PHOTOREACTIVE NITRILE HYDRATASE - THE PHOTOREACTION SITE IS LOCATED ON THE ALPHA-SUBUNIT

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 exists in active and inactive forms. The inactive NHase is immediately activated by lig ht irradiation and changes to the active form, To characterize the pho toreactive center, the inactive NHase was denatured by 6M urea, and tw o kinds of subunits (alpha and beta) were separated and purified by an ion-exchange chromatography, In a manner similar to the native NHase, the isolated alpha subunit showed two absorption peaks at 280 and 370 nm, which were diminished by light irradiation. However, irradiation f ailed to elicit the appearance of absorption peaks at around 400 nm an d at 710 nn, which were characteristic of the activated enzyme, The be ta subunit seemed not to possess any photoreactive chromophore because its absorption spectrum was not altered by light irradiation. Neither of the subunits showed NHase activity before or after light irradiati on, but the inactive NHase was reconstituted by incubating the two sub units together in the dark at 4 degrees C for 1 h. Light irradiation o f the beta subunit did not affect subsequent complex formation or NHas e activity. However, the irradiated alpha subunit could not assemble w ith the beta subunit, and no activity was recovered. These results dem onstrate that the chromophore(s) responsible for the photoactivation o f NHase are entirely located on the alpha subunit, and imply that ligh t irradiation induces conformational change of the alpha subunit.