Dye-decolorizing peroxidase (DyP) is a heme-containing enzyme that catalyzes the degradation of anthraquinone dyes. In our previous study, we characterized DyP from Vibrio cholerae (VcDyP) and found that the dye-decolorizing activity of VcDyP depends on the pH with an optimal pH of 4.5. In this study, we tried to identify the decisive factor in the pH dependence of VcDyP.
A crystal structure of VcDyP revealed that a polar amino acid residue, Asp144, is located in the heme distal site. Therefore, we replaced Asp144 with His, but this mutation resulted in a significant loss of dye-decolorizing activity. Introduction of His at a different position from Asp144 resulted in partial restoration of activity, but no shift in the optimal pH.
His178 is located near heme and forms hydrogen bonds with Asp138 and Thr278. While mutation of His178 and Thr278 did not affect the pH dependence, activity at pH 6.5 was significantly increased when Asp138 was replaced with Val. Based on these results, we propose that neither distal amino acid residues, including Asp144, nor hydrogen bonds between His178 and Thr278 are responsible, while the hydrogen bond between His178 and Asp138 plays a key role in the pH dependence of activity.
During the expression of VcDyP, we found that this protein was purified with protoporphyrin IX (PPIX) but not with heme. This observation led us to examine whether the protein possesses deferrochelatase activity . The formation of PPIX from heme was monitored by fluorescence spectroscopy. When VcDyP was reduced anaerobically with sodium dithionite, a demetallation reaction was observed. The yield of PPIX was the highest at pH 6.5-7.5. VcDyP is the first enzyme with confirmed demetallation activity at neutral pH.
Our results show that VcDyP is a bifunctional protein that degrades anthraquinone dyes and demetallates heme.