PURIFICATION AND CHARACTERIZATION OF THE 3-CHLORO-4-HYDROXY-PHENYLACETATE REDUCTIVE DEHALOGENASE OF DESULFITOBACTERIUM HAFNIENSE

The membrane-bound 3-chloro-3-hydroxyphenylacetate (Cl-OHPA) reductive dehalogenase from the chlorophenol-reducing anaerobe Desulfitobacteri um hafniense was purified 11.3-fold to apparent homogeneity in the pre sence of the detergent CHAPS. The purified dehalogenase catalyzed the reductive dechlorination of Cl-OHPA to 4-hydroxyphenylacetate with red uced methyl viologen as the electron donor at a specific activity of 1 03.2 nkat/mg protein, SDS-PAGE revealed a single protein band with an apparent molecular mass of 46.5 kDa, The enzyme contained 0.68 +/- 0.2 mol corrinoid, 12.0 +/- 0.7 mol iron, and 13.0 +/- 0.7 mol acid-labil e sulfur per mol subunit, The N-terminal amino acid sequence of the en zyme was determined and no significant similarity was found to any pro tein present in the gene bank, (C) 1998 Federation of European Biochem ical Societies.