Kinetics of the Respiration of Cyanide‐Insensitive Mitochondria from the Yeast Saccharomycopsis lipolytica

Abstract

In cyanide‐insensitive mitochondria from Saccharomvcopsis lipolytica, the respiration can proceed either via the cytochrome c oxidase or via an alternative oxidase. This alternative, mitochondrial, cyanide‐insenstive respiration can be intergrated in the respiratory kinetic model of the mobile ubiquinone pool, originally designed by Kröger and Klingenberg. In the proposed kinetic model, three groups of electron carriers, a donor, catalyzing the electron transfer from the oxidation substrate to ubiquione, and two acceptors, catalyzing the electron transfer from ubiquinol to oxygen, via either the cytochrome c oxidase or the alternative oxidase, are linked together by a mobile ubiquinone pool. The donor and acceptors are charcterized by their respective activites. It is assumed, in the simplest way, that ubiquinol gives off its reducing equivalents randomly to either cytochrome or alternative acceptors. The kinetic model is expressed mathematically by a set of three equations relating the respiration rates to the activities. The proposed model, an alternative to Bahr and Bonner's kinetic model, does not necessarily require a modulation factor, that is, a variable protion of the alternative acceptors being, at times, inactive. As three respiration rates can be experimentally measured in a independent way, the set of three equations can be solved unambiguously for the donor and acceptor activities. The proposed model was experimentally assessed by kinetic analysis, after modifying the cytochrome acceptor activity by either ADP, antimycin or added cytochrome c and/or the alternative acceptor activity by either benzhydroxamic acid or submission of the mitochondria to an osmotic shock. The kinetic data fitted the proposed model and hence, more generally, provide further evidence in favor of the respiratory model of the mobile ubiquinone pool and also in favor of the branching point of the alternative respiratory pathway on the main respiratory pathway at the level of ubiquinone. That the donor and acceptor activities corresponded to their definition in the Kröger and Klingenberg model was strengthened by the observation that the stimulatory or inhibiory agents only affected the acceptor activity characterizing the group of electron carriers where the site of action of these agents is known to be located; also by the demonstration that the ratios of donor to cytochrome acceptor activities derived from the proposed model, corresponded to the same ratios computed independently from antimycin titration data; and furthermore by the fact that the cytochrome acceptor activity varied linearly with the number of inhibited sites for antimycin. The model also explains why the total respiration rate is always inferior to the mathematical sum of the respiration rates via the cytochrome c oxidase, and via the alternative oxidase, measured separately, and why upon titration by hydroxamic acids, a linear relation exists between the total respiration rate and the respiration rates via the alternative oxidase, both first observed by Bahr and Bonner.