Intra- and intermolecular transfers of protein radicals in the reactions of sperm whale myoglobin with hydrogen peroxide
Intra- and intermolecular transfers of protein radicals in the reactions of sperm whale myoglobin with hydrogen peroxide
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DOI：
10.1074/jbc.m304726200
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发表时间：
2003-09-19
影响因子：
4.8
通讯作者：
de Montellano, PRO
中科院分区：
文献类型：
--
作者：
Lardinois, OM;de Montellano, PRO
抹香鲸高铁肌红蛋白(SwMb)与H_2O_2反应生成铁基(MbFe(IV)=O)和蛋白质自由基，并导致低聚产物的形成。二聚体(28%)和三聚体(17%)的最大产率为1或2等当量。SwMb Y151F突变株与天然apoSwMb和H_2O_2共孵育产生二聚体产物，这需要从非二聚突变株Y151F向apoSwMb进行自由基转移。铁基SwMb的自动还原为铁态是两相的，t=3.4和25.9min。在低蛋白质浓度下存在分子内自动还原过程，但在蛋白质浓度较高时，齐聚作用会降低铁基物种的寿命。部分蛋白质仍然是单体。这种抗二聚化的蛋白质处于铁基状态，但在自动还原后，它与过氧化氢进行了正常的二聚化。蛋白水解性消化证实了低聚蛋白质中同时存在双酪氨酸和异剂量酪氨酸交联链，其中等剂量酪氨酸链主要由Tyr(151)-Tyr(151)偶联形成。二聚体中的酪氨酸含量降低了47%，回收单体中的酪氨酸含量降低了14%，但二聚体中异剂量酪氨酸和二羟甲基酪氨酸的产率仅为原始酪氨酸含量的15.2%和6.8%。因此，大约23%的丢失的酪氨酸有另一种但未知的命运。结果清楚地证明了涉及Mb蛋白自由基的分子内和分子间电子转移过程是同时发生的。在旁观者蛋白上产生蛋白质自由基的分子间电子转移可能会传播由金属蛋白与过氧化氢反应引发的细胞损伤。
Reaction of sperm whale metmyoglobin (SwMb) with H2O2 produces a ferryl (MbFe(IV)=O) species and a protein radical and leads to the formation of oligomeric products. The ferryl species is maximally formed with one equivalent of H2O2, and the maximum yields of the dimer (28%) and trimer (17%) with 1 or 2 eq. Co-incubation of the SwMb Y151F mutant with native apoSwMb and H2O2 produced dimeric products, which requires radical transfer from the nondimerizing Y151F mutant to apoSwMb. Autoreduction of ferryl SwMb to the ferric state is biphasic with t = 3.4 and 25.9 min. An intramolecular autoreduction process is implicated at low protein concentrations, but oligomerization decreases the lifetime of the ferryl species at high protein concentrations. A fraction of the protein remained monomeric. This dimerization-resistant protein was in the ferryl state, but after autoreduction it underwent normal dimerization with H2O2. Proteolytic digestion established the presence of both dityrosine and isodityrosine cross-links in the oligomeric proteins, with the isodityrosine links primarily forged by Tyr(151)-Tyr(151) coupling. The tyrosine content decreased by 47% in the dimer and 14% in the recovered monomer, but the yields of isodityrosine and dityrosine in the dimer were only 15.2 and 6.8% of the original tyrosine content. Approximately 23% of the lost tyrosines therefore have an alternative but unknown fate. The results clearly demonstrate the concurrence of intra- and intermolecular electron transfer processes involving Mb protein radicals. Intermolecular electron transfers that generate protein radicals on bystander proteins are likely to propagate the cellular damage initiated by the reaction of metalloproteins with H2O2.