Characterization of conserved active site residues in class I nitronate monooxygenase

Propionate 3-nitronate (P3N) is a natural toxin that irreversibly inhibits mitochondrial succinate dehydrogenase. P3N poisoning leads to a variety of neurological disorders and even death. Nitronate monooxygenase (NMO) from Pseudomonas aeruginosa PAO1 was the first NMO characterized in bacteria and serves as a paradigm for Class I NMO. Here, we hypothesized that the carboxylate group of P3N might form a hydrogen bond with one or more of the four tyrosine or a lysine residues that are conserved in the active site of the enzyme. In the wild-type enzyme, the k_cat value was pH independent between pH 6.0 and 11.0, while the k_cat/K_P3N value decreased at high pH, suggesting that a protonated group with a pK_a value of 9.5 is required for binding the anionic substrate. A pH titration of the UV-visible absorption spectrum of the enzyme showed an increased absorbance at 297 nm with increasing pH, defining a pK_a value of 9.5 and a Δε_{297 nm} of 2.4 M^-1cm^-1, consistent with a tyrosine being important for substrate binding. The N3 atom of the oxidized flavin, instead, did not ionize likely because its pK_a was perturbed by the ionization of a tyrosine in the active site of the enzyme. The Y109F, Y254F, Y299F, Y303F, and K307 M, substitutions had small effects (i.e., <3.5-fold) on the steady-state kinetic parameters of the enzyme. With all mutated enzymes, the k_cat/K_P3N value was less than 2.5-fold different from the wild-type enzyme, suggesting that none of the residues is solely essential for substrate binding.