Vibrational Spectro-Electrochemistry of Heme Proteins
Authors: Todorovic Smilja , Barbosa Catarina , Zuccarello Lidia , Silveira Celia M 
Published: July 2022

Heme proteins perform a plethora of distinct cellular functions, including e.g. electron transport (ET), energy conversion, detoxification, catalysis, signaling, and gene regulation, and as such inspire a myriad of biotechnological applications. We use resonance Raman (RR) and Surface enhanced RR (SERR) spectroscopies to probe the architecture of the heme pocket in diverse heme proteins and enzymes, which is essential for the understanding of their physiological properties as well as for the evaluation of their potential for development of the 3rd generation bioelectronic devices (1,2). Moreover, plasmonic metal that gives origin to the surface enhancement of the Raman signal of the molecules found in its close proximity can serve as an electrode, thus driving electrochemical processes. In the case of heme proteins attached to plasmonic Ag electrodes, SERR spectra selectively show vibrational bands originating from the heme moiety only, which are sensitive to spin, coordination and redox state and of the heme iron. These properties that govern the catalytic performance of heme enzymes can be monitored in potential dependent manner by SERR spectro-electrochemistry.
We have demonstrated that SERR spectro-electrochemistry possesses unique capacity of to i) disentangle ET processes in multi hemic proteins, such as 28 heme containing nitrite reductase, which represent a challenge for all other experimental approaches and ii) detect subtle immobilization induced structural changes in enzymes of biotechnological interest, which e.g. in the case of cytochrome P450 may prevent their successful applications (1-4). Here we show that SERRS monitoring of electrocatalytic processes by immobilized heme peroxidases, can provide information on catalytically relevant species in situ. Several members of a recently discovered family of heme dye-decolorizing peroxidases (DyPs) that possess remarkable catalytic properties in solution and high biotechnological potential, have been immobilized on biocompatible Ag electrodes. Their structural and electrocatalytic properties studied by RR, SERR spectro-electrochemistry and electrochemistry (2). The immobilized DyP from Pseudomonas putida (PpDyP), in particular, shows native structure and outstanding analytical and catalytic parameters, and hence an exceptional potential for development of 3rd generation biosensors for H2O2 detection. In terms of sensitivity, the bioelectrodes carrying immobilized PpDyP outperform HRP based counterparts reported in the literature (2,4). The biosensor based on a PpDyP variant that harbors mutations at the second shell of the heme cavity reveals further improved storage. Our work highlights the importance of integrated, multidisciplinary approach to simultaneously evaluate the structure and catalytic properties of the enzymes, which ensures faster identification and optimization of the promising candidates for biotechnological applications.
References:
1. Silveira, C. M.; Moe, E.; Fraaije, M.; Martins, L. O.; Todorovic, S. (2020). Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases. RSC Advances 10, 11095. https://doi.org/10.1039/D0RA00950D

2 Barbosa, C.; Silveira, C. M.; Silva, D.; Brissos, V.; Hildebrandt, P.; Martins, L. O.; Todorovic, S. (2020). Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing. Biosensors and Bioelectronics 153. https://doi.org/10.1016/j.bios.2020.112055

3 Zuccarello, L.; Barbosa, C.; Galdino, E.; Lončar, N.; Silveira, C.M.; Fraaije, M.W.; Todorovic, S. (2021) SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices. Int. J. Mol. Sci. 22, 7998. https://doi.org/10.3390/ijms22157998

4 Zuccarello, L.; Barbosa, C.; Todorovic, S., Silveira, C.M. (2021) Electrocatalysis by Heme Enzymes-Applications in Biosensing. Catalysts 11, 218. https://doi.org/10.3390/catal11020218