IDENTIFICATION AND LOCALIZATION OF A STABLE SULFENIC ACID IN PEROXIDE-TREATED TETRACHLOROHYDROQUINONE DEHALOGENASE USING ELECTROSPRAY MASS-SPECTROMETRY

Background: Tetrachlorohydroquinone dehalogenase catalyzes the reducti ve dehalogenation of tetrachlorohydroquinone to trichlorohydroquinone and then to 2,6-dichlorohydroquinone. This enzyme undergoes oxidative damage during purification which causes it to form aberrant products. The damage is reversible by treatment with dithiothreitol. Possible ty pes of oxidative damage include an inappropriate disulfide bond, a cys teine sulfenic acid, or a methionine sulfoxide. Results: Using electro spray liquid chromatography/mass spectrometry, we have demonstrated th at oxidation of tetrachlorohydroquinone dehalogenase with H2O2 results in formation of a sulfenic acid at Cys13. Further oxidation to a sulf inic acid was also observed. Conclusions: Oxidation of Cys13 to a sulf enic acid prevents the normal reductive dehalogenation reaction from b eing completed. This finding is consistent with previous work which su ggested that Cys13 acts as a nucleophile during the conversion of tetr achlorohydroquinone to trichlorohydroquinone. The technique described for identification and localization of the cysteine sulfenic acid shou ld be applicable to a wide variety of biological systems.