Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases, in which a catalytic distal aspartate is involved in H2O2 activation to catalyze oxidations in acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial TcDyP from Thermomonospora curvata is among the most active DyPs and shows activity toward phenolic lignin model compounds (J. Biol. Chem. 2015, 290, 23447). Based on the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Blue 19 (RB19), and formation of compound II-like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the TcDyP catalyzes oxidation of large-size substrates via multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in TcDyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the crosslinking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class TcDyP is minimal possibly due to its extremely small solvent accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin.

The formation of hydroxylated intermediates is the first step in the persulfate-based advanced oxidation processes (AOPs) and depends directly on the oxidation degree of recalcitrant pollutants. Therefore, the monitoring of hydroxylated intermediates plays a vital role in persulfate-based AOPs for evaluating the oxidation degree and understanding the degradation mechanism. In this work, based on the attractive amplification of luminol chemiluminescence (CL) signals by hydroxylated intermediates, we developed a rapid, sensitive, and simple CL-based method to monitor hydroxylated intermediate (i.e., 3,5-diethyl-1,4-benzenediol) generated in persulfate-based AOPs of 1,2-divinylbenzene (DVB). The possible mechanism of the proposed CL system was that hydroxylated intermediates could be oxidized by persulfate in alkaline solution to form superoxide anion radicals and thus lead to an increase in the luminol CL. Finally, the proposed CL system was successfully applied in monitoring hydroxylated intermediates generated during persulfate-based AOPs under different degradation conditions, whose validity and reliability were verified by liquid chromatography–mass spectrometry (LC-MS) and Fourier-transform infrared spectroscopy (FT-IR). The generality of the proposed CL system was verified by monitoring hydroxylated intermediates generated in persulfate-based AOPs of methylbenzene and benzoic acid. These results suggested that this present CL system provided a promising indicator for monitoring hydroxylated intermediates in persulfate-based AOPs.