A Theoritical And Experimental Research On Interactions Between Uranyl Ions And Cytochrome B₅

The stablest state of ranium is U （VI）, forming water soluble ion, UO₂²⁺. UO₂²⁺has a high biological toxicity, which is due to the ability of UO₂²⁺forming stablecomplexes with a series of proteins, whereby disrupting the normal biologicalfunctions of these protein.Cytochrome b₅（Cyt b₅） is a heme protein, which is widely distributed invarious biological microsomal and mitochondrial outer membrane, and functions asan electron transfer. Addtionally, Cyt b₅likely plays a crucial role in apoptosis.Provious study showed that UO₂²⁺interacts with protein major throughcoordination interactions with a series of acidic surface residues such as Glutamate.Moreover, there are many acidic residues on the surface of cyt b₅, forming so-called“acidic cluster”. Based on these information, we relized that UO₂²⁺has a largepossibility to interact with cyt b₅,and impacts on the structure and function of cyt b₅.Therefore, we herein performed such a study, both theretically and experimentally.Theoretically,we investigated the interactions of UO₂²⁺with cyt b₅as well as itsmutant cyt b₅H39S by molecular dynamics simulation, and obtained the theoreticalsturture of UO₂²⁺-cyt b₅and UO₂²⁺-cyt b₅H39S complexes that otherwise can hardlybe obtained experimentally, which thus provides theoretical guide for experiment.Experimently, we first monitored the effect of UO₂²⁺binding on proteinstructure of cyt b₅and its mutant cyt b₅H39S, using UV-Vis and fluorescencespectroscopies. In UV-vis spectra, although only a sligh spectral change wasobserved for cyt b₅in the process of uranyl titration, the spectrum of the mutant cytb₅H39S decreased obviously, which suggests that UO₂²⁺does interact with cyt b₅surface to some extent. In fluorescence spectra, the inherent fluorescence （Trp22） of cyt b₅deceased in the process of uranyl titration, whereas that of cyt b₅H39Sincreased slightly, suggesting that UO₂²⁺has a more greater effect on the structure ofcyt b₅H39S, resulting in a more exposure of Trp22to protein solution. Second, wedetermined the catalytic and electron transfer properties of cyt b₅as well as its cyt b₅H39S mutant, using kinetic spectra and cyclic voltammetry （CV） studies. The resultsshowed that the addition of UO₂²⁺will decrease the peroxidase activity of cyt b₅,H39S, suggesting that UO₂²⁺will alters the structure of the active site of cyt b₅,H39S.Furthermore, the interaction of UO₂²⁺and cyt b₅will affect the electron transferproperty of cyt b₅,as the reduction potential will increase as a result of addition ofUO₂²⁺in cyt b₅solution.The theoritial and experimental research on interactions between uranyl ionsand cyt b₅in this study will provide an insight ino the fact that uranyl inducesapoptosis, which also provides valuable information for biological toxicity of UO₂²⁺.