Aono S, Ohkubo K, Matsuo T, Nakajima H
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Nomi-gun, Ishikawa 923-1292, Japan.
J Biol Chem. 1998 Oct 2;273(40):25757-64. doi: 10.1074/jbc.273.40.25757.
The transcriptional activator CooA from Rhodospirillum rubrum contains a b-type heme that acts as a CO sensor in vivo. CooA is the first example of a transcriptional regulator containing a heme as a prosthetic group and of a hemeprotein in which CO plays a physiological role. In this study, we constructed an in vivo reporter system to measure the transcriptional activator activity of CooA and prepared some CooA mutants in which a mutation was introduced at Cys, His, Met, Lys, or Tyr. Only the mutations of Cys75 and His77 affected the electronic absorption spectra of the heme in CooA. The electronic absorption spectra, EPR spectra, and the transcriptional activator activity of the wild-type and mutant CooA proteins indicate that 1) the thiolate derived from Cys75 is the axial ligand in the ferric heme, but it is not coordinated to the CO-bound ferrous heme; 2) Cys75 is protonated or displaced in the ferrous heme; and 3) His77 is the proximal ligand in the CO-bound ferrous heme and probably also in the ferrous heme, but it is not coordinated to the ferric heme. NMR spectra reveal that the conformational change around the heme, which will trigger the activation of CooA by CO, takes place upon the binding of CO to the heme.
来自红螺菌的转录激活因子CooA含有一个b型血红素，它在体内作为一氧化碳（CO）传感器发挥作用。CooA是第一个含有血红素作为辅基的转录调节因子的例子，也是第一个CO在其中发挥生理作用的血红素蛋白的例子。在本研究中，我们构建了一个体内报告系统来测量CooA的转录激活活性，并制备了一些在半胱氨酸（Cys）、组氨酸（His）、甲硫氨酸（Met）、赖氨酸（Lys）或酪氨酸（Tyr）处引入突变的CooA突变体。只有Cys75和His77的突变影响了CooA中血红素的电子吸收光谱。野生型和突变型CooA蛋白的电子吸收光谱、电子顺磁共振（EPR）光谱以及转录激活活性表明：1）来自Cys75的硫醇盐是高铁血红素中的轴向配体，但它不与结合CO的亚铁血红素配位；2）Cys75在亚铁血红素中被质子化或被取代；3）His77是结合CO的亚铁血红素以及可能也是亚铁血红素中的近端配体，但它不与高铁血红素配位。核磁共振（NMR）光谱显示，当CO与血红素结合时，血红素周围会发生构象变化，这将触发CooA被CO激活。
