Insertion of an H-Bonding Residue into the Distal Pocket of the Ferriheme Protein Nitrophorin 4: Effect on Nitrite?Iron Coordination and Nitrite Disproportionation
Insertion of an H-Bonding Residue into the Distal Pocket of the Ferriheme Protein Nitrophorin 4: Effect on Nitrite?Iron Coordination and Nitrite Disproportionation
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DOI：
10.1002/cbdv.201100401
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发表时间：
2012-09-01
影响因子：
2.9
通讯作者：
Knipp, Markus
中科院分区：
文献类型：
--
作者：
He, Chunmao;Ogata, Hideaki;Knipp, Markus
血红素蛋白是亚硝酸盐代谢的重要实体。对所报告的血红素蛋白亚硝酸盐晶体结构的结构特征的检查表明，除硝基磷酸蛋白 4 (NP4) 外，与亚硝酸盐配体的氢键合是通过组氨酸或精氨酸残基完成的。这些氢键可能对亚硝酸盐配位和/或反应性发挥重要作用。在催化亚硝酸盐歧化反应的硝基传递蛋白中，缺少这样的残基。在这里，我们报告了 NP 同蛋白 NP4 的 L130R 突变体，它提供了 Arg130 残基作为柔性 G?H 环的一部分，作为远端血红素口袋中潜在的氢键残基。与野生型蛋白质类似，亚硝酸盐在 NP4(L130R) 的晶体结构中保持 N 键合。然而，光谱研究表明，在溶液中，存在第二配体旋转方向，它与正常的平行配体方向处于快速交换平衡。此外，NP4(L130R)中的亚硝酸歧化作用受到抑制。与另一个活性较低的突变体 NP4(D30N) 的比较表明，H2O 分子从血红素腔中的置换阻止了通过 Asp30 的质子捐赠途径。
Heme proteins are important entities for the metabolism of nitrite. Inspection of the structural features of the reported hemoprotein?nitrite crystal structures reveals that, except for nitrophorin 4 (NP4), H-bonding to the nitrite ligand is accomplished via histidine or arginine residues. These H-bonds probably play an important role for the nitrite coordination and/or reactivities. In nitrophorins, which catalyze the nitrite disproportionation reaction, such a residue is missing. Here, we report on the L130R mutant of the NP isoprotein NP4 that provides the Arg130 residue as part of the flexible G?H loop as a potential H-bonding residue in the distal heme pocket. Similar to the wild-type protein, nitrite remains N-bonded in the crystal structure of NP4(L130R). However, spectroscopic investigations show that, in solution, a second ligand-rotational orientation exists, which is in fast-exchange equilibrium with the normal, parallel ligand orientation. Moreover, the nitrite disproportionation is inhibited in NP4(L130R). Comparison with another, also less active mutant NP4(D30N) suggests that the displacement of H2O molecules from the heme cavity prevents the proton donation pathway through Asp30.