Modulation of the kinetics and the steady-state level of intermediates of mitochondrial coupled reactions by inhibitors and uncouplers

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Abstract
In oxidative phosphorylation and ATP-driven uphill electron transfer from succinate to NAD, double-reciprocal plots of rates vs. substrate concentrations of the energy-driven reactions are a family of parallel lines at several fixed subsaturating concentrations of the substrates, or at several moderate concentrations of the inhibitors of the energy-yielding reactions. Partial uncoupling decreases the .**GRAPHIC**. and increases the .**GRAPHIC**. of the substrates of the energy-driven reactions, resulting in a decrease of Vmax/Km as a function of increased uncoupling. However, partial limitation of the flow rates of the energy-yielding reactions decreases both the .**GRAPHIC**. and the .**GRAPHIC**. of the substrates of the energy-driven reactions, resulting in no change in Vmax/Km. This is true as long as the rate limitation is moderate (e.g., < 60%), under which conditions the steady-state membrane potential (.DELTA..psi.) remains essentially unchanged. At high inhibition of the energy-yielding reactions, or at moderate inhibition in the presence of low levels of an uncoupler to cause partial uncoupling, then the family of double-reciprocal plots is no longer parallel and tends to converge toward the left. Under these conditions, steady-state .DELTA..psi. and Vmax/Km also decrease as inhibition is increased. The relationship between the magnitude of steady-state .DELTA..psi. and the rate of the energy-driven reaction was studied in oxidative phosphorylation, ATP-driven electron transfer from succinate to NAD, and respiration-driven uniport Ca transport by intact [beef heart] mitochondria. The rates of the driven reactions were modulated by partial uncoupling or partial inhibition of the energy-yielding reactions. Addition of increasing amounts of an uncoupler caused parallel decreases in the rates of the energy-driven reactions and the magnitude of .DELTA..psi.. When the rates were diminished by addition of increasing amounts of an inhibitor of the energy-yielding reactions, then the correlation between steady-state .DELTA..psi. and the rate of the energy-driven reaction depended on the nature of the latter reaction. The correlation was excellent for uniport Ca transport (a), partial for oxidative phosphorylation (b) and poor for ATP-driven uphill electron transfer (c). It was noted that in (a) the excellent correlation was between 2 bulk phase phenomena and that in (b) and (c) the more energy-demanding reaction (i.e., uphill electron transfer) showed the poorer correlation. In uncoupled submitochondrial particles the steady-state redox level of a known intermediate (cytochromes c + c1) was studied as a function of partial inhibition of electron flow, once at the dehydrogenase and a 2nd time at the oxidase ends of the respiratory chain. These experiments were also repeated in the presence of added cytochrome c to increase the pool size of the intermediate (i.e., cytochromes c + c1) up to 33-fold. In all cases, there was an excellent correlation between the rate of electron flow and the steady-state redox level of cytochromes c + c1. The above results have been discussed with regard to the premise that .DELTA.~.mu.H+ (or .DELTA..psi. in the case of the submitochondrial particles) is the sole or the principal form of protonic energy transfer.