Journal
BIOCHEMICAL JOURNAL
Volume 466, Issue -, Pages 253-262Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20141211
Keywords
catalytic protein radical; dye-decolorizing peroxidase; EPR spectroscopy; molecular docking; QM/MM; site-directed mutagenesis
Categories
Funding
- INDOX European Union [KBBE-2013-7-613549]
- PELE European Union [ERC-2009-Adg 25027]
- Spanish Ministry of Economy and Competitiveness (MINECO) [BIO2011-26694, CTQ2013-48287, BFU2011-24615]
- Italian Ministry of Education, Universities and Research (MIUR) [PRIN 2009-STNWX3]
- EU
- Ramon y Cajal contract of the Spanish Ministry of Economy and Competitiveness (MINECO)
- ICREA Funding Source: Custom
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Dye-decolorizing peroxidase (DyP) of Auricularia auriculajudae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H2O2-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high-and low-turnover sites. The high-turnover site for oxidation of RB19 (k(cat)> 200 s(-1)) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 k(cat) similar to 20 s(-1)) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant.
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