Tracking of proton flow during transition from anaerobiosis to steady state in rat liver mitochondria

Abstract

(1) The hydrophobic pH indicator Bromthymol blue and the hydrophilic pH indicator Phenol red have been used to follow the redox-pump-linked proton flows during transition from anaerobiosis to static head. The domains monitored by the pH indicators, whether external or internal, and the localization of the dye, whether free or membrane bound, have been identified by recording the absorbance changes following addition of nigericin or valinomycin to anaerobic or aerobic mitochondria and the effects of permeant and impermeant buffers. (2) After addition of the H +/K + exchanger, nigericin, to anaerobic mitochondria, Phenol red and Bromthymol blue record an alkalinization and an acidification, respectively, indicating that while the hydrophilic pH indicator faces an external domain, the hydrophobic pH indicator faces, at least partly, an internal domain. The latter effect is sensitive to phosphate and to phosphate carrier inhibitors. On the other hand, addition of nigericin to aerobic mitochondria leads to an increased Bromthymol blue absorbance, which reflects an alkalinization, indicating that the pH indicator faces an external domain. The reorientation of the dye from the internal to the external domain is a function of the uncoupler concentration and thus of the membrane potential (cf. Mitchell et al. (1968) Eur. J. Biochem. 4, 9–19). (3) The amount of oxygen required for the transition from anaerobiosis to static head has been determined by following in parallel the extent of oxidation of cytochrome aa 3 and the rise of Δ \\ ̃ gm H + . With succinate as substrate, 50% levels of cytochrome oxidation are obtained at 0.125 ngatom oxygen/mg and 50% of Safranine response at about 0.2 ngatom oxygen/mg. These amounts of oxygen correspond to an H + displacement of about 0.8–1.2 ngatom/mg on the basis of the H +/O stoichiometry. It is concluded that mitochondria are in presteady state below, and in static head above, displacement of 2–3 ngatom H +/mg. This figure is very close to the original calculation of Mitchell (Mitchell, P. (1966) Biol. Rev. 41, 445–502). (4) Transition, by oxygen pulses, of EGTA-supplemented mitochondria from anaerobiosis to either presteady state or static head state results in a response of the hydrophilic pH indicator, Phenol red, which is negligible in amount and/or kinetically unrelated to the Δ \\ ̃ gm H + rise. The fact that H + extrusion in the bulk aqueous phase is negligible also in presteady state excludes proton cycling as an explanation. Addition of oxygen pulses to Sr 2+-supplemented anaerobic mitochondria results in an H + extrusion whose amount and rate is proportional to the Sr 2+ concentration. The increase in rate and amount of the H + extrusion is accompanied by a proportional depression of Δ \\ ̃ gm H + . (5) Transition, by oxygen pulses, of EGTA- and Mg 2+-supplemented mitochondria from anaerobiosis to static head state results in a decreased absorbance of Bromthymol blue. The decrease in absorbance is insensitive to impermeant buffers but partially sensitive to phosphate and the effect of phosphate is abolished by N-ethylmaleimide. The decrease in absorbance is proportional to the amount of oxygen in the range between zero and 0.6 ngatom O/mg and fully sensitive to the addition of protonophores. (6) It appears that hydrophilic and hydrophobic pH indicators may be used to record pH changes occurring at the two sides of the hydrophobic barrier and at the membrane level. It is further proposed that the redox pump-linked proton extrusion takes place at the membrane level.