Electron donation to the flavoprotein NifL, a redox-sensing transcriptional regulator.
Electron donation to the flavoprotein NifL, a redox-sensing transcriptional regulator.
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向氧化还原感应转录调节因子黄素蛋白 NifL 提供电子。
DOI：
10.1042/bj3320413
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
R. Dixon
中科院分区：
文献类型：
--
作者：
P. Macheroux;S. Hill;S. Austin;T. Eydmann;T. Jones;Sung;R. Poole;R. Dixon
固氮调节蛋白 L (NifL) 介导固氮 (nif) 基因响应氧气的转录控制，固氮调节蛋白 L (NifL) 是一种调节黄素蛋白，可调节转录激活剂固氮调节蛋白 A (NifA) 的活性。纯化的 NifL 的 CD 光谱表明 FAD 在不对称环境中与 NifL 结合，并且该蛋白质主要是 α 螺旋。在适当介体存在的情况下，通过黄嘌呤氧化酶/黄嘌呤对 NifL 的酶促还原测定，NifL 的氧化还原电位在 pH 8 时为 -226 mV。黄嘌呤氧化酶对 NifL 的还原阻止了 NifL 作为 NifA 抑制剂的作用。在没有电子介体的情况下，NifL 也可以被大肠杆菌黄血蛋白 (Hmp) 以 NADH 作为还原剂还原。 Hmp 包含一个以血红素 B 作为辅基的球蛋白样结构域和一个包含 FAD 的氧化还原酶模块。 Hmp 的羧基铁血红素形式能够还原 NifL，这表明向 NifL 提供的电子源自 Hmp 中的黄素，而不是通过直接从血红素转移电子。菠菜铁氧还蛋白：NAD(P)氧化还原酶采用与Hmp的FAD和NAD结合结构域相似的折叠，也以NADH作为还原剂还原NifL。在空气存在的情况下，NifL 的再氧化会迅速发生，这增加了 NifL 感知细胞内氧气的可能性。我们提出了一种生理氧化还原循环，其中 NifL 被氧氧化并因此其抑制特性的激活迅速发生，而 NifL 从活性形式转变为还原形式则发生得更慢。
Transcriptional control of the nitrogen fixation (nif) genes in response to oxygen in Azotobacter vinelandii is mediated by nitrogen fixation regulatory protein L (NifL), a regulatory flavoprotein that modulates the activity of the transcriptional activator nitrogen fixation regulatory protein A (NifA). CD spectra of purified NifL indicate that FAD is bound to NifL in an asymmetric environment and the protein is predominantly alpha-helical. The redox potential of NifL is -226 mV at pH 8 as determined by the enzymic reduction of NifL by xanthine oxidase/xanthine in the presence of appropriate mediators. The reduction of NifL by xanthine oxidase prevented NifL from acting as an inhibitor of NifA. In the absence of electron mediators NifL could also be reduced by Escherichia coli flavohaemoprotein (Hmp) with NADH as reductant. Hmp contains a globin-like domain with haem B as prosthetic group and an FAD-containing oxidoreductase module. The carboxyferrohaem form of Hmp was competent to reduce NifL, suggesting that electron donation to NifL originates from the flavin in Hmp rather than by direct electron transfer from the haem. Spinach ferredoxin:NAD(P) oxidoreductase, which adopts a folding similar to the FAD- and NAD-binding domains of Hmp, also reduced NifL with NADH as reductant. Re-oxidation of NifL occurs rapidly in the presence of air, raising the possibility that NifL might sense intracellular oxygen. We propose a physiological redox cycle in which the oxidation of NifL by oxygen and hence the activation of its inhibitory properties occurs rapidly, in contrast with the switch from the active to the reduced form of NifL, which occurs more slowly.