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

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 M iron/subunit/s) than rH-1 (0.59 ؎ 0.07 M iron/subunit/s) and rH-2 (0.48 ؎ 0.04 M 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 ϳ10 times larger than those of rH-1 and rH-2. This helps elucidate the widespread occurrence of heteropolymeric ferritins in plants.Iron and oxygen chemistry, in a variety of non-heme diiron proteins, has drawn considerable attention because of their various roles in reversible O 2 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 ferritin (1-6). Diiron centers have similar structural motifs containing a combination of carboxylate and histidine ligands that either bind or bridge the two metal ions of the dinuclear active site. Although the dinuclear centers of the protein are very similar in structure, each of the proteins fulfills a different biological function that appears to be mediated by the nature of the first and second coordination sphere of the diiron center.Ferritins are a special class of diiron proteins that play a role in both iron housekeeping and iron detoxification. They are widely distributed in animals, plants, and bacteria (but not in yeast) and are typically composed of 24 similar or identical subunits assembled into a shell-like structure. In vertebrates, ferritins consist of two types of subunits, H and L, respectively. The two types have about 55% identity in amino acid sequence.

Soybean Ferritin Expression in Saccharomyces cerevisiae Modulates Iron Accumulation and Resistance to Elevated Iron Concentrations

“…The present work actually showed that soybean ferritins protected ccc1⌬ yeast cells from iron toxicity, despite previous studies that used human ferritins not being able to show a protective effect when expressed in a ccc1⌬ strain (63). Previous in vitro studies reported that SFerH1/SFerH2 heteropolymers display greater iron oxidation activity and DNA protection from oxidative damage during iron oxidative deposition than soybean ferritin homopolymers (30,64). Here, it was observed that simultaneously expressed SFerH1 and SFerH2 assembled into heteropolymers in yeast.…”

“…However, in pea seeds, Ͼ90% of iron is stored in phytoferritin, which is required for plant germination and early growth (27,28). In vitro work has shown that the SFerH1 and SFerH2 subunits synergistically interact during iron mineralization by exhibiting greater iron oxidation activity than separate homopolymers (29,30). Soybean ferritin has been used to enrich cereals with iron (31)(32)(33).…”

Isolation and characterization of a new phytoferritin from broad bean (Vicia faba) seed with higher stability compared to pea seed ferritin

“…So far, the amino acids involved in the definition of the ferroxidase center are highly conserved in all reported plant ferritins, but these ferritins are different in the catalyzing activity with each other, suggesting that amino acid residues nearby the ferroxidase centers or other amino acids have an effect on such activity. Consistent with this idea, our recent studies found that recombinant H-1 soybean seed ferritin (rH-1) exhibited a stronger catalytic activity than recombinant H-2 soybean seed ferritin (rH-2), although their ferroxidase centers are identical (Deng, Liao, Yang, Zhang, Hua, Masuda, Goto, Yoshihara, & Zhao, 2010). Another possible reason for the large difference in the catalytic activity between BBSF and PSF is stemmed from their different subunit composition (Fig.…”

“…5). Agreeing with this view, it was observed that, at low iron loading of protein (48 Fe 2 + /protein shell), rH-1 exhibited a stronger iron oxidation activity than its analogs, rH-2, which was attributed to the larger catalytic ability of the ferroxidase centers within H-1 subunit than those in H-2 (Deng, Liao, Yang, Zhang, Hua, Masuda, Goto, Yoshihara, & Zhao, 2010). BBSF contains more H-2 subunits than PSF, so this might be an important reason why it exhibits a slower kinetics as compared to PSF.…”

“…At low iron loading of apoferritin (48 Fe 2 + /protein shell), the initial rate of BBSF (0.068 ± 0.008 μM/subunit/s) is about 6-fold slower than that of PSF (0.35 ± 0.03 μM/subunit/s). The iron oxidation rate of BBSF is likewise much lower than that of soybean seed ferritin (0.47 ± 0.04 μM/subunit/s) under the same experimental conditions (Deng, Liao, Yang, Zhang, Hua, Masuda, Goto, Yoshihara, & Zhao, 2010). The UV absorbance at 300 nm as a function of the Fe 2 + /ferritin ratio was plotted in Fig.…”