Figure 4.

Kinetic parameters for FET and RET by bovine complex I in proteoliposomes. (A) Dependence of the rates of NADH oxidation (FET, blue) and NAD⁺ reduction (RET, red) on Q₁₀ concentration in the membrane (1 mM Q₁₀ (membrane) = 1 nmol Q₁₀ per mg of phospholipid). RET activities were recorded with 100 μg mL^–1 GlpD. The V_max values are 24.4 ± 0.85 and 0.50 ± 0.04 μmol min^–1 (mg CI_out)⁻¹ (± S.E. of the fit). (B) Estimation of the level of Q-pool reduction for proteoliposomes reduced by NADH or catalyzing RET. For NADH measurements, no AOX was present and the Q-pool in 5 μg mL^–1 CI_out proteoliposomes was reduced by addition of 200 μM NADH. For RET measurements, GlpD was present at 100 μg mL^–1 and RET was initiated as described in Experimental Procedures before being quenched at the time specified. (C) Dependence of the rate of NAD⁺ reduction on NAD⁺ concentration. RET was measured under standard conditions with proteoliposomes reconstituted with 10 nmol Q₁₀ (mg lipid)⁻¹. The V_max value was 0.36 ± 0.01 μmol min^–1 (mg CI_out)⁻¹. (D) Dependence of the rates of NADH oxidation (FET) and NAD⁺ reduction (RET) on piericidin A concentration. All measurements performed with 2 μg mL^–1 CI_out. See Experimental Procedures for standard assay conditions. All data are mean averages with error (± S.D.) values from triplicate technical replicates (including propagated error from underlying measurements in (A)).