Purification and Characterization of Glutathione Conjugate Reductase: A Component of the Tetrachlorohydroquinone Reductive Dehalogenase System from Phanerochaete chrysosporium

Abstract

A membrane-bound glutathione S-transferase and a soluble glutathione conjugate reductase constitute the reductive dehalogenase system of P. chrysosporium. This enzyme system reductively removes chlorine substituents from tetrachlorohydroquinone, a metabolite of pentachlorophenol. The membrane-bound glutathione S-transferase converts tetrachlorohydroquinone to S-glutathionyltrichloro-1,4-hydroquinone, which is subsequently reduced to 3,5,6-trichlorohydroquinone by the soluble glutathione conjugate reductase (GCR). This GCR can accept glutathione, dithiothreitol, cysteine, or β-mercaptoethanol as cosubstrates. GCR was purified to apparent homogeneity by ion-exchange and covalent chromatography. The enzyme exhibits optimum activity at pH 6.0 and 55°C and appears to be a homodimer with a Mr of ∼60 kDa. Activity increases as the number of chlorine substituents on the hydroquinone ring is increased. GCR has an apparent Km of ∼33 μM and an apparent kcat of ∼3.43 s−1 for 2-S-glutathionyl-3,5,6-trichloro-1,4-hydroquinone. Inhibitors of GCR include Cd2+, Fe2+, Mn2+, iodoacetic acid, and p-chloromercuribenzoic acid, suggesting the presence of a catalytic cysteine thiol(s) at the active site. When glutathione is used as a cosubstrate, reduction of S-glutathionyltrichloro-1,4-hydroquinone is accompanied by the production of trichlorohydroquinone and oxidized glutathione in a 1:1 ratio. A mechanism for this novel enzyme is proposed.