Coniferyl Alcohol Radical Detection by the Dirigent Protein AtDIR6 Monitored by EPR

Plant dirigent proteins (DIRs) control the stereoselectivity of the monolignol coniferyl alcohol radical coupling. The main mechanistic hypothesis on this chemo‐ and stereoselective reaction invokes a binding of coniferyl alcohol radical substrates in the dirigent protein active site so that only one enantiomeric form can be produced. We have studied the influence of the Arabidopsis thaliana AtDIR6 protein on the transient coniferyl alcohol radical by EPR. Herein, we show that AtDIR6 stabilizes coniferyl alcohol radicals prior to directing their coupling towards the formation of (−)‐pinoresinol. Stabilization of CA• by DIR has been observed experimentally. Using controlled photo‐irradiation of coniferyl alcohol to generate a CA• and room temperature EPR and HPLC to monitor its fate, we demonstrate that the dirigent protein AtDIR6 stabilizes the formed radical prior to directing the coupling of radicals towards the formation of (−)‐pinoresinol.