Glutathione (GSH) is the most abundant thiol in mammalian cells and plays a crucial role in maintaining redox cellular homeostasis. The thiols of two GSH molecules can be oxidized to the disulfide GSSG. The cytosolic GSH/GSSG ratio is very high (>100), and its reduction can lead to apoptosis or necrosis, which are of interest in cancer research. Cu II ions are very efficient oxidants of thiols, but with an excess of GSH, Cu I n (GS) m clusters are formed, in which Cu I is very slowly reoxidized by O 2 at pH 7.4 and even more slowly at lower pH. Here, the aerobic oxidation of GSH by Cu II was investigated at different pH values in the presence of the anticancer thiosemicarbazone Dp44mT, which accumulates in lysosomes and induces lysosomal membrane permeabilization in a Cu-dependent manner. The results showed that Cu II -Dp44mT catalyzes GSH oxidation faster than Cu II alone at pH 7.4 and hence accelerates the production of very reactive hydroxyl radicals. Moreover, GSH oxidation and hydroxyl radical production by Cu II -Dp44mT were accelerated at the acidic pH found in lysosomes. To decipher this unusually faster thiol oxidation at lower pH, density functional theory (DFT) calculations, electrochemical and spectroscopic studies were performed. The results suggest that the acceleration is due to the protonation of Cu II -Dp44mT on the hydrazinic nitrogen, which favors the ratelimiting reduction step without subsequent dissociation of the Cu I intermediate. Furthermore, preliminary biological studies in cell culture using the proton pump inhibitor bafilomycin A1 indicated that the lysosomal pH plays a role in the activity of Cu II -Dp44mT.
Some microorganisms, like the aerobic soil bacteria, Oligotropha carboxidovorans, have the capability to oxidize the highly toxic atmospheric gas carbon monoxide (CO) into CO 2 through CO dehydrogenase enzymes, whose active site contains a bimetallic MoCu center. Over the last decades, a number of experimental and theoretical investigations were devoted to understanding the mechanism of CO oxidation and, in particular, the role of a very stable thiocarbonate intermediate that may be formed during the catalytic cycle. The occurrence of such an intermediate was reported to make the CO 2 release step kinetically difficult. In this work, by using an accurate QM/MM approach and energy refinement by means of the BigQM method, we were able to determine the role of such an intermediate and propose a novel mechanism for the oxidation of CO into CO 2 by Mo/Cu CO dehydrogenase. Surprisingly, we found that the detachment of CO 2 occurs directly from the product of the MoO nucleophilic attack reaction on the carbon of CO aided by the transient coordination of the active site glutamate to the Mo ion. The estimated activation barrier is in good agreement with the experimental one, while the thiocarbonate turned out to not interfere with the CO-oxidation catalytic cycle. The results highlight the importance of the environmental effects in the assembly of the molecular model and in the choice of the computational protocol. Our accurate modeling of the enzyme also allowed us to exclude the involvement of a frustrated Lewis pair in the CO-oxidation mechanism, which has recently been suggested based on an analysis of structural and electronic features of synthetic mimics of the Mo/Cu CO dehydrogenase active site.
α-Pyridyl thiosemicarbazones (TSC) such as Triapine (3AP) and Dp44mT are a promising class of anticancer agents. Contrary to Triapine, Dp44mT showed a pronounced synergism with Cu II , which may be due to the generation of reactive oxygen species (ROS) by Dp44mT-bound Cu II ions. However, in the intracellular environment, Cu II complexes have to cope with glutathione (GSH), a relevant Cu II reductant and Cu Ichelator. Here, aiming at rationalizing the different biological activity of Triapine and Dp44mT, we first evaluated the ROS production by their Cu II -complexes in the presence of GSH, showing that Cu II -Dp44mT is a better catalyst than Cu II -3AP. Furthermore, we performed density functional theory (DFT) calculations, which suggest that a different hard/soft character of the complexes could account for their different reactivity with GSH.
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