REDOX STATES OF RESPIRATORY-CHAIN COMPONENTS IN RAT-LIVER MITOCHONDRIA .I. EFFECT OF VARYING SUBSTRATE CONCENTRATION AND OF AZIDE

1. Increasing amounts of substrate cause an increasing degree of reduction of nicotinamide nucleotides, cytochrome b and cytochrome c in rat-liver mitochondria in the presence of ADP (State 3). In State 4 (after consumption of ADP), however, the redox state is only slightly changed by varying the substrate concentration in the same range. 2. Increasing concentrations of azide cause increasing reduction of cytochromes a, b and c in State 3, but have no effect on the redox state in State 4, except at high concentration. 3. Oligomycin has no effect on the degree of reduction of NAD in State 4 with succinate, and only slightly increases it with NAD-linked substrates. 4. The effect of uncoupling on the redox state of the respiratory chain is best studied under conditions in which uncoupling and competition between uncoupler and substrate do not cause the redox state to move in the same direction. This is conveniently achieved by adding azide. Under these conditions, uncoupling leads to an increased reduction of cytochrome c, whereas competition for penetration of substrate leads to an increased oxidation. 5. It is concluded that the redox state of an electron carrier in State 3 represents a kinetic steady state governed by the relative activities of those portions of the chain responsible for reduction and oxidation of the carrier. In State 4, however, the respiratory chain approaches thermodynamic equilibrium with ADP, ATP and Pi. © 1969.