Purification and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase of Desulfitobacterium hafniense.

The membrane-bound 3-chloro-4-hydroxyphenylacetate (Cl-OHPA) reductive dehalogenase from the chlorophenol-reducing anaerobe Desulfitobacterium hafniense was purified 11.3-fold to apparent homogeneity in the presence of the detergent CHAPS. The purified dehalogenase catalyzed the reductive dechlorination of Cl-OHPA to 4-hydroxyphenylacetate with reduced methyl viologen as the electron donor at a specific activity of 103.2 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-labile sulfur per mol subunit. The N-terminal amino acid sequence of the enzyme was determined and no significant similarity was found to any protein present in the gene bank.