A CRITIQUE OF THE USE OF INHIBITORS TO ESTIMATE PARTITIONING OF ELECTRONS BETWEEN MITOCHONDRIAL RESPIRATORY PATHWAYS IN PLANTS

The contribution of individual plant mitochondrial respiratory pathway s to total respiration is commonly assessed by titration with specific inhibitors of different components in the branched electron transport chain. A pathway's contribution is equal to the activity when the oth er branch is blocked by an inhibitor multiplied by the degree (0-1.0) to which this activity is engaged when both pathways are operating. Ac cording to Bahr and Bonner (1973. J. Biol. Chem. 218: 3441-3445) the p lot of the activities of identical titrations, one performed in the ab sence and the other in the presence of a specific inhibitor of the oth er branch of the respiratory chain, yields a straight line whose slope indicates the engagement of the titrated pathway during uninhibited r espiration. An initial slope of zero may occur if electron flux is div erted between pathways during titrations. However, beyond the breakpoi nt (representing the point of pathway saturation), a straight line is obtained with a slope representing engagement. This technique assumes that the kinetics of inhibiting a specific component of the respirator y chain are independent of the absolute rate of electron flux through the total pathway. To test this assumption, the activity of respirator y pathways in isolated soy bean (Glycine max [L]. Merr. cv. Stevens) m itochondria was titrated with specific inhibitors of the cytochrome an d alternative oxidases. Under these conditions, the electron flux thro ugh a given pathway was manipulated by poising the rate of succinate o xidation with the succinate dehydrogenase inhibitor malonate. Construc tion of activity plots in the presence versus absence of malonate fail ed to result in straight lines for either KCN (when titrating the cyto chrome pathway) or salicylhydroxamic acid (when titrating the alternat ive pathway). Rather, the resultant plots were always curvilinear when ever the activity in the presence of malonate divided by the activity in the absence of malonate was less than 1.0. In no case could the rea l engagement of the pathway be precisely estimated from the titration data. Titrations of cytochrome pathway activity in isolated potato tub er (Solanum tuberosum L. cv. Sabago and Canabex) mitochondria (which l ack the alternative oxidase) showed that as the inhibitor concentratio n was increased, so did the reduction status of the ubiquinone pool, t o a new steady state. The dependence of inhibition kinetics on the rat e of flux through the pathway, and the increase in ubiquinone pool red uction upon KCN addition, are explained in terms of the elasticity of component enzymes as outlined in the theory of metabolic control analy sis. The implications of this finding for the use of titrations to est imate engagement of plant respiratory pathways are discussed.