Insights into the Efficacy and Binding Mode of 1,4-Disubstituted 1,2,3-Triazoles─A New Class of Agricultural Nitrification Inhibitors

Recently, 1,4-disubstituted 1,2,3-triazoles were reported by us as a new class of nitrification inhibitors, which can outperform the commercial compound 3,4-dimethylpyrazole phosphate (DMPP) in soil incubations. In this work, the mechanism of inhibition of five 1,2,3-triazoles with different substitution patterns was explored using a bacterial assay based on the measurement of nitrite (NO₂^-) production by pure cell cultures of Nitrosomonas europaea and Nitrosospira multiformis. While polar functional groups, such as amines, esters, and alkoxy residues, were detrimental to inhibiting production of NO₂^-, triazoles carrying only aliphatic substituents showed the highest inhibition of up to 98%. The observed correlation between lipophilicity and inhibitory activity suggests that more lipophilic compounds could more easily access the membrane-bound ammonia monooxygenase (AMO), which catalyzes the first step of the nitrification process. Measurement of the Michaelis-Menten kinetics suggests that the disubstituted 1,2,3-triazoles studied in this work act as reversible, noncompetitive inhibitors. Real-time measurements of the oxygen (O₂) consumption showed that the O₂ uptake rate by AMO follows zero-order kinetics in the presence of the triazoles, confirming the nonmechanistic mode of inhibition.