Five non-symbiotic hemoglobins (nsHb) have been identified in rice (Oryza sativa). Previous studies have shown that stress conditions can induce their overexpression, but the role of those globins is still unclear. To better understand the functions of nsHb, the reactivity of rice Hb1 toward hydrogen peroxide (H₂O₂) has been studied in vitro. Our results show that recombinant rice Hb1 dimerizes through dityrosine cross-links in the presence of H₂O₂. By site-directed mutagenesis, we suggest that tyrosine 112 located in the FG loop is involved in this dimerization. Interestingly, this residue is not conserved in the sequence of the five rice non-symbiotic hemoglobins. Stopped-flow spectrophotometric experiments have been performed to measure the catalytic constants of rice Hb and its variants using the oxidation of guaiacol. We have shown that Tyrosine112 is a residue that enhances the peroxidase activity of rice Hb1, since its replacement by an alananine leads to a decrease of guaiacol oxidation. In contrast, tyrosine 151, a conserved residue which is buried inside the heme pocket, reduces the protein reactivity. Indeed, the variant Tyr151Ala exhibits a higher peroxidase activity than the wild type. Interestingly, this residue affects the heme coordination and the replacement of the tyrosine by an alanine leads to the loss of the distal ligand. Therefore, even if the amino acid at position 151 does not participate to the formation of the dimer, this residue modulates the peroxidase activity and plays a role in the hexacoordinated state of the heme.

Graphical abstract

中文翻译：

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水稻 (Oryza sativa) 血红蛋白的过氧化物酶活性：酪氨酸 112 和 151 的独特作用

水稻 ( Oryza sativa )中已鉴定出五种非共生血红蛋白 (nsHb) 。先前的研究表明，应激条件可以诱导它们的过度表达，但这些珠蛋白的作用仍不清楚。为了更好地了解 nsHb 的功能，在体外研究了水稻 Hb1 对过氧化氢 (H ₂ O _{2 ) 的反应性。}我们的结果表明，在 H ₂ O ₂存在的情况下，重组水稻 Hb1 通过二酪氨酸交联二聚化。通过定点诱变，我们认为位于 FG 环中的酪氨酸 112 参与了这种二聚化。有趣的是，该残基在五种水稻非共生血红蛋白的序列中并不保守。已进行停流分光光度实验，利用愈创木酚的氧化来测量大米 Hb 及其变体的催化常数。我们已经证明酪氨酸 112 是一种增强水稻 Hb1 过氧化物酶活性的残基，因为它被丙氨酸取代会导致愈创木酚氧化减少。相反，酪氨酸 151（埋藏在血红素口袋内的保守残基）会降低蛋白质反应性。事实上，变体 Tyr151Ala 表现出比野生型更高的过氧化物酶活性。有趣的是，该残基影响血红素配位，并且用丙氨酸替代酪氨酸导致远端配体的丢失。因此，即使151位的氨基酸不参与二聚体的形成，该残基也调节过氧化物酶活性并在血红素的六配位状态中发挥作用。

图形概要