Dynamics of iron release from transferrin N-lobe studied by electrospray ionization mass spectrometry

Transferrins constitute a class of metalloproteins that are involved in cir culatory iron transport in a variety of species. The metal ion-binding prop erties of these proteins have been die focus of extensive research efforts in the past decade due to their extreme importance in a variety of biologic al and healthcare-related fields. The large size of these proteins, as well as the presence of high-spin metal ions (e.g,, Fe3+), limits the use of NM R, In this work, we report on the use of electrospray ionization mass spect rometry (ESI MS) to study dynamics of the transferrin system in vitro under conditions that are designed to mimic the endosomal environment. ESI MS is shown to provide valuable insights into the mechanistic aspects of metal i on-binding/release by transferrins and is complementary to other spectrosco pic techniques. Conformational stability of the complex is evaluated based on the appearance of the charge-state distribution of protein ions, while t he composition of the protein-ligand complex is determined based on the mas s of the protein ions. In the absence of iron chelators, a stepwise dissoci ation of the ternary complex (protein-metal ion-synergistic anion) is obser ved as the solution pH is gradually decreased. Although the release of syne rgistic anion from the complex is initiated at typical endosomal pH levels (i.e., 5.5), metal ion remains largely bound to the protein until the pH is lowered to a level of similar to4.5. Under these conditions, a significant fraction of the protein populates unfolded conformations, In stark contras t to this behavior, addition of an iron chelating agent (citrate) to the pr otein solution results in facile iron release at typical endosomal pH level s without any detectable unfolding of the protein. The mass spectral data l ends further credibility to the notion that the holoprotein samples conform ations that are specific to the apo form (e.g, "open conformation"), from w hich iron dissociation most likely occurs. The results of the present study demonstrate that ESI MS can be used to model metal ion release from transf errin under conditions that are designed to mimic the physiological environ ment.