Direct Electrochemical Generation of Catalytically Competent Oxyferryl Species of Classes I and P Dye Decolorizing Peroxidases

Abstract

This work introduces a novel way to obtain catalytically competent oxyferryl species for two different dye-decolorizing peroxidases (DyPs) in the absence of H2O2 or any other peroxide by simply applying a reductive electrochemical potential under aerobic conditions. UV-vis and resonance Raman spectroscopies show that this method yields long-lived compounds II and I for the DyPs from Bacillus subtilis (BsDyP; Class I) and Pseudomonas putida (PpDyP; Class P), respectively. Both electrochemically generated high valent intermediates are able to oxidize ABTS at both acidic and alkaline pH. Interestingly, the electrocatalytic efficiencies obtained at pH 7.6 are very similar to the values recorded for regular catalytic ABTS/H2O2 assays at the optimal pH of the enzymes, ca. 3.7. These findings pave the way for the design of DyP-based electrocatalytic reactors operable in an extended pH range without the need of harmful reagents such as H2O2.

Electrochemical Actuation of a DyP Peroxidase: A Facile Method for Drastic Improvement of the Catalytic Performance

“…The RR spectra also present some clear changes, both in the position and relative intensities of the heme marker bands, particularly of the normal modes ν 4 , ν 28 , ν 3 , ν 19 , ν 2 , ν 37 , ν 10 , and ν CC that appear at 1373, 1428, 1505, 1554, 1563, 1580, 1600, 1619, and 1636 cm –1 , respectively (Figure and Table S1). These spectral features are consistent with the formation of CompI, as discussed previously. ,,,, The component analysis of the RR spectrum reveals an almost quantitative conversion of the resting enzyme to CompI, with only a very minor additional contribution of the Fe 2+ enzyme as revealed by the weak ν 3 and ν 4 bands at 1355 and 1473 cm –1 , respectively. At pH > 4.5, the absorption spectra of the electrolyzed samples exhibit more drastic changes, which include a 13 nm upshift of the Soret band and well-resolved Q-bands at 532, 556, and 622 nm.…”

“…Deconvolution of the absorption spectra with Gaussian bands confirms this assignment. As shown in Figure D for the spectrum recorded after 4 min of electrolyzation, spectra can be quantitatively simulated with bands mainly originating in the resting enzyme (shown in gray), along with bands at 543 and 532 nm, which are the most prominent reporters of compounds III and II, respectively. ,,,,, Deconvolution of the RR spectra measured under similar conditions confirms the coexistence of CompIII and CompII at the initial stages of electrolyzation (Figure S7). The time evolution of the CompIII/CompII relative contributions is displayed in Figure F through their absorptions at 543 and 532 nm, respectively.…”

“…3 cm –1 , with the characteristic ν 4 , ν 28 , ν 3 , ν 19 , ν 2 , ν 37 , ν 10 , and ν CC bands appearing at 1377, 1430, 1509, 1553, 1564, 1582, 1602, 1621, and 1640 cm –1 , respectively (Figure and Table S1). These spectral changes are indicative of the formation of CompII. ,,,, …”

Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase

“…In recent years, we have devoted efforts to the investigation of Bs DyP (class I) from Bacillus subtilis and Pp DyP (class P) from Pseudomonas putida MET94 [55] , providing mechanistic insight into the catalytic cycle and reaction intermediates [9] , [43] , [73] , electro-catalytic properties and interactions modulating redox properties [42] , [58] , [59] , [60] . Additionally, directed evolution approaches allowed for identifying engineered variants with improved catalytic efficiency for the lignin-related phenolic 2,6-dimethoxyphenol (DMP) [6] , [9] , [54] .…”

Detection of Glucose Based on Noble Metal Nanozymes: Mechanism, Activity Regulation, and Enantioselective Recognition

“…For example, the color of TMB, ODP and ABTS can change from colorless to blue, red, and green with absorbance at ≈450 nm, [72] ≈652 nm, [73] and ≈418 nm. [74,75]…”