Insight into the structural deterioration of biosynthesized holoferritin upon thermal treatment.

Holoferritin is considered a promising iron supplement, yet its preparation is challenging due to low extraction efficiencies from natural sources and the potential for structural damage during in vitro mineralization. This study reported the in vivo biosynthesis of a highly stable holoferritin (bs-holoFt) in Escherichia coli a high iron-loading capacity (1213 Fe atoms/protein) and systematically characterized the impact of heat treatments (70-100 °C) on the protein's multi-level structure and dual functions. Results showed a clear, temperature-dependent degradation pathway, initiated by the loss of α-helical content (decreased from 77.3 % to 33 %) and unfolding of the tertiary structure. This denaturation drove the subsequent disassembly of the quaternary shell and promoted aggregation, culminating in large, non-native aggregates (~260 nm) at 100 °C. Critically, this structural decay was directly correlated with a severe loss of function. Both the rate of reductive iron release and the peroxidase-like nanozyme activity decreased in a strikingly parallel manner, retaining 17.7 % and 15.3 % of their initial capacity after treatment at 100 °C, respectively. We conclude that this functional impairment is primarily caused by the collapse and blockage of the protein's molecular channels upon unfolding. This work not only presents a robust method for producing highly stable holoferritin but also provides a fundamental understanding of its denaturation mechanism, offering a theoretical basis for optimizing its use in iron supplements and other nanotechnology applications.