Effect of Ca²⁺ ions on the slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase

Slow active/inactive transition of the membrane-bound mitochondrial NADH-ubiquinone reductase (Kotlyar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys. Acta 1019, 151-158) is sensitive to Ca²⁺ and other divalent cations. Millimolar concentrations of Ca²⁺ drastically reduce the rate of the turnover-dependent activation of NADH-ubiquinone reductase. When NADH oxidase, the rotenone-sensitive NADH-ubiquinone reductase or the succinate-supported Δ_μH⁺-dependent NAD⁺ reduction were initiated by the deactivated enzyme preparations all the three activities were strongly inhibited by Ca²⁺; no sensitivity of these reactions to Ca²⁺ was observed when the assays were started by the activated enzyme preparations. The affinity of the deactivated enzyme to polyvalent cations was in the following order: Ni²⁺>Co²⁺>La³⁺>Mn²⁺>Ca²⁺≈Mg²⁺>Ba²⁺. Monovalent metal cations had no effect on the slow turnover-dependent enzyme activation. The apparent affinity of the deactivated enzyme to Ca²⁺ was strongly pH-dependent. The K_ca²⁺ values of 5.7mM and 0.6 mM at pH 7.5 and 8.5 were determined from the presteady-state kinetics parameters. The spontaneous temperature-dependent deactivation of the enzyme was insensitive to Ca²⁺. Ca²⁺ increases the reactivity of the enzyme sulfhydryl group in the deactivated preparations towards N-ethylmaleimide. This effect was also used to quantitate Ca²⁺ affinity for the enzyme. The K_ca²⁺ values of 1.2 mM and 0.4 mM at pH 8.0 and 9.0, respectively, were determined. The data obtained suggest that Ca²⁺ content in the mitochondrial matrix may play an important role in the control of NADH oxidation by the respiratory chain.