(497f) A Kinetic Platform to Determine the Fate of Nitric Oxide in Bacteria

Nitric oxide (NO•) is generated by cells of the innate immune response (e.g., macrophages) to neutralize pathogens. NO• has an extensive biochemical reaction network that includes reactions with iron-sulfur clusters, DNA, thiols, tyrosine residues, and hemes. The fate of NO• inside a pathogen depends on the kinetic competition among its many targets. This competition is governed by microbial parameters such as intracellular metabolite and enzyme concentrations, and is of critical importance as it determines the fitness of an organism in NO•-stressed environments. Many NO• reaction products are difficult or impossible to directly measure (e.g., NO₂•, N₂O₃, ONOO⁻), and their terminal pathway products are non-unique (e.g., NO₂⁻, NO₃⁻, N₂O). Therefore, dynamic kinetic models have been developed to predict concentrations of NO• and its products in aqueous solutions and physiological environments, such as inflamed tissue.^1,2 These efforts have laid a foundation for modeling NO• reaction networks, but have focused primarily on non-enzymatic, small-molecule reactions and not addressed the fundamental question of how NO• distributes within microbes. Here, we have constructed a comprehensive kinetic model that unites and extends existing treatments of NO• chemistry to encompass the broad reactivity of NO• in E. coli. The incorporation of spontaneous and enzymatic reactions, as well as damage and repair of biomolecules, allows for a detailed analysis of intracellular NO• distribution and its dependence on various microbial parameters. Interestingly, a parametric analysis of our dynamic model identified a dependency in the utility of NO• dioxygenase (Hmp) on the rate of NO• delivery to the cell. Hmp is the major enzyme responsible for aerobic NO• detoxification in E. coli, but when NO• is delivered at a rapid rate, even for short periods of time, the distribution and removal of NO• become unaffected by the presence of Hmp. This observation was experimentally confirmed, as an Δhmp mutant exhibited wild-type sensitivity toward rapidly delivered NO•, whereas a considerable increase in sensitivity for Δhmp compared to wild-type was observed at slower delivery rates. Our model serves as a platform to predict the fate of NO• within bacteria and offers a framework to identify novel NO•-potentiating therapeutics.

1. Lancaster, J. R., Jr. Chem. Res. Toxicol. 2006.

2. Lim, C. H. et al. Chem. Res. Toxicol. 2008.