentration respectively.

Figure 4. Relative inhibitory efficiency of palmitate on the catalytic activity (line 1) and on the activation rate (line 2) of NADH oxidase at 25 °C

Line 1, half-maximal inhibitory concentrations of palmitate on the rate of NADH oxidation were determined as depicted in Figure 1(B, curve 1) at different protein concentrations in the assay mixture. Line 2, the concentrations of palmitate required to decrease the rate constant k_a by 50% as described in Figure 2 were determined at different protein concentrations. The values of IC₅₀ extrapolated to zero enzyme concentration were 9 and 3 μM for lines 1 and 2 respectively.

Figure 5. Time-dependent inactivation of NADH oxidase by palmitate at different temperatures

(A) Actual tracing of NADH oxidation at 37 °C. The reaction was initiated by the addition of NADH (0.5 mM) and gramicidin D (0.05 μg/ml) to SMP (10 μg/ml) in the standard reaction mixture. Palmitate (15 μM) and BSA (2 mg/ml) were added at the time indicated by arrows. (B) Time course of the palmitate-induced inhibition at different temperatures measured as shown in (A). The activity at zero time (∼45% of that measured in the absence of palmitate) corresponds to instant inhibition caused by the addition of fatty acid. No albumin was present in the assay mixture.

Figure 6. Inhibitory effect of palmitate on inside-out SMP and right-side-out permeabilized mitochondria

(A) Rat heart mitochondria (10 μg/ml) were added to the standard assay mixture at 25 °C containing alamethicin (20 μg/ml) and 1 mM MgCl₂. After 1 min preincubation, 2 mM EDTA was added and the reaction was initiated by the addition of 0.1 mM NADH. (B) SMP_A (10 μg/ml) were added to the standard assay mixture at 25 °C and the reaction was started by the addition of 0.1 mM NADH and 0.05 μg/ml gramicidin D.

Figure 7. Effect of palmitate on the spontaneous thermal de-activation of NADH oxidase

SMP_A (100 μg/ml) were incubated at 37 °C in 0.25 mM sucrose, 50 mM Tris/HCl (pH 8.0) and 0.2 mM EDTA in the presence or absence of 65 μM palmitate (the concentration which inhibits NADH oxidase activity by 70% at 25 °C at this protein content). The samples were diluted 10 times in the standard assay mixture (25 °C) containing BSA (2 mg/ml) to remove palmitate (see Figure 1A), 1 mM N-ethylmaleimide was added to inhibit the de-activated enzyme irreversibly [14,17] and after 1 min incubation the residual activity initiated by the addition of 0.1 mM NADH was determined.