Directly measured ferritin cavity acidification during iron oxidation and hydrolysis.

Demonstrated that ferritin cavity acidification can regulate iron biomineralization.

Developed fluorescence methods for measuring pH inside and outside ferritin in the presence of iron.

Proposed a kinetic model of directional proton handling in ferritin biomineralization.

Studies of ferritin biomineralization have elucidated iron loading/egress pathways and mechanisms of iron oxidation, while paying less attention to the spatiotemporal details of proton generation during iron hydrolysis. Here, we performed ferroxidase reactions with Archaeoglobus fulgidus ferritin (AfFtn) 24mer cage, which allows site-specific labeling as a dimer at low ionic strength and reassembly into a 24mer at high ionic strength. Cysteines engineered on the ferritin interior surface were covalently labeled with fluorescein-5-maleimide (F5M), which reported the dynamic changes in proton activity during ferroxidase chemistry. F5M labeled at D61C was highly responsive to proton generation and release, without being vulnerable to fluorescence quenching by Fe2+/3+. C61-F5M fluorescence quenching was maximal within 15 s of stoichiometric Fe2+ addition, and corresponded to an apparent pH value of 5.5 in the cavity. C61-F5M recovered ∼25% of the original “pre‑iron” fluorescence signal on the 5-min timescale but did not recover further with longer incubation. A complementary fluorescein-labeled peptide in bulk solution showed immediate fluorescence quenching, consistent with direct proton release from the ferroxidase center. Solution pH measurements revealed additional acidification on the 5-min timescale, consistent with the kinetics of proton egress from the ferritin cavity. The external and internal pH probes indicated that ferritin releases into solution a total of 1.6H+ for each Fe2+ oxidation, while retaining 0.4H+. This agrees with prior measurements of 2 total H+ per Fe2+ oxidation, and now reveals ferritin's propensity to accumulate protons within the protein cavity, which serves as a “brake” on iron biomineralization.

Ferroxidase activity

pH sensing

Fluorescein

Proton transfer

Iron hydrolysis

Archaeoglobus fulgidus ferritin

fluorescein-5-maleimide

size exclusion chromatography

dynamic light scattering

tetramethylrhodamine-5-maleimide

6-carboxyfluorescein

penta-alanine peptide with 6-carboxyfluorescein conjugated at the C-terminal Lys

© 2026 The Authors. Published by Elsevier Inc.