Allostery in the nitric oxide dioxygenase mechanism of flavohemoglobin.
Allostery in the nitric oxide dioxygenase mechanism of flavohemoglobin.
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DOI：
10.1074/jbc.ra120.016637
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Gardner PR
中科院分区：
文献类型：
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作者：
Gardner AM;Gardner PR
底物O2和NO协同激活大肠杆菌黄素血红蛋白的NO双加氧酶功能。稳态和瞬态动力学测量支持基于结构的机制模型，其中O2和NO的运动和保守的氨基酸在E11，G8，E2，E7，B10和F7的位置内的珠蛋白域控制激活。在合作和变构机制，O2迁移到催化血红素网站通过一个长的疏水隧道和取代LeuE 11远离三价铁，这迫使打开一个短隧道的ValG 8/IleE 15对和LeuE 11门控的催化位点。NO渗透该通道并利用触发CD环的门控侧链以卷起，其移动E和F-螺旋并切换由LysF 7、GlnE 7和水形成的电子转移门。这允许FADH 2还原三价铁，其形成稳定的铁-超氧化物-TyrB 10/GlnE 7复合物。这种复合物与内化的NO反应，双分子速率常数为1010 M−1 s−1，形成硝酸盐，硝酸盐迁移到CD环并展开弹簧状结构。为了重新开始循环，LeuE 11切换回三价铁。通过O2和NO运动激活电子转移，避免了不可逆的NO中毒和酶的还原失活。结构快照和动力学常数一起提供了中间构象状态、运动时间尺度和相关能量的一瞥。
The substrates O2 and NO cooperatively activate the NO dioxygenase function of Escherichia coli flavohemoglobin. Steady-state and transient kinetic measurements support a structure-based mechanistic model in which O2 and NO movements and conserved amino acids at the E11, G8, E2, E7, B10, and F7 positions within the globin domain control activation. In the cooperative and allosteric mechanism, O2 migrates to the catalytic heme site via a long hydrophobic tunnel and displaces LeuE11 away from the ferric iron, which forces open a short tunnel to the catalytic site gated by the ValG8/IleE15 pair and LeuE11. NO permeates this tunnel and leverages upon the gating side chains triggering the CD loop to furl, which moves the E and F-helices and switches an electron transfer gate formed by LysF7, GlnE7, and water. This allows FADH2 to reduce the ferric iron, which forms the stable ferric–superoxide–TyrB10/GlnE7 complex. This complex reacts with internalized NO with a bimolecular rate constant of 1010 M−1 s−1 forming nitrate, which migrates to the CD loop and unfurls the spring-like structure. To restart the cycle, LeuE11 toggles back to the ferric iron. Actuating electron transfer with O2 and NO movements averts irreversible NO poisoning and reductive inactivation of the enzyme. Together, structure snapshots and kinetic constants provide glimpses of intermediate conformational states, time scales for motion, and associated energies.