Control of respiration by cytochrome c oxidase in intact cells: role of the membrane potential

Abstract

Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Delta psi) and chemical (Delta pH) component of the mitochondrial transmembrane proton electrochemical gradient (Delta mu(H(+))) was investigated. The overall O(2) consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Delta psi and Delta pH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (C(IV) = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (C(IV) = 0.85) was also measured in the presence of nigericin, i.e. when Delta psi is maximal, and in the presence of nigericin and valinomycin (C(IV) = 0.77), when Delta mu(H(+)) is abolished. In contrast, CcOX displayed a markedly lower control coefficient (C(IV) = 0.30) upon addition of valinomycin, when Delta psi is converted into Delta pH. These results show that Delta psi is responsible for the tight control of CcOX over respiration in actively phosphorylating cells.

FIGURE 1.

Cyanide titration of O₂ consumption by HepG2 cells: effect of valinomycin. Oxygraphic traces (thick line refers to the left axis scale) shown together with their first derivative curves, indicating the rate of oxygen consumption, (thin (stepwise) line refers to the right axis scale, 100% taken as the activity measured in the absence of the inhibitor). Endogenous respiration (A and C) and CcOX specific activity (B and D) measured in untreated (top panels) or valinomycin (VAL)-treated (bottom panels) cells in the presence of different cyanide concentrations (from a to l): 2.7 μm; 5.3 μm; 10.7 μm; 16 μm; 26.7 μm; 80 μm; 133.3 μm; 189.4 μm; 295.7 μm; and 829.4 μm. Cell density (cells·ml⁻¹): 3.3 × 10⁶ (A); 3.9 × 10⁶ (B) and 2.3 × 10⁶ (C and D). CTR, control.

FIGURE 2.

Cyanide titration profiles. Cyanide titration profiles of global (open circle) and CcOX-specific (closed circle) activity, as measured in intact HepG2 cells under the following conditions: control (CTR), n = 8 (A); valinomycin (VAL), n = 9 (B); nigericin (NIG), n = 9 (C); valinomycin and nigericin, n = 7 (D). Data points ± S.D. Insets show data in the low cyanide concentration range, together with the linear regression analysis.

FIGURE 3.

Threshold plots. Threshold plots of the data shown in Fig. 2. Control (1, closed circles); valinomycin (2, open squares); nigericin (3, open circles); valinomycin and nigericin (4, open triangles). Data points ± S.D.