A spectroscopic study of uranyl-cytochrome b5/cytochrome c interactions

Uranium is harmful to human health due to its radiation damage and the ability of uranyl ion (UO22+) to interact with various proteins and disturb their biological functions. Cytochrome b(5) (cyt b(5)) is a highly negatively charged heme protein and plays a key role in mediating cytochrome c (cyt c) signaling in apoptosis by forming a dynamic cyt b(5)-cyt c complex. In previous molecular modeling study in combination with UV-Vis studies, we found that UO22+ is capable of binding to cyt b(5) at surface residues, Glu37 and Glu43. In this study, we further investigated the structural consequences of cyt b(5) and cyt c, as well as cyt b(5)-cyt c complex, upon uranyl binding, by fluorescence spectroscopic and circular dichroism techniques. Moreover, we proposed a uranyl binding site for cyt c at surface residues, Glu66 and Glu69, by performing a molecular modeling study. It was shown that uranyl binds to cyt b(5) (K-D = 10 mu M), cyt c (KD = 87 mu M and cyt b(5)-cyt c complex (KD = 30 mu M) with a different affinity, which slightly alters the protein conformation and disturbs the interaction of cyt b(5)-cyt c complex. Additionally, we investigated the functional consequences of uranyl binding to the protein surface, which decreases the inherent peroxidase activity of cyt c. The information of uranyl-cyt b(5)/cyt c interactions gained in this study likely provides a clue for the mechanism of uranyl toxicity. (C) 2013 Elsevier B.V. All rights reserved.