Purification and characterization of glutathione conjugate reductase: A component of the tetrachlorohydroquinone reductive dehalogenase system from Phanerochaete chrysosporium

A membrane-bound glutathione S-transferase and a soluble glutathione conjug ate reductase constitute the reductive dehalogenase system of P. chrysospor ium, This enzyme system reductively removes chlorine substituents from tetr achlorohydroquinone, a metabolite of pentachlorophenol, The membrane-bound glutathione S-transferase converts tetraehlorohydroquinone to S-glutathiony ltrichloro-1,4-hydroquinone, which is subsequently reduced to 3,5,6-trichlo rohydroquinone by the soluble glutathione conjugate reductase (GCR), This G CR can accept glutathione, dithiothreitol, cysteine, or P-mercaptoethanol a s cosubstrates, GCR was purified to apparent homogeneity by ion-exchange an d covalent chromatography, The enzyme exhibits optimum activity at pH 6.0 a nd 55 degreesC and appears to be a homodimer with a M-r of similar to 60 kD a, Activity increases as the number of chlorine substituents on the hydroqu inone ring is increased, GCR has an apparent K-m of similar to 33 muM and a n apparent k(cat) of similar to3.43 s(-1) for 2-S-glutathionyl-3,5,6-trichl oro-1,4-hydroquinone. Inhibitors of GCR include Cd2+, Fe2+ Mn2+, iodoacetic acid, and p-chloromercuribenzoic acid, suggesting the presence of a cataly tic cysteine thiol(s) at the active site, When glutathione is used as a cos ubstrate, reduction of S-glutathionyltrichloro-1,4-hydroquinone is accompan ied by the production of trichlorohydroquinone and oxidized glutathione in a 1:1 ratio. A mechanism for this novel enzyme is proposed. (C) 2001 Academ ic Press.