Direct measurement by laser flash photolysis of intramolecular electron transfer in a two-domain construct of murine inducible nitric oxide synthase
Direct measurement by laser flash photolysis of intramolecular electron transfer in a two-domain construct of murine inducible nitric oxide synthase
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DOI：
10.1021/ja0578606
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发表时间：
2006-03-22
影响因子：
15
通讯作者：
Enemark, JH
中科院分区：
文献类型：
--
作者：
Feng, CJ;Thomas, C;Enemark, JH
从 FMN 到血红素的亚基间分子内电子转移 (IET) 对于血红素结构域中 O-2 激活以及随后由 NO 合酶 (NOS) 合成一氧化氮 (NO) 所需的电子传递至关重要。先前的晶体结构和功能研究主要涉及酶构象，该构象充当还原酶结构域中来自 NADPH 和 FAD 的电子还原 FMN 的输入状态。为了有利于加氧酶结构域中从 FMN 到血红素的后续 IET 的输出状态的形成，设计并表达了一种新型截短的双结构域 oxyFMN 构建体鼠诱导型一氧化氮合酶 (iNOS)，其中仅存在 FMN 和血红素结构域。在部分还原的 oxyFMN 和单域血红素加氧酶构建体的比较研究中，使用 CO 解离的激光闪光光解直接确定了该构建体中 FMN 和血红素结构域之间的 IET 动力学。
Intersubunit intramolecular electron transfer (IET) from FMN to heme is essential in the delivery of electrons required for O-2 activation in the heme domain and the subsequent nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation that serves as the input state for reduction of FMN by electrons from NADPH and FAD in the reductase domain. To favor formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of the IET between the FMN and heme domains in this construct was directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single domain heme oxygenase constructs.