Native Electron Capture Dissociation Maps to Iron-Binding Channels in Horse Spleen Ferritin

Native electron capture
dissociation (NECD) is a process during
which proteins undergo fragmentation similar to that from radical
dissociation methods, but without the addition of exogenous electrons.
However, after three initial reports of NECD from the cytochrome c dimer complex, no further evidence of the effect has been
published. Here, we report NECD behavior from horse spleen ferritin,
a ∼490 kDa protein complex ∼20-fold larger than the
previously studied cytochrome c dimer. Application
of front-end infrared excitation (FIRE) in conjunction with low- and
high-m/z quadrupole isolation and
collisionally activated dissociation (CAD) provides new insights into
the NECD mechanism. Additionally, activation of the intact complex
in either the electrospray droplet or the gas phase produced c-type fragment ions. Similar to the previously reported
results on cytochrome c, these fragment ions form
near residues known to interact with iron atoms in solution. By mapping
the location of backbone cleavages associated with c-type ions onto
the crystal structure, we are able to characterize two distinct iron
binding channels that facilitate iron ion transport into the core
of the complex. The resulting pathways are in good agreement with
previously reported results for iron binding sites in mammalian ferritin.