Two-dimensional infrared spectroscopic study of cytochrome c peroxidase activity in deep eutectic solvent

Deep eutectic solvents (DESs) prepared by mixing hydrogen-bond donor and acceptor molecules have been found to be of use in several applications. Recently, it was shown that DESs can enhance the peroxidation activity of cytochrome c. Here, to elucidate the effects of DESs on the peroxidase activity of cytochrome c, we carried out linear and nonlinear infrared spectroscopic studies of the CO stretch mode of carbon monoxide cytochrome c (COCytc) in ethylammonium chloride (EAC)/urea DES. The FTIR spectrum of COCytc shows a significant spectral shift upon addition of the DES. The broadening and red-shifting of the CO band are observed in both urea and DES solutions, which are induced by the change of the distal ligands around the heme. Although the FTIR study is sensitive to structural changes in the active site, it does not provide quantitative information about structural dynamics related to the catalytic activity itself. Thus, we carried out two-dimensional IR spectroscopy of the CO mode, which suggests that there is a different conformer that could be related to the enhanced catalytic activity in DES. In particular, the spectral diffusion dynamics of that conformer exhibits quite different behavior. The experimental results lead us to propose a hypothesis that the DES increases the population of the conformer with distal ligand lysines close to the reaction center through the combining effect of urea and EAC, which results in the enhancement of the peroxidase activity of cytochrome c. We anticipate that the present experimental work stimulates future investigations of the effects of DES on biocatalysis.