How Difficult Azide ion / Hydrazoic Acid Passes Biological Membranes: Experimental and Computational Study

Abstract Hydrazoic acid (HN3) and its deprotonated form azide ion (N3ˉ) (AHA) are toxic since they inhibit cytochrome c oxidase complex IV (CoX IV) embedded in the inner mitochondrial membrane as part of the enzyme complexes of the respiratory chain. Especially critical for its toxicology is inhibition of CoX IV in the central nervous system and cardiovascular system. Hydrazoic acid is an ionizable species and its affinity toward membranes and therewith associated permeabilities depend on the pH values of aqueous media on both sides of the membranes. In article, we addressed permeability of AHA through the biological membrane. In order to understand affinity of the membrane for neutral and ionized form of azide we measured octanol-water partition coefficients at pH values of 2.0 and 8.0, which are 2.01 and 0.00034, respectively. By using Parallel Artificial Membrane Permeability Assay (PAMPA) experiment, we measured effective permeability through the membrane, which is logPe -4.97 and -5.26 for pH values of 7.4 and pH 8.0, respectively. Experimental permeability was used to validate theoretical permeability, which was estimated by numerically solving a diffusion equation for AHA diffusion through the membrane. We demonstrated that the rate of permeation through the cell membrane of 8.46 ·104 s-1 is much higher than the rate of chemical step of CoX IV inhibition by azide of 200 s-1. The results of this study show that transport through the membranes does not represent the rate-limiting step and therefore does not control the rate of CoX IV inhibition in the mitochondria. Observed dynamics of azide poisoning is however controlled by circulation transport that takes place on a minute time scale.