Toxicity of peroxynitrite and related reactive nitrogen species toward Escherichia coli.

The toxicity of peroxynitrite toward Escherichia coli (expressed as LD50, the concentration required to kill 50% of the bacteria) was found to be independent of bacterial cell densities over a wide experimental range, spanning 10(6)-10(10) colony-forming units/mL; the magnitude of LD50 was also pH-independent over the range pH 5.9-8.3. This highly unusual behavior can be quantitatively reproduced by a dynamical model in which (i) ONO2H is identified as the toxic form of the oxidant and (ii) the bulk of the added peroxynitrite decays to nitrate ion under these conditions. From the model, one estimates that 10(6)-10(7) ONO2H molecules are required to kill a bacterium, indicating a very high intrinsic toxicity (cf. HOCl, for which LD50 = 10(7)-10(8) molecules/cell of E. coli). Nearly complete protection was observed when bicarbonate ion was added to the buffer, even when concentrations of peroxynitrite exceeded 50 times the LD50 measured in the absence of bicarbonate. Consistent with previous reports, combinations of H2O2 and NO and, in weakly acidic media, H2O2 and NO2- were found to exhibit enhanced toxicities relative to the individual reactants. Protection by bicarbonate was utilized to assess the potential role of intermediary formation of ONO2H in bacterial killing in these systems. Approximately 25% protection by bicarbonate was observed for media containing H2O2 and NO2-, consistent with a minor contribution to killing by ONO2H under the experimental conditions. No protection was observed for media containing H2O2 and *NO in both anaerobic and aerobic environments, excluding extracellularly generated ONO2H as a participant in these bactericidal reactions.

Mechanism of carbon dioxide-catalyzed oxidation of tyrosine by peroxynitrite.

Peroxynitrite ion (ONO2-) reacted rapidly with CO2 to form a short-lived intermediate provisionally identified as the ONO2CO2- adduct. This adduct was more reactive in tyrosine oxidation than ONO2- itself and produced 3-nitrotyrosine and 3,3'-dityrosine as the major oxidation products. With tyrosine in excess, the rate of 3-nitrotyrosine formation was independent of the tyrosine concentration and was determined by the rate of formation of the ONO2CO2- adduct. The overall yield of oxidation products was also independent of the concentration of tyrosine and medium acidity; approximately 19% of the added ONO2- was converted to products under all reaction conditions. However, the 3-nitrotyrosine/3,3'-dityrosine product ratio depended upon the pH, tyrosine concentration, and absolute reaction rate. These data are in quantitative agreement with a reaction mechanism in which the one-electron oxidation of tyrosine by ONO2CO2- generates tyrosyl and NO2 radicals as intermediary species, but are inconsistent with mechanisms that invoke direct electrophilic attack on the tyrosine aromatic ring by the adduct. Based upon its reactivity characteristics, ONO2CO2- has a lifetime shorter than 3 ms and a redox potential in excess of 1 V, and oxidizes tyrosine with a bimolecular rate constant greater than 2 x 10(5) M-1 s-1. In comparison, in CO2-free solutions, oxidation of tyrosine by peroxynitrite was much slower and gave significantly lower yields (approximately 8%) of the same products. When tyrosine was the limiting reactant, 3,5-dinitrotyrosine was found among the reaction products of the CO2-catalyzed reaction, but this compound was not detected in the uncatalyzed reaction.

Role of compartmentation in promoting toxicity of leukocyte-generated strong oxidants.

A rigorous mathematical model is developed to describe the distribution of respiration-generated oxidants among reactive sites within the phagolysosomes of leukocytic cells. Reaction parameters include the diffusion coefficient of the oxidant, the intrinsic rate constants for its reaction with the phagosomal membrane and the cell envelopes of entrapped bacteria, the overall rate constant for its reaction with solution components of the phagosomal fluid, and the phagosomal dimensions. The model is used to describe the dynamics of randomly generated .OH and HCO3. radicals within the phagosome. These radicals were chosen because the necessary rate parameters either have been measured or could be reasonably estimated. The calculations show that .OH radical cannot be an effective bactericide unless generated in the immediate vicinity of the bacterial surface because its extreme reactivity precludes any significant diffusion. The HCO3. radical, however, is predicted to be a very effective toxin, even when relatively high concentrations of oxidant scavengers are present in the phagosomal fluid. In the absence of complicating features, the reactivity patterns of other less reactive oxidants (e.g., metal oxo or peroxo complex ions) are predicted to be very similar, although quantitative analysis is precluded by the lack of relevant rate data. For these oxidants, the predicted intraphagosomal toxicities differ markedly from toxicities measured in dilute bacterial suspensions because differences in mean diffusion lengths of the oxidants are unimportant in environments with the dimensions of phagosomes, but very important under in vitro conditions. The model is general and can be applied to other reactions occurring in similar microheterogeneous cellular and subcellular environments.