Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding
Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding
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DOI：
10.1074/jbc.m109.098269
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发表时间：
2010-06-04
影响因子：
4.8
通讯作者：
Marletta, Michael A.
中科院分区：
文献类型：
--
作者：
Derbyshire, Emily R.;Deng, Sarah;Marletta, Michael A.
一氧化氮（NO）是哺乳动物血红素蛋白可溶性鸟苷酸环化酶（sGC）的生理相关激活剂。α 1 β 1 sGC的血红素辅因子对NO具有高亲和力，但从未观察到与氧形成复合物。在sGC血红素结合结构域β 1（1-385）中引入关键的酪氨酸残基足以产生氧结合蛋白，但全长酶中的这种突变并没有改变氧亲和力。为了评估全长sGC中的配体结合特异性，我们突变了几个保守的远端血红素口袋残基（β 1瓦尔-5、Phe-74、Ile-145和Ile-149）以在血红素配体附近引入氢键供体。我们发现，NO配位状态，NO解离，和酶的活化显着影响的存在下，酪氨酸在远端血红素口袋，但是，稳定性的减少卟啉和蛋白质对氧的亲和力不变。最近，来自果蝇的非典型sGC，Gyc-88 E，被证明与氧形成稳定的复合物。该蛋白质的序列分析确定了两个残基的预测血红素口袋（酪氨酸和谷氨酰胺），可能发挥作用，以稳定在非典型环化酶的氧结合。将这些残基引入大鼠β 1远端血红素口袋（Ile-145 -> Tyr和Ile-149 -> Gln）导致sGC构建体通过在417 nm处具有最大吸光度的中间体氧化。该吸收最大值与球蛋白Fe-II-O-2复合物一致，并且可能是在全长α 1 β 1蛋白中首次观察到FeII-O-2复合物。此外，这些数据表明，非典型sGC通过涉及酪氨酸和谷氨酰胺的氢键网络稳定O-2结合。
Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of alpha 1 beta 1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain beta 1(1-385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (beta 1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat beta 1 distal heme pocket (Ile-145 -> Tyr and Ile-149 -> Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe-II-O-2 complexes and is likely the first observation of a FeII-O-2 complex in the full-length alpha 1 beta 1 protein. Additionally, these data suggest that atypical sGCs stabilize O-2 binding by a hydrogen bonding network involving tyrosine and glutamine.