Four-Dimensional Protein Crystallography of Nitrile Hydratase Reaction

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Nitrile hydratase from Rodococcus sp. N-771 is the enzyme that catalyzes the hydration of nitriles to the corresponding amides, and contains a mononuclear non-heme iron as the reaction center (Fe-type NHase). The center is photo-reactive, inactivated by nitrosylation and activated by photo-driven NO release. The photo-activated Fe-type NHase loses the activity within 24 hours under aerobic conditions. Previous studies have revealed that the post-translationally modified cystein sulfenate (aCys114-SO-) of active enzyme is further oxidized under the aerobic conditions to cystein sulfinate (aCys114-SO_2-).In order to avoid the further oxidation, a crystallization system was constructed under anaerobic conditions of less than 0.1% (v/v) oxygen concentration. The really active structure of intact Fe-type NHase was studied by X-ray crystallography, including complex structures with butyric acid as an inhibitor/stabilizer and with cyclohexyl-isocyanide (ch-NC) as a substrate analogue. We also crystallized the inactive nitrosylated NHase under the anaerobic conditions in the complex form with ch-NC. The dynamic structure changes were traced by using the large-angle oscillation technique (LOT) after photo-activation at a time-resolution of 30min at a RIKEN beamline: BL45XU, SPring-8, the data collection system of which was remodeled for our purpose. Based on the results obtained, the role of aCys 114-SO- in the nitrile hydration mechanism of Fe-type NHase was revealed.