PURIFICATION AND CHARACTERIZATION OF HYDROXYQUINOL 1,2-DIOXYGENASE FROM AZOTOBACTER SP STRAIN GP1

Hydroxyquinol 1,2-dioxygenase was purified from cells of the soil bact erium Azotobacter sp. stain GP1 grown with 2,4,6-trichlorophenol as th e sole source of carbon. The presumable function of this dioxygenase e nzyme in the degradative Pathway of 2,4,6-trichlorophenol is discussed . The enzyme was highly specific for 6-chlorohydroxyquinol (6-chloro-1 ,2,4-trihydroxybenzene) and hydroxyquinol (1,2,4-trihydroxybenzene) an d was found to perform ol tho cleavage of the hydroxyquinol compounds, yielding chloromaleylacetate and maleylacetate, respectively. With th e conversion of 1 mol of 6-chlorohydroxyquinol, the consumption of 1 m ol of O-2 and the formation of 1 mol of chloromaleylacetate were obser ved. Catechol was not accepted as a substrate. The enzyme has to be in duced, and no activity was found in cells grown on succinate. The mole cular weight of native hydroxyquinol 1,2-dioxygenase was estimated to 58,000, with a sedimentation coefficient of 4,32. The subunit molecula r weight of 34,250 indicates a dimeric structure of the dioxygenase en zyme. The addition of Fe2+ ions significantly activated enzyme activit y, and metal chelating agents inhibited it. Electron paramagnetic reso nance data are consistent with high-spin iron(III) in a rhombic enviro nment. The NH2-terminal amino acid sequence was determined for up to 4 0 amino acid residues and compared with sequences from literature data for other catechol and chlorocatechol dioxygenases.