A role for highly conserved carboxylate, aspartate-140, in oxygen activation and heme degradation by heme oxygenase-1
A role for highly conserved carboxylate, aspartate-140, in oxygen activation and heme degradation by heme oxygenase-1
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DOI：
10.1021/ja010490a
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发表时间：
2001-07-11
影响因子：
15
通讯作者：
Yoshida, T
中科院分区：
文献类型：
--
作者：
Fujii, H;Zhang, XH;Yoshida, T
血红素加氧酶（HO）通过三个连续的单加氧酶反应催化血红素的氧依赖性降解为胆绿素IX α、CO和游离铁离子。虽然HO与细胞色素P450家族不同的活性位点结构表明其具有独特的远端蛋白质机制来激活分子氧，但其机制和关键氨基酸尚不清楚。为了研究HO-1远端螺旋中高度保守的极性氨基酸的功能，我们制备了丙氨酸突变体：T135 A，R136 A，D140 A和S142 A，并发现D140 A的血红素降解反应发生了剧烈变化。在本文中，我们报告了D140参与HO-1中氧活化机制的第一个证据。血红素复合物HO突变体在这项研究中检查折叠和绑定血红素正常。氧型铁结合水酸自氧化速率的pK（a）值随R136 A、D140 A和S142 A突变而增加，但随T135 A突变而不改变。与野生型一样，T135 A、R136 A和S142 A用H2 O2将血红素降解为绿血红素IX α，用NADPH还原酶系统将其降解为胆绿素IXa。另一方面，D140 A血红素络合物与H2 O2形成化合物II，并且不发生血红素降解。对于NADPH还原酶系统，D140 A血红素复合物的氧化形式在反应中积累，并且只有50%的血红素被降解。停流实验表明，D140 A不能适当地活化铁结合的双氧和氢过氧化物。为了研究D140的羧酸酯官能度，我们进一步用谷氨酸（D140 E）、苯丙氨酸（D140 F）和天冬酰胺（D140 N）替换D140。D140 E正常降解血红素，但D140 N显示出与D140 A相似的反应性。D140 F完全丧失血红素降解活性。所有这些结果表明，在位置140的羧酸是必不可少的激活铁结合的二氧和氢过氧化物。在此基础上，我们提出了一种氧活化机制，该机制涉及通过桥连水和D140侧链形成的氢键网络。
Heme oxygenase (HO) catalyzes the oxygen-dependent degradation of heme to biliverdinIX alpha, CO, and free iron ion via three sequential monooxygenase reactions. Although the distinct active-site structure of HO from cytochrome P450 families suggests unique distal protein machinery to activate molecular oxygen, the mechanism and the key amino acid for the oxygen activation have not been clear. To investigate the functionality of highly conserved polar amino acids in the distal helix of HO-1, we have prepared alanine mutants: T135A, R136A, D140A, and S142A, and found drastic changes in the heme degradation reactions of D140A. In this paper, we report the first evidence that D140 is involved in the oxygen activation mechanism in HO-1. The heme complexes of HO mutants examined in this study fold and bind heme normally. The pK(a) values of the iron-bound water acid autoxidation rates of the oxy-form are increased with R136A, D140A, and S142A mutations, but are not changed with T135A mutation. As the wild-type, T135A, R136A, and S142A degrade heme to verdohemeIX alpha with H2O2 and to biliverdinIXa with the NADPH reductase system. On the other hand, D140A heme complex forms compound II with H2O2, and no heme degradation occurs. For the NADPH reductase system, the oxy-form of D140A heme complex is accumulated in the reaction, and only 50% of heme is degraded. The stopped flow experiments suggest that D140A cannot activate iron-bound dioxygen and hydroperoxide properly. To investigate the carboxylate functionality of D140, we further replaced D140 with glutamic acid (D140E), phenylalanine (D140F), and asparagine (D140N). D140E degrades heme normally, but D140N shows reactivity similar to that of D140A. D140F loses heme degradation activity completely. All of these results indicate that the carboxylate at position 140 is essential to activate the iron-bound dioxygen and hydroperoxide. On the basis of the present findings, we propose an oxygen activation mechanism involving the hydrogen-bonding network through the bridging water and D140 side chain.