Surface enhanced resonance Raman detection of a catalytic intermediate of DyP-type peroxidase

Abstract

We report herein the vibrational spectroscopic characterisation of a catalytic intermediate formed by the reaction of H 2 O 2 with DyP-type peroxidase immobilised on a biocompatible coated metal support.The SERR spectroscopic approach is of general applicability to other peroxidases which form relatively stable catalytic intermediates.Dye-decolourising peroxidases (DyPs) are novel heme peroxidases, the primary sequence, structural and mechanistic properties of which are unrelated to those of other known peroxidases. [1][2][3][4][5][6][7][8][9][10] They lack the distal histidine that is highly conserved in classical peroxidases and which acts as an acid/base catalyst in the reduction of hydrogen peroxide to water. DyPs are classified into four phylogenetically distinct classes (A-D); their physiological role is not fully established yet and it appears to be subfamilydependent. 1-10 DyP from Pseudomonas putida MET94 (PpDyP) is an extremely versatile B-type DyP, capable of efficient oxidation of a wide range of anthraquinonic and azo dyes, phenolic substrates, the non-phenolic veratryl alcohol and even manganese and ferrous ions. 8 In the reaction with H 2 O 2 , PpDyP forms a stable Compound I (Cpd I) at a rate of 1.4 AE 0.3 Â 10 6 M À1 s À1 , comparable to those of classical peroxidases and other DyPs. 8 The intermediate is, like in other DyPs, surprisingly long-living, with a half-life of B60 min, as demonstrated by electronic absorption spectroscopy. 9 Upon immobilisation on biocompatible metal supports, PpDyP is capable of efficient electrocatalytic activity in the presence of hydrogen peroxide. The enzyme is therefore a promising candidate for the design of bio-electronic devices with unique DyP-type peroxidase substrate specificity. 9,10 Resonance Raman (RR) spectroscopy has been extensively used in the detection and characterisation of peroxidase and catalase-peroxidase catalytic intermediates, and RR spectroscopic fingerprints of Cpd I (two equivalent oxidised resting ferric state) and Cpd II (one equivalent oxidised resting ferric state) are well established. [11][12][13][14][15][16] However, a strong fluorescence impeded the RR studies of PpDyP intermediates. Here, we have probed catalytic intermediate species of immobilised PpDyP by surface enhanced RR (SERR) spectroscopy. The SERR spectra of heme proteins immobilised on nanostructured Ag surfaces exclusively display the cofactor signals of the adsorbed molecules. In addition, close proximity of the molecule to the metal surface can efficiently quench fluorescence. 17 Like in RR spectroscopy, SERR signals include, inter alia, the core-size marker bands (e.g. n 4 , n 3 , n 2 , n 10 ) which are specifically enhanced upon excitation in resonance with the Soret absorption band of the porphyrin. The frequencies of these modes are indicative of the redox and spin states and the coordination pattern of the heme iron.We have previously shown that the high frequency region of RR spectra of the resting PpDyP in solution displays broad marker bands, indicative of t...

Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases

“…21 It is noteworthy that Raman spectroscopy provided the rst unambiguous evidence that the unusual QS species is actually catalytically competent. 25 Surface enhanced RR experiments (SERR), employing PpDyP attached to biocompatible plasmonic metal, revealed that the QS species is capable of H 2 O 2 binding and formation of the catalytic intermediate Compound I, which has a distinct (SERR) ngerprint. 25 Apart from RR band shis, a temperature decrease induces a spin transition from the 5cHS to the 6cLS state at a xed temperature (À45 C) in PpDyP.…”

Protein Film Infrared Electrochemistry Demonstrated for Study of H₂ Oxidation by a [NiFe] Hydrogenase

“…Potentials quoted in V vs SHE. Reproduced with permission from Hidalgo et al 35 Please click here to view a larger version of this figure.…”

“…Spectrum b ii , recorded at a later time during the −0.074 V step, is reported as a difference spectrum relative to b i and shows that no change in distribution of active site states occurs, consistent with the stability of the potential at −0.074 V. Spectra b iii and b iv are also reported as difference spectra relative to b i and show gradual conversion of Ni-SI to Ni-B during anaerobic inactivation at +0.356 V vs SHE. Reproduced with permission from Hidalgo et al 35 Please click here to view a larger version of this figure.…”

“…Several spectroelectrochemical methods have been reported for studying immobilized redox proteins at a range of different electrodes: UV-visible spectroscopy at transparent metal oxide electrodes; 25 26 27 fluorescence spectroscopy at gold electrodes; 28 29 surface enhanced infrared absorption (SEIRA) spectroscopy at gold electrodes; 30 31 32 33 and surface enhanced Raman spectroscopic techniques, principally at silver electrodes. 34 35 …”

Synchrotron-Based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control

“…This means that only a small number of active sites are being addressed in comparison to surface science studies of small molecule adsorbates on metal electrodes, for example. Several spectroelectrochemical approaches have been developed for immobilized proteins, including UV–visible spectroscopy at transparent metal oxide electrodes, − fluorescence , or surface enhanced infrared absorption (SEIRA) spectroscopy , at gold electrodes, and various surface enhanced Raman spectroscopies, , most commonly at silver electrodes. We recently demonstrated an attenuated total reflectance IR (ATR-IR) approach for studying redox enzymes immobilized on high surface area carbon electrodes, , exploiting the ease of adsorption of many proteins onto carbon surfaces.…”