Redox-dependent change of nucleotide affinity to the active site of the mammalian complex I

Abstract

A very potent and specific inhibitor of mitochondrial NADH:ubiquinone oxidoreductase (complex I), a derivative of NADH (NADH-OH) has recently been discovered (Kotlyar, A. B., Karliner, J. S., and Cecchini, G. (2005) FEBS Lett. 579, 4861-4866). Here we present a quantitative analysis of the interaction of NADH-OH and other nucleotides with oxidized and reduced complex I in tightly coupled submitochondrial particles. Both the rate of the NADH-OH binding and its affinity to complex I are strongly decreased in the presence of succinate. The effect of succinate is completely reversed by rotenone, antimycin A, and uncoupler. The relative affinity of ADP-ribose, a competitive inhibitor of NADH oxidation, is also shown to be significantly affected by enzyme reduction (KD of 30 and 500 microM for oxidized and the succinate-reduced enzyme, respectively). Binding of NADH-OH is shown to abolish the succinate-supported superoxide generation by complex I. Gradual inhibition of the rotenone-sensitive uncoupled NADH oxidase and the reverse electron transfer activities by NADH-OH yield the same final titration point (approximately 0.1 nmol/mg of protein). The titration of NADH oxidase appears as a straight line, whereas the titration of the reverse reaction appears as a convex curve. Possible models to explain the different titration patterns for the forward and reverse reactions are briefly discussed.

Figure 1

Inhibition of the NADH oxidase activity by NADH-OH. (A) SMPs (5 μg of protein/mL, about 0.5 nM complex I) were incubated with 6 (■), 12 (○), and 24 (●, □) nM NADH-OH for the time indicated on the abscissa in the standard reaction mixture (0.25 M sucrose, 50 mM Tris/Cl⁻, 0.2 mM EDTA, pH 8.0). The residual NADH oxidase activity was initiated by the addition of 0.1 mM NADH and gramicidin D (0.05 μg/mL). Potassium succinate (2.5 mM) was present during incubation with NADH-OH in the sample marked as □ (reduced complex I), and 2.5 mM potassium malonate was present in the NADH oxidase assay. The continuous lines are the computer-generated curves for the simple bimolecular irreversible enzyme–inhibitor interaction with the second-order rate constant of 1.6 × 10⁸ (●, ○, ■) and 1.5 × 10⁷ (□, reduced complex I) M⁻¹ min⁻¹. (B) Pseudo-first-order rate logarithmic anamorphosis of the data shown in (A). The inset shows the linear dependence of the apparent first-order rate constant on the concentration of NADH-OH.

Figure 2

Rapid irreversible decomposition of NADH-OH by HAR and the resistance of the enzyme-bound inhibitor to the electron acceptor. Actual tracings of the NADH:HAR reductase activities after preincubation with NADH-OH and HAR are shown. (Curve 1) SMPs (25 μg/mL) were incubated for 1 min in the standard reaction mixture containing 0.5 mM HAR, and the reaction was initiated by the addition of 100 μM NADH and 5 μM rotenone as indicated. (Curve 2) NADH-OH (28 nM) was preincubated in the standard mixture containing 0.5 mM HAR for 1 min, 25 μg/mL of SMPs were then added following 1 min of incubation, and the reaction was initiated by the addition of NADH and rotenone. (Curve 3) SMPs (25 μg/mL) were incubated for 1 min with 28 nM NADH-OH, 0.5 mM HAR was added following 1 min of incubation, and the reaction was initiated by the addition of NADH and rotenone. (Curve 4) Same as curve 3. The reaction was initiated by simultaneous addition of NADH, HAR, and rotenone.

Figure 3

Dissociation rates of the enzyme–inhibitor complex. (A) (Lines 1 and 2 (controls, ●, ■)) SMPs (25 μg/mL) were incubated in the standard reaction mixture containing either 0.5 mM HAR (■) or 5 mM succinate and 150 μM NADH (●) for the time indicated on the abscissa, and NADH oxidation was initiated by the addition of 100 μM NADH and 5 μM rotenone (■) or 5 mM potassium malonate and gramicidin D (0.05 μg/mL) (●). (Curve 3 (○)) SMPs (1 mg/mL) preincubated with 100 nM NADH-OH for 15 min at room temperature were added at zero time to the reaction mixture (final concentration of 25 μg/mL) and incubated and assayed as described for line 1 (●). (Curve 4 (□)) SMPs (1 mg/mL) preincubated with 100 nM NADH-OH for 15 min at room temperature were added at zero time to the reaction mixture (final concentration of 25 μg/mL) and incubated and assayed as described for line 2 (■). (B) Logarithmic anamorphosis of curves 3 and 4. The activities shown in (A, lines 1 and 2) were taken as v_∞ to construct lines 3 and 4, corresponding to the first-order rate constants of 0.05 min⁻¹ (oxidized enzyme) and 0.09 min⁻¹ (succinate-reduced enzyme). Activity of 100% in (A) corresponds to the specific activities of 0.8 (μmol/min)/mg (●) and 2.4 (μmol/min)/mg (■).

Figure 4

Effect of ADP-ribose on irreversible inhibition of complex I by NADH-OH. (A) SMPs (25 μg/mL) were incubated for 1 min in the standard reaction mixture containing 28 nM NADH-OH and ADP-ribose (concentrations are indicated on the abscissa). The residual NADH oxidase activities (100 μM NADH and 0.05 μg/mL gramicidin D) were determined. The pseudo-first-order rate constants for NADH-OH-induced inhibition were calculated as k_obs = ln(v₀/v _t) and plotted as a function of ADP-ribose concentration. v₀ corresponds to the rate of NADH oxidation in the absence of NADH-OH, and v_t is the residual activity after 1 min of incubation with NADH-OH. (B) Secondary plot of 1/k_obs versus ADP-ribose concentration used to determine K_D for ADP-ribose.

Figure 5

Inhibition of the complex I-mediated superoxide generation by tightly coupled submitochondrial particles. Superoxide generation (superoxide dismutase-sensitive acetylated cytochrome c reduction) was measured as described in the Materials and Methods. SMPs (1 mg/mL) were preincubated in the standard mixture for 15 min with or without 100 nM NADH-OH (I), and the rates of succinate-supported or NADH (50 μM)-supported superoxide (in the presence of 5 μM rotenone) generation were measured in the standard assay mixture containing 0.1 M potassium phosphate (pH 8.0) and 20 μM acetylated cytochrome c.

Figure 6

Titration curves for inhibition of uncoupled NADH oxidation and succinate-supported aerobic reverse electron transfer by NADH-OH. SMPs were preincubated in the standard reaction mixture with the indicated concentrations of NADH-OH for 15 min. The residual NADH oxidase (●, 100 μM NADH and 0.05 μg/mL gramicidin D) and reverse electron transfer (■, 1 mM NAD⁺ and 5 mM potassium succinate) activities were determined. The final concentration of SMPs in the assay mixture was 25 μg/mL. The SMP content in the preincubation mixture was 2 mg/mL. An activity of 100% corresponds to the specific activity (initial rate) of 0.8 and 0.1 (μmol/min)/mg of protein for NADH oxidase and reverse electron transfer, respectively. Longer preincubation of SMPs with NADH-OH did not change the titration curves.