Distal-proximal crosstalk in the heme binding pocket of the NO sensor DNR
Distal-proximal crosstalk in the heme binding pocket of the NO sensor DNR
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DOI：
10.1007/s10534-014-9770-3
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发表时间：
2014-08-01
期刊：
影响因子：
3.5
通讯作者：
Rinaldo, Serena
中科院分区：
文献类型：
--
作者：
Cutruzzola, Francesca;Arcovito, Alessandro;Rinaldo, Serena
在条件致病菌铜绿假单胞菌中，反硝化过程是由一氧化氮(NO)触发的，对慢性感染部位的微生物-厌氧生物膜的生存起着至关重要的作用。这一呼吸途径是由DNR转录诱导的，DNR是一种基于血红素的气体传感器，对NO有积极的反应。我们最近报道了一项体外研究，首次解剖了血红素-铁环境，并确定了一个与NO反应至关重要的血红素轴向配体(即His187)。然而，第二个血红素轴向配体的鉴定并不成功，因为在DNR中可能发生了一种特殊的配体围绕血红素铁交换的现象。柔红素与血红素结合的不同寻常的性质可能是由于柔红素本身在溶液中具有显著的灵活性，而这反过来又是传感活性的关键；蛋白质的灵活性和动力学确实代表了基于血红素的氧化还原传感器所采用的共同策略，呈现出与典型的血红素蛋白截然不同的特征。通过对H167A/H187A DNR双突变体的光谱表征，证明了DNR在血红素-铁周围进行深度重排的能力，这表明了NO和CO的异常结合动力学。此外，我们还表明，血红素口袋远端的改变(如组氨酸突变)是由近端的改变感知的，可能是通过DNR蛋白链。
In the opportunistic pathogen Pseudomonas aeruginosa the denitrification process is triggered by nitric oxide (NO) and plays a crucial role for the survival in chronic infection sites as a microaerobic-anaerobic biofilm. This respiratory pathway is transcriptionally induced by DNR, an heme-based gas sensor which positively responds to NO. Molecular details of the NO sensing mechanism employed by DNR are now emerging: we recently reported an in vitro study which dissected, for the first time, the heme-iron environment and identified one of the heme axial ligand (i.e. His187), found to be crucial to respond to NO. Nevertheless, the identification of the second heme axial ligand has been unsuccessful, given that a peculiar phenomenon of ligand switching around the heme-iron presumably occurs in DNR. The unusual heme binding properties of DNR could be due to the remarkable flexibility in solution of DNR itself, which, in turns, is crucial for the sensing activity; protein flexibility and dynamics indeed represent a common strategy employed by heme-based redox sensors, which present features deeply different from those of "canonical" hemeproteins. The capability of DNR to deeply rearrange around the heme-iron as been here demonstrated by means of spectroscopic characterization of the H167A/H187A DNR double mutant, which shows unusual kinetics of binding of NO and CO. Moreover, we show that the alteration (such as histidines mutations) of the distal side of the heme pocket is perceived by the proximal one, possibly via the DNR protein chain.