Structural evidence of the conversion of nitric oxide (NO) to nitrite ion (NO2‾) by lactoperoxidase (LPO): Structure of the complex of LPO with NO2‾ at 1.89Å resolution

Abstract

Abstract Lactoperoxidase (LPO, E.C. 1.11.1.7) is a heme containing enzyme of the innate immune system which uses hydrogen peroxide (H2O2) to catalyze the conversion of substrates into antimicrobial products. It is found in the body extracellular fluids including plasma, saliva, airway epithelial lining fluids, nasal lining fluid, milk, tears and gastric juices. Previously, we had shown structurally that LPO converted substrates, thiocyanate (SCN‾) and iodide (I‾) ions into oxidized products hypothiocyanite (OSCN‾) and hypoiodite (IO‾) ions respectively. We report here a new structure of the complex of LPO with NO2‾ showing that LPO converts nitric oxide (NO) into an oxidized product nitrite (NO2‾) ion. NO2‾ was produced using the two step reaction of LPO by adding hydrogen peroxide (H2O2) in the first step and then introducing the NO gas in 0.1M phosphate buffer at pH 6.8. The resulting mixture was crystallized using 20% (w/v) PEG-3350 and 0.2M ammonium iodide at pH 6.8. The structure determination showed the presence of NO2‾ ion in the distal heme cavity of the substrate binding channel. The structure further revealed that the propionate group attached to pyrrole ring C of the heme moiety was split into two positions. Similarly, the side chain of the covalently linked Asp108 was also split into two components. As a result of these changes, the conformation of the side chain of Arg255 also changed to gain interactions with the disordered carboxylic group of propionate moiety. The observed structural changes indicate the presence of an intermediate in the reaction pathway.