A dye-decolorizing peroxidase from Vibrio cholerae can demetallate heme.

Iron is an essential element for bacterial survival. Bacterial pathogens have therefore developed methods to obtain iron. Vibrio cholerae, the intestinal pathogen that causes cholera, utilizes heme as an iron source. DyP from V. cholerae (VcDyP) is a dye-decolorizing peroxidase. When VcDyP was expressed in Escherichia coli and purified, it was found to contain protoporphyrin IX (PPIX) but not heme, indicating that the protein possesses deferrochelatase activity. Here, we examined the demetallation reaction of VcDyP using fluorescence spectroscopy. Treatment of heme-reconstituted VcDyP with sodium dithionite under anaerobic conditions led to an increase in the fluorescence intensity at 624 nm, suggesting the formation of PPIX. Although the same reaction was conducted using myoglobin, horseradish peroxidase and hemin, no increase in the fluorescence was observed. Therefore, demetallation of heme is specific to VcDyP. This reaction was faster at lower pH, but the amplitudes of the fluorescence increase were larger at pH 6.5-7.5, in clear contrast to the dye-decolorizing activity with the optimal pH of 4.5. In contrast to HutZ from V. cholerae, which is a heme-degrading enzyme that cleaves the heme macrocycle to release iron, VcDyP can remove iron from heme without degradation. To our knowledge, VcDyP is the first enzyme whose demetallation activity has been confirmed at neutral pH. Our results show that VcDyP is a bifunctional protein that degrades anthraquinone dyes and demetallates heme.