Role of H-1 and H-2 Subunits of Soybean Seed Ferritin in Oxidative Deposition of Iron in Protein

Xiangyu Zhang,Toshihiro Yoshihara,Haixia Yang,Fumiyuki Goto,Guanghua Zhao,Jianjun Deng,Zichun Hua,Taro Masuda,Xiayun Liao

doi:10.1074/jbc.m110.130435

Abstract

Naturally occurring phytoferritin is a heteropolymer consisting of two different H-type subunits, H-1 and H-2. Prior to this study, however, the function of the two subunits in oxidative deposition of iron in ferritin was unknown. The data show that, upon aerobic addition of 48-200 Fe(2+)/shell to apoferritin, iron oxidation occurs only at the diiron ferroxidase center of recombinant H1 (rH-1). In addition to the diiron ferroxidase mechanism, such oxidation is catalyzed by the extension peptide (a specific domain found in phytoferritin) of rH-2, because the H-1 subunit is able to remove Fe(3+) from the center to the inner cavity better than the H-2 subunit. These findings support the idea that the H-1 and H-2 subunits play different roles in iron mineralization in protein. Interestingly, at medium iron loading (200 irons/shell), wild-type (WT) soybean seed ferritin (SSF) exhibits a stronger activity in catalyzing iron oxidation (1.10 ± 0.13 μm iron/subunit/s) than rH-1 (0.59 ± 0.07 μm iron/subunit/s) and rH-2 (0.48 ± 0.04 μm iron/subunit/s), demonstrating that a synergistic interaction exists between the H-1 and H-2 subunits in SSF during iron mineralization. Such synergistic interaction becomes considerably stronger at high iron loading (400 irons/shell) as indicated by the observation that the iron oxidation activity of WT SSF is ∼10 times larger than those of rH-1 and rH-2. This helps elucidate the widespread occurrence of heteropolymeric ferritins in plants.

Highlights

Iron and oxygen chemistry, in a variety of non-heme diiron proteins, has drawn considerable attention because of their various roles in reversible O2 binding for respiration in hemerythrin, oxidation and desaturation of organic substrates in methane monooxygenase, R2 subunit of ribonucleotide reductase, stearoyl-acyl carrier protein ⌬-9 desaturase, as well as the detoxification and concentration of iron in Dps and fer
Recent studies of our group reveal the role of the extension peptide (EP) during iron oxidative deposition in phytoferritin as the second binding and ferroxidase center that contributes to mineralization of the iron core at high iron loading of ferritin (Ͼ48 irons/shell) (13)
To shed light on the role of the H-1 and H-2 subunits in SSF for iron oxidative deposition in phytoferritin, we first conducted dynamic and stopped-flow light scattering experiments with H-1 soybean seed ferritin and its analog, rH-2

Summary

Introduction

In a variety of non-heme diiron proteins, has drawn considerable attention because of their various roles in reversible O2 binding for respiration in hemerythrin, oxidation and desaturation of organic substrates in methane monooxygenase, R2 subunit of ribonucleotide reductase, stearoyl-acyl carrier protein ⌬-9 desaturase, as well as the detoxification and concentration of iron in Dps and fer-. The light scattering intensity increased markedly because of protein aggregation when the aporH-2 was rapidly mixed aerobically with a series of Fe2ϩ solutions to give ratios from 96 to 200 Fe2ϩ/shell (Fig. 1A), and the rise in the initial rate, ␯0, is first order with respect to iron concentration (Table 1).

Results

Conclusion