Iron entry route in horse spleen apoferritin. Involvement of the three-fold channels as probed by selective reaction of cysteine-126 with the spin label 4-maleimido-tempo.

The research article investigates how iron enters the protein structure of apoferritin in horse spleen, with primary attention placed on the role of the three-fold channels. Cysteine-126, located in these channels, was selectively labeled with a particular derivative and then titrated with iron. They found that at lower iron-to-protein ratios, a magnetic dipolar interaction occurs, reducing the intensity of the label’s electron paramagnetic resonance (EPR) signal, with the distance between the label and metal estimated to be 8-12 Angstrom. These results suggest the iron binds within these channels in higher oxidation states, signifying them as the method of metal entry.

Methodology

• The team used apoferritin, a protein primarily found in horse spleen, for the experiment.
• They labeled Cysteine-126, located in the hydrophilic 3-fold channels of this protein, with a maleimide nitroxide derivative. The aim of using this derivative was to track iron’s interaction within the protein.
• A titration process with Fe(II), an ionic version of iron that forms the initial Fe(III)-apoferritin complex, was conducted.

Results

• Upon titration, the researchers observed that the intensity of the EPR signal from the derivative label decreased. This decline was due to the magnetic dipolar interaction at lower metal-to-protein ratios.
• The distance between the label and metal was estimated, through titrations done with VO(IV), to be between 8-12 Angstrom. VO(IV) was used because it is known to bind in the 3-fold channels as well and also results in a decrease of the EPR signal from the label.

Conclusions

• The decrease in the EPR signal from the label and the magnetic dipolar interaction indicate that iron binds in the hydrophilic channels of the protein in a higher oxidation state.
• The observation suggests that these channels function as the routes of metal entry into the protein structure, at least when the metal-to-protein ratios are low.