Cavity acidification limits ferritin iron biomineralization

Area of Science:

• Biochemistry
• Biomineralization
• Protein Engineering

Background:

• Ferritin biomineralization studies focus on iron pathways and oxidation mechanisms.
• Spatiotemporal details of proton generation during iron hydrolysis are less understood.

Purpose of the Study:

• Investigate proton generation and release during ferroxidase reactions within Archaeoglobus fulgidus ferritin (AfFtn).
• Utilize site-specific labeling and fluorescence probes to monitor internal proton activity.

Main Methods:

• Engineered cysteines on AfFtn interior surface for covalent labeling with fluorescein-5-maleimide (F5M).
• Performed ferroxidase reactions and monitored F5M fluorescence quenching and bulk solution pH.
• Reassembled AfFtn 24mer cage from dimers at high ionic strength.

Main Results:

• F5M labeled at D61C (C61-F5M) effectively reported proton activity without Fe ion quenching.
• Internal cavity pH dropped to apparent 5.5 within 15s of Fe2+ addition.
• Ferritin released 1.6H+ per Fe2+ oxidized, retaining 0.4H+, indicating internal proton accumulation.

Conclusions:

• Ferritin accumulates protons within its cavity, acting as a regulatory brake on iron biomineralization.
• Internal proton buffering influences the kinetics of iron oxidation and release.
• The study provides novel insights into the spatiotemporal dynamics of proton activity in ferritin.