Redox-Active Sites in Auricularia auricula-judae Dye-Decolorizing Peroxidase and Several Directed Variants: A Multifrequency EPR Study
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- 作者：Maria Camilla Baratto ; Adalgisa Sinicropi ; Dolores Linde ; Ver贸nica S谩ez-Jim茅nez ; Lorenzo Sorace ; Francisco J. Ruiz-Duenas ; Angel T. Martinez ; Riccardo Basosi ; Rebecca Pogni
- 刊名：Journal of Physical Chemistry B
- 出版年：2015
- 出版时间：October 29, 2015
- 年：2015
- 卷：119
- 期：43
- 页码：13583-13592
- 全文大小：482K
- ISSN：1520-5207
文摘
Peroxide-activated Auricularia auricula-judae dye-decolorizing peroxidase (DyP) forms a mixed Trp377 and Tyr337 radical, the former being responsible for oxidation of the typical DyP substrates (Linde et al. Biochem. J., 2015, 466, 253鈥?62); however, a pure tryptophanyl radical EPR signal is detected at pH 7 (where the enzyme is inactive), in contrast with the mixed signal observed at pH for optimum activity, pH 3. On the contrary, the presence of a second tyrosine radical (at Tyr147) is deduced by a multifrequency EPR study of a variety of simple and double-directed variants (including substitution of the above and other tryptophan and tyrosine residues) at different freezing times after their activation by H2O2 (at pH 3). This points out that subsidiary long-range electron-transfer pathways enter into operation when the main pathway(s) is removed by directed mutagenesis, with catalytic efficiencies progressively decreasing. Finally, self-reduction of the Trp377 neutral radical is observed when reaction time (before freezing) is increased in the absence of reducing substrates (from 10 to 60 s). Interestingly, the tryptophanyl radical is stable in the Y147S/Y337S variant, indicating that these two tyrosine residues are involved in the self-reduction reaction.