Uptake, speciation, and uncoupling activity of substituted phenols in energy transducing membranes

Phenolic compounds are toxic to many organisms in that they may affect the energy production in cells by inhibition of the electron transport or by destroying the electrochemical proton gradient built up across membranes. This latter mode of toxic action is commonly referred to as uncoupling of oxidative phosphorylation or photophosphorylation. In this study, the relationship between uncoupling activity, total concentration, and speciation in the photosynthetic membrane (chromatophores) of the purple bacterium Rhodobacter sphaeroides has been evaluated for 18 nitro- and chlorophenols covering a wide range of hydrophobicity and acidity. The uncoupling activity has been determined by time-resolved spectroscopy and is quantified by a pseudo-first-order rate constant, k(obs), which is a measure for the increased decay rate of the membrane potential in the presence of a certain amount of a given phenol. The experimental data can be described by an extended ''shuttle mechanism'' model in which it is assumed that the rate of diffusion of the phenoxide and/or a phenoxide/phenol-heterodimer species through the lipid bilayer of the membrane determines the rate of decay of the electrochemical 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 estimated from membrane-water partitioning experiments), k(1) is a measure of the mobility of the phenoxide in the lipid bilayer; and K-2 is a lumped parameter describing both the tendency of the compound to form a heterodimer in the membrane as well as the mobility of this heterodimer in the lipid bilayer. To our knowledge, this is the first study in which, for a given class of ionogenic organic compounds, a direct quantitative 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 demonstrates 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 relationships (QSARs). Furthermore, the approach taken in this study offers the possibility to evaluate quantitatively synergistic and antagonistic effects of different phenolic compounds on energy transduction when such compounds are present in mixtures

Purification and characterization of a two-component monooxygenase that hydroxylates nitrilotriacetate from "Chelatobacter" strain ATCC 29600.

An assay based on the consumption of nitrilotriacetate (NTA) was developed to measure the activity of NTA monooxygenase (NTA-Mo) in cell extracts of "Chelatobacter" strain ATCC 29600 and to purify a functional, NTA-hydroxylating enzyme complex. The complex consisted of two components that easily dissociated during purification and upon dilution. Both components were purified to more than 95% homogeneity, and it was possible to reconstitute the functional, NTA-hydroxylating enzyme complex from pure component A (cA) and component B (cB). cB exhibited NTA-stimulated NADH oxidation but was unable to hydroxylate NTA. It had a native molecular mass of 88 kDa and contained flavin mononucleotide (FMN). cA had a native molecular mass of 99 kDa. No catalytic activity has yet been shown for cA alone. Under unfavorable conditions, NADH oxidation was partly or completely uncoupled from hydroxylation, resulting in the formation of H2O2. Optimum hydroxylating activity was found to be dependent on the molar ratio of the two components, the absolute concentration of the enzyme complex, and the presence of FMN. Uncoupling of the reaction was favored in the presence of high salt concentrations and in the presence of flavin adenine dinucleotide. The NTA-Mo complex was sensitive to sulfhydryl reagents, but inhibition was reversible by addition of excess dithiothreitol. The Km values for Mg(2+)-NTA, FMN, and NADH were determined as 0.5 mM, 1.3 microM, and 0.35 mM, respectively. Of 26 tested compounds, NTA was the only substrate for NTA-Mo