Uranyl-cytochrome B₅and Nitrite-myoglobin Interactions

Uranium is harmful to human health due to its radiation damage and the abilityof uranyl ion (UO22+) to interact with various proteins and disturb their biologicalfunctions. Cytochrome b5(cyt b5) is a highly negatively charged heme protein andplays a key role in mediating cytochrome c(cyt c) signaling in apoptosis by forming adynamic cyt b5-cyt c complex. In previous molecular modeling study in combinationwith UV–Vis studies, we found that UO22+is capable of binding to cyt b5at surfaceresidues, Glu37and Glu43. In this study, we further investigated the structuralconsequences of cyt b5and cyt c, as well as cyt b5-cyt c complex, upon uranyl binding,by fluorescence spectroscopic and circular dichroism techniques. Moreover, weproposed a uranyl binding site for cyt c at surface residues, Glu66and Glu69, byperforming a molecular modeling study. It was shown that uranyl binds to cyt b5(KD=10μM), cyt c (KD=87μM), and cyt b5-cyt c complex (KD=30μM) with a differentaffinity, which slightly alters the protein conformation and disturbs the interaction ofcyt b5-cyt c complex. Additionally, we investigated the functional consequences ofuranyl binding to the protein surface, which decreases the inherent peroxidase activityof cyt c. The information of uranyl-cyt b5/cyt c interactions gained in this study likelyprovides a clue for the mechanism of uranyl toxicity.Although with a distinct heme active site, both myoglobin (Mb) andcytochrome c oxidase (CcO) were found to function as a nitrite reductase (NIR) underhypoxic conditions. To probe the mediation effects of introducing distal histidine (His)and/or tyrosine (Tyr) in heme active site on the NIR activity of Mb, we preparedL29H Mb, F43H Mb, F43Y Mb, L29H/F43H Mb and L29H/F43YMb mutants, andsolved the X-ray crystal structure of L29H Mb and L29H/F43Y Mb. By making bothstructure and NIR activity comparisons of these mutants, we found that introduction of His and/or Tyr in the heme active site of Mb leads to the formation of a differentdistal hydrogen network, and inhibits the NIR activity to a different extent.Meanwhile, the reductase activity of both L29H Mb and L29H/F43H Mb areregulated by metal ions such as Cu(II) or Zn(II) binding to the distal histidines,i.e.,Cu(II) enhances the reactivity while Zn(II) inhibits the reactivity. These findingsprovide valuable insights into the structure and function relationship for Mbfunctioning as a NIR in biological system. Moreover, this study shows that thestructure and function of a heme protein can be regulated by design of a hydrogennetwork in the active site and metal ions.