UPTAKE, SPECIATION, AND UNCOUPLING ACTIVITY OF SUBSTITUTED PHENOLS INENERGY-TRANSDUCING MEMBRANES

Phenolic compounds are toxic to many organisms in that they may affect the energy production in cells by inhibition of the electron transpor t or by destroying the electrochemical proton gradient built up across membranes. This latter mode of toxic action is commonly referred to a s uncoupling of oxidative phosphorylation or photophosphorylation. In this study, the relationship between uncoupling activity, total concen tration, and speciation in the photosynthetic membrane (chromatophores ) of the purple bacterium Rhodobacter sphaeroides has been evaluated f or 18 nitro- and chlorophenols covering a wide range of hydrophobicity and acidity. The uncoupling activity has been determined by time-reso lved spectroscopy and is quantified by a pseudo-first-order rate const ant, k(obs), which is a measure for the increased decay rate of the me mbrane potential in the presence of a certain amount of a given phenol . The experimental data can be described by an extended ''shuttle mech anism'' model in which it is assumed that the rate of diffusion of the phenoxide and/or a phenoxide/phenol-heterodimer species through the l ipid bilayer of the membrane determines the rate of decay of the elect rochemical proton gradient: k(obs) = k(1)C(cph)(A-) + k(2)'C-cph(A-) C -cph(HA), where C-cph(A-) and C-cph(HA) are the concentrations of the phenoxide and phenol, respectively, in the chromatophores (both estima ted from membrane-water partitioning experiments), k(1) is a measure o f the mobility of the phenoxide in the lipid bilayer; and K-2 is a lum ped parameter describing both the tendency of the compound to form a h eterodimer in the membrane as well as the mobility of this heterodimer in the lipid bilayer. To our knowledge, this is the first study in wh ich, for a given class of ionogenic organic compounds, a direct quanti tative measure of a specific toxic effect (i.e., uncoupling) has been successfully related to the actual concentration and speciation of the compounds at the target site (i.e., in the membrane). This study demo nstrates that it is possible to separate the contributions of uptake, speciation, and actual activity (expressed by k(1) and/or k(2)) to the overall uncoupling potency of a given phenol, which is necessary for the derivation of improved quantitative structure-activity relationshi ps (QSARs). Furthermore, the approach taken in this study offers the p ossibility to evaluate quantitatively synergistic and antagonistic eff ects of different phenolic compounds on energy transduction when such compounds are present in mixtures.