ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 273, No. 1, August 15, pp. 14%15’7,1989 Regulation of Alternative Pathway Activity in Plant Mitochondria: Nonlinear Relationship between Electron Flux and the Redox Poise of the Quinone Pool IAN B. DRY,*,’ ANTHONY L. MOORE,? DAVID A. DAY,* AND JOSEPH T. WISKICH* *Department of Botany, University of Adelaide, Adelaide, South Australia 5001, Australia; ~Department of Biochemistry, University of Sussa, Falwwr, Brighton BNl9QG, United Kingdom; and $Department of Botany, Australian National University, Canberra, Australian Capital Terrikny 2601,Australia Received December 30,1988, and in revised form April 3,1989 The dependence of respiratory flux via the alternative pathway on the redox poise of the ubiquinone (Q) pool was investigated in soybean cotyledon mitochondria. A marked nonlinear relationship was observed between Q-pool reduction level and O2 uptake via the alternative oxidase. Significant engagement of the alternative pathway was not ap- parent until Q-pool reduction level reached 35-40% but increased disproportionately on further reduction. Similar results were obtained with electron donation from either Complex 1 or Complex 2. Close agreement was obtained over a range of experimental conditions between the estimated contribution of the alternative pathway to total respi- ratory flux, as measured with salicylhydroxamic acid, and that predicted from the redox poise of the Q-pool. These results are discussed in terms of existing models of the regula- tion of respiratory flux via the alternative pathway. Q 1989 Academic Press, lne. Mitochondria from many plant species possess a branched respiratory electron transport chain with two terminal oxi- dases: cytochrome oxidase and the cya- nide-resistant alternative oxidase (1). The alternative pathway branches from the main respiratory chain at the level of ubi- quinone (Q)’ which acts as a mobile elec- tron carrier linking Complex 1, succinate dehydrogenase (Complex II), and the ex- ternal NADH dehydrogenase to both the cytochrome be1 complex and the alterna- tive oxidase (2-4). Thus, the partitioning of electron flux between these two pathways will be a function of the relative kinetic 1 To whom correspondence should be addressed. ’ Abbreviations used: BSA, bovine serum albumin; PVP, polyvinylpyrrolidone; Q, ubiquinone; Q-l, ubi- quinone 5; Q-Z, ubiquinone 10; Qt, total redox-active ubiquinone; Qr, ubiquinone reduced under steady- state conditions; SCM, soybean cotyledon mitochon- dria; SHAM, salicylhydroxamic acid; Tes, N-tris(hy- droxymethyl)-2-aminoethanesulfonic acid. and thermodynamic behavior of the cyto- chrome bcl complex and the alternative ox- idase, with respect to the oxidation of re- duced ubiquinone. Kroger and Klingenberg (5,6) have pre- viously shown the oxidation of reduced ubiquinone by cytochrome bc, complex, in bovine heart mitochondria, to be a first-or- der process with respect to the substrate; i.e., the rate of oxygen uptake was directly proportional to the amount of the total quinone pool in the reduced state. Using a new technique for the measurement of the redox state of the Q-pool in situ, we have recently been able to demonstrate that a similar linear relationship exists between the level of reduced Q and electron flux in certain plant mitochondria (7). However, there was some evidence of a deviation from linearity in pea leaf mitochondria under state 4 conditions, possibly due to the engagement of alternative pathway activity. Bahr and Bonner (8, 9) have previously shown alternative pathway activity to be 0003-9861/89 $3.00 Copyright0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved. 148