Intramolecular electron transfer from biopterin to FeII-O2 complex in nitric oxide synthases occurs at very different rates between bacterial and mammalian enzymes: Direct observation of a catalytically active intermediate
Intramolecular electron transfer from biopterin to FeII-O2 complex in nitric oxide synthases occurs at very different rates between bacterial and mammalian enzymes: Direct observation of a catalytically active intermediate
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DOI：
10.1016/j.jinorgbio.2022.112035
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发表时间：
2022-10-27
影响因子：
3.9
通讯作者：
Kozawa，Takahiro
中科院分区：
文献类型：
--
作者：
Kobayashi，Kazuo;Ito，Yuko Tsutsui;Kozawa，Takahiro
一氧化氮合酶 (NOS) 是一种细胞色素 P450 型单加氧酶，可催化 L-精氨酸氧化为一氧化氮。我们之前使用脉冲放射分解观察到耐辐射奇球菌 NOS (DrNOS) 中从生物蝶呤到 Fe2+-O2 的分子内电子转移。然而，DrNOS 中的电子转移速率 (2.2 × 103s−1) 与使用快速冷冻猝灭 (RFQ) EPR 测定的哺乳动物 NOS 中报道的相应速率 (11 s−1) 形成对比。我们将脉冲放射分解应用于枯草芽孢杆菌 NOS (bsNOS) 和大鼠神经 NOS 加氧酶结构域 NOS (mNOS)。同时，RFQ EPR 用于在酶的单周转酶反应过程中捕获蝶呤自由基。通过使用脉冲放射分解法，水合电子 (eaq−) 还原了 NOS 酶的血红素铁。随后，亚铁血红素与O2反应形成Fe2+-O2中间体。在蝶呤存在的情况下，bsNOS 中间体被发现在几毫秒的时间范围内转化为其他中间体。在蝶呤结合的 mNOS 脉冲放射分解后，确定发生了类似的过程，尽管速度要慢得多。所有NOS酶的中间体在数秒的时间内进一步转化为原始的三价铁形式。当使用RFQ方法时，蝶呤自由基在DrNOS和bsNOS中在毫秒的时间范围内非常迅速地形成。相比之下，mNOS 中的蝶呤自由基形成缓慢，速率约为 20 s−1。
Nitric oxide synthase (NOS) is a cytochrome P450-type mono‑oxygenase that catalyzes the oxidation of L-arginine to nitric oxide. We previously observed that intramolecular electron transfer from biopterin to Fe2+-O2in Deinococcus radiodurans NOS (DrNOS) using pulse radiolysis. However, the rate of electron transfer in DrNOS (2.2 × 103s−1) contrasts with a reported corresponding rate (11 s−1) in a mammalian NOS determined using rapid freeze-quench (RFQ) EPR. We applied pulse radiolysis to Bacillus subtilis NOS (bsNOS) and to rat neural NOS oxygenase domain NOS (mNOS). Concurrently, RFQ EPR was used to trap a pterin radical during single-turnover enzyme reactions of the enzymes. By using the pulse radiolysis method, hydrated electrons (eaq−) reduced the heme iron of NOS enzymes. Subsequently, ferrous heme reacted with O2to form a Fe2+-O2intermediate. In the presence of pterin, the intermediate of bsNOS was found to convert to other intermediate in the time range of milliseconds. A similar process was determined to have occurred after pulse radiolysis of the pterin-bound mNOS, though the rate was much slower. The intermediates of all of the NOS enzymes further converted to the original ferric form in the time range of seconds. When using the RFQ method, pterin radicals were formed very rapidly in both DrNOS and bsNOS in the time range of milliseconds. In contrast, the pterin radical in mNOS was observed to form slowly, at a rate of ∼20 s−1.