Stability and iron oxidation properties of a novel homopolymeric plant ferritin from adzuki bean seeds: a comparative analysis with recombinant soybean seed H-1 chain ferritin.

Background: All reported plant ferritins are heteropolymers comprising two different H-type subunits. Whether or not homopolymeric plant ferritin occurs in nature is an open question.

Methods: A homopolymeric phytoferritin from adzuki bean seeds (ASF) was obtained by various protein purification techniques for the first time, which shares the highest identity (89.6%) with soybean seed H-1 ferritin (rH-1). Therefore, we compared iron oxidation activity and protein stability of ASF with those of rH-1 by stopped-flow combined with light scattering or UV/Vis spectrophotography, SDS- and native- PAGE analyses. Additionally, a new rH-1 variant (S68E) was prepared by site-directed mutagenesis approach to elucidate their difference in protein stability.

Results: At high iron loading of protein, the extension peptide (EP) of plant ferritin was involved in iron oxidation, and the EP of ASF exhibited a much stronger iron oxidative activity than that of rH-1. Besides, ASF is more stable than rH-1 during storage, which is ascribed to one amino acid residue, Ser68.

Conclusions: ASF exhibits a different mechanism in iron oxidation from rH-1 at high iron loading of protein, and a higher stability than rH-1. These differences are mainly stemmed from their different EP sequences.

General Significance: This work demonstrates that plant cells have evolved the EP of phytoferritin to control iron chemistry and protein stability by exerting a fine tuning of its amino acid sequence.