The reaction of iron(ii) and hydrogen peroxide, namely the Fenton reaction, is well-known for its strong oxidizing capability. While the Fenton reactions are ubiquitous and have wide applications in many areas, the detailed mechanism, especially the nature of the reactive intermediates responsible for oxidation, is not completely clear. In this work, the performances of various density functional theory (DFT) methods on the relative energies of key Fenton intermediates are evaluated. The DFT method selected from the benchmark study is then exploited to investigate the aqueous Fenton reactions in different pH conditions. The results show that at pH > 2.2, the major Fenton oxidants are high-valent oxoiron(iv) aquo complexes. However, depending on the pH conditions, these complexes can exist in three protonation states that display quite different oxidation reactivities. The oxidizing power of FeIV[double bond, length as m-dash]O is found to be principally determined by the total charge of the ligands and is less influenced by the axial ligand effect. Moreover, the calculations reveal that the presence of the hydronium ion can stabilize the intermediate of the hydroxyl radical and further inhibit oxoiron(iv) formation via proton transfer. The contribution of hydroxyl radicals could compete with the oxoiron(iv) species at pH below 2.2. In addition, high-level ab initio calculations question the existence of the iron(iv)-dihydroxo intermediate suggested in the literature. The implications of the computational results for the Fenton oxidation process, cytochrome P450, and catalyst design are discussed.

BackgroundChemoresistance is a major obstacle that limits the benefits of 5-Fluorouracil (5-Fu)-based chemotherapy for colon cancer patients. Autophagy is an important cellular mechanism underlying chemoresistance. Recent research advances have given new insights into the use of natural bioactive compounds to overcome chemoresistance in colon cancer chemotherapy. As one of the multitargeted and safer phytomedicines, curcumin has been reported to work as cancer-specific chemosensitizer, presumably via induction of autophagic signaling pathways. The precise therapeutic effect of curcumin on autophagy in determining tumorous cells’ fate, however, remains unclear. This study was conducted to investigate the differential modulations of the treatments either with 5-Fu alone or 5-Fu combined with curcumin on cellular autophagic responses and viabilities in the human colon cancer cells HCT116 and HT29, and explore molecular signaling transductions underlying the curcumin-mediated autophagic changes and potentiation of 5-Fu’s cytotoxicity in vitro and in vivo.MethodsCell proliferation assay and morphology observation were used to identify the cytotoxicity of different combinations of curcumin and 5-Fu in HCT116 and HT29 cells. Cell immunofluorescence assay, Flow cytometry and Western blot were employed to detect changes of autophagy and the autophagy-related signaling pathways in the colon cancer cells and/or xenograft mice.ResultsCurcumin could significantly augment the cytotoxicity of 5-Fu to the tumorous cells, and the pre-treatment with curcumin followed by 5-Fu (pre-Cur) proved to be the most effective one compared to other two combinations. The chemosensitizing role of curcumin might attribute to the autophagy turnover from being activated in 5-Fu mono-treatment to being inhibited in the pre-Cur treatment as indicated by the changes in expression of beclin-1, p62 and LC3II/LC3I and the intensity of Cyto-ID Green staining. The autophagic alterations appeared to be contributed by down-regulation of not only the phospho-Akt and phospho-mTOR expressions but the phospho-AMPK and phospho-ULK1 levels as well. The cellular activation of AMPK by addition of A-769662 to the pre-Cur combination resulted in reversed changes in expressions of the autophagy protein markers and apoptotic status compared to those of the pre-Cur combination treatment. The findings were validated in the xenograft mice, in which the tumor growth was significantly suppressed in the mice with 25-day combination treatment, and meanwhile expressions of the autophagy markers, P-AMPK and P-ULK1 were all reversely altered in line with those observed in HCT116 cells.ConclusionPre-treatment with curcumin followed by 5-Fu may mediate autophagy turnover both in vitro and in vivo via AMPK/ULK1-dependent autophagy inhibition and AKT modulation, which may account for the increased susceptibility of the colon cancer cells/xenograft to the cytotoxicity of 5-Fu.Electronic supplementary materialThe online version of this article (10.1186/s13046-017-0661-7) contains sup...

The benchmark study of DFT methods on the activation energies of phosphodiester C3'-O and C5'-O bond ruptures and glycosidic C1'-N bond ruptures induced by electron attachment was performed. While conventional pure and hybrid functionals provide a relatively reasonable description for the C1'-N bond rupture, they significantly underestimate the energy barriers of the C-O bond ruptures. This is because the transition states of the later reactions, which are characterized by an electron distribution delocalized from the nucleobase to sugar-phosphate backbone, suffer from a severe self-interaction error in common DFT methods. CAM-B3LYP, M06-2X, and ωB97XD are the top three methods that emerged from the benchmark study; the mean absolute errors relative to the CCSD(T) values are 1.7, 1.9, and 2.2 kcal/mol, respectively. The C-O bond cleavages of 3'- and 5'-dXMP(•-), where X represents four nucleobases, were then recalculated at the M06-2X/6-31++G**//M06-2X/6-31+G* level, and it turned out that the C-O bond cleavages do not proceed as easily as previously predicted by the B3LYP calculations. Our calculations revealed that the C-O bonds of purine nucleotides are more susceptible than pyrimidine nucleotides to the electron attachment. The energies of electron attachment to nucleotides were calculated and discussed as well.

Light-driven sodium ion pump rhodopsin (NaR) is a new functional class of microbial rhodopsin. A previous flash photolysis study of Krokinobacter eikastus rhodopsin 2 (KR2) revealed the presence of three kinetically distinct intermediates: K, L/M, and O. Previous low-temperature Fourier-transform infrared (FTIR) spectroscopy of KR2 showed that photoisomerization from the all-trans to the 13-cis form is the primary event of the Na+ pumping photocycle, but structural information on the subsequent intermediates is limited. Here, we applied step-scan time-resolved FTIR spectroscopy to KR2 and Nonlabens dokdonensis rhodopsin 2 (NdR2). Both low-temperature static and time-resolved FTIR spectra resolved a K-like intermediate, and the corresponding spectra showed few differences. Strong hydrogen-out-of-plane (HOOP) vibrations, which appeared in the K intermediate, are common among other rhodopsins. It is, however, unique for NaR that such HOOP bands are persistent in late intermediates, such as L and O intermediates. This observation strongly suggests similar chromophore structures for the K, L, and O intermediates. In fact, an isotope-labeled study that used 12,14-D2 retinal revealed that the chromophore configuration of the O intermediate in NaR is 13-cis. In contrast to the vibrations of the chromophore, those of the protein differ among intermediates, and this is related to the sodium-pumping function. The molecular mechanism of the light-driven sodium pump is discussed on the basis of the present time-resolved FTIR results.