Determining and understanding the control of flux - An illustration in submitochondrial particles of how to validate schemes of metabolic control

Two complementary methods were used to determine how the rate of respiratio n and that of ATP hydrolysis were controlled in rat liver submitochondrial particles. In the first, 'direct control analysis' method, respiration was titrated with malonate, antimycin or cyanide at 20, 30 and 37 degrees C, to determine the flux control exerted by succinate dehydrogenase, cytochrome bc(1) complex and cytochrome c oxidase, respectively. Together, the three r espiratory complexes only controlled the flux by about 50%, leaving the oth er 50% of flux control to the H+ leak. In the second, 'elasticity based' me thod, the elasticity coefficients of the respiratory chain or the H+-ATPase and the H+ leak towards the H+ gradient were determined. Then, the flux co ntrol coefficients were calculated using the connectivity and summation law s of metabolic control theory. The correspondence between the flux control coefficients determined in the two ways validated the two methods. This all owed us to use the second method to analyse what was the kinetic origin of the observed distribution of control. Control of ATP hydrolysis by the ATPa se decreased with increasing ATPase activity; hence, the control exerted by the H+ leak increased with increasing ATPase activity, due to a diminishin g elasticity towards the H+ gradient. Reverse electron transport was mainly controlled by the ATPase; the sum of flux control coefficients of succinat e dehydrogenase, NADH-CoQ oxidoreductase, and H+-ATPase yielded a value gre ater than one, indicating that the H+ leak exerted a significant negative c ontrol on this pathway.