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 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.

FIGURE 1.

A, time course of rH-2 aggregation during Fe²⁺ oxidation. B, time course of rH-2 or EP-deleted rH-2 aggregation upon the addition of Fe²⁺ or Fe³⁺. The curve represents an average of six experimental measurements. C, relative scattered light intensity distribution curves for aporH-2 and aporH-2 plus Fe²⁺ ion. Conditions were as follows: [aporH-2] = 0.5 μm in 100 mm Mops (pH 7.0), 24–100 μm FeSO₄, 25 °C.

FIGURE 2.

A, time course of rH-1 aggregation during Fe²⁺ oxidation. B, time course of rH-1 or EP-deleted rH-1 aggregation upon the addition of Fe²⁺ or Fe³⁺. The curve represents an average of six experimental measurements. C, relative scattered light intensity distribution curves for aporH-1 and aporH-1 plus Fe²⁺ ion. Conditions were as follows: [aporH-1] = 0.5 μm in 100 mm Mops (pH 7.0), 24–250 μm FeSO₄, 25 °C.

FIGURE 3.

MS profiles of EP-1 (A) and EP-2 (B) treated with FeCl₃ (Fe³⁺/EP ratio = 10:1) acquired by MALDI-TOF-MS. Conditions were as follows: [EP-1 or EP-2] = 140 μm in 5 mm Mops, pH 7.0.

FIGURE 4.

A, time course of WT SSF aggregation during Fe²⁺ oxidation. B, time course of WT SSF or EP-deleted SSF aggregation upon the addition of Fe²⁺ or Fe³⁺. The curve represents an average of six experimental measurements. C, relative scattered light intensity distribution curves for apoSSF and apoSSF plus Fe²⁺ ion. Conditions were as follows: [WT SSF] = 0.5 μm in 100 mm Mops (pH 7.0), 24–100 μm FeSO₄, 25 °C.

FIGURE 5.

A, regeneration of oxidation activities in rH-1, rH-2, and WT SSF as normalized initial rates. Each injection is half-saturation of the ferroxidase sites, 1 Fe²⁺/site. Conditions were as follows: [aporH-1, aporH-2, or WT apoSSF] = 2.0 μm in 0.15 m NaCl and 100 mm Mops, pH 7.0, 25 °C, time intervals between injections 3 min. Error bars, S.E. B, change of relative fluorescence intensity of apoferritin after the aerobic addition of 48 Fe²⁺ atoms/shell. Conditions were as follows: λ_ex = 280 nm, λ_em = 325 nm, [aporH-1, aporH-2, or WT apoSSF] = 0.5 μm in 0.15 m NaCl and 0.1 m Mops, pH 7.0, 25 °C.

FIGURE 6.

Time course of the formation and dissociation of rH-1 (A), rH-2 (B), and WT SSF (C) aggregates during the oxidative deposition of iron in the protein shell. The curve represents an average of six independent experimental measurements. Conditions were as follows: [aporH-1, aporH-2, or WT apoSSF] = 0.5 μm in 100 mm Mops (pH 7.0), 200 μm Fe²⁺, 25 °C.

FIGURE 7.

Kinetic curves of the formation and dissociation of rH-1 (A), rH-2 (B), and WT SSF (C) aggregates during the oxidative deposition of iron in the protein shell. The curve represents an average of six independent experimental measurements. Conditions were as follows: 0.5 μm apoferritin in 100 mm Mops (pH 7.0), 72 μm Fe³⁺, 25 °C.

FIGURE 8.

Kinetic curves of Fe²⁺ oxidation by O₂ in aporH-1, aporH-2, WT apoSSF, and aporH-1H-2 at different Fe²⁺ concentrations, 48 (A), 200 (B), and 400 Fe²⁺/shell (C). Conditions were as follows: final [aporH-1, aporH-2, aporH-1H-2, or WT apoSSF] = 0.5 μm in 100 mm Mops (pH 7.0), 24–200 μm FeSO₄, 25 °C.

REACTION 1