Abstract：
Cytochromes c\'-α are nitric oxide (NO)-binding heme proteins derived from bacteria that can thrive in a wide range of temperature environments. Studies of mesophilic Alcaligenes xylosoxidans cytochrome c\'-α (AxCP-α) have revealed an unusual NO-binding mechanism involving both heme faces, in which NO first binds to form a distal hexa-coordinate Fe(II)-NO (6cNO) intermediate and then displaces the proximal His to form a proximal penta-coordinate Fe(II)-NO (5cNO) final product. Here we characterize a thermally stable cytochrome c\'-α from thermophilic Hydrogenophilus thermoluteolus (PhCP-α) to understand how protein thermal stability affects NO binding. Electron paramagnetic and resonance Raman spectroscopies reveal the formation of a PhCP-α 5cNO product, with time-resolved (stopped-flow) UV-visible absorbance indicating the involvement of a 6cNO intermediate. Relative to AxCP-α, the rates of 6cNO and 5cNO formation in PhCP-α are ∼11-fold and ∼13-fold lower, respectively. Notably, X-ray crystal structures of PhCP-α in the presence and absence of NO suggest that the sluggish formation of the proximal 5cNO product results from conformational rigidity: the Arg-132 residue (adjacent to the proximal His ligand) is held in place by a salt bridge between Arg-75 and Glu-135 (an interaction not present in AxCP-α or a psychrophilic counterpart). Overall, our data provide fresh insights into structural factors controlling NO binding in heme proteins, including 5cNO complexes relevant to eukaryotic NO sensors.
摘要：
细胞色素c'-α是来自细菌的一氧化氮（NO）结合血红素蛋白，可以在广泛的温度环境中茁壮成长。嗜温的产碱菌木氧化素细胞色素c'-α（AxCP-α）的研究揭示了涉及两个血红素面的不寻常的NO结合机制，其中NO首先结合以形成远端六坐标Fe（II）-NO（6cNO）中间体，然后置换近端His以形成近端五坐标Fe（II）-NO（5cNO）最终产物。在这里，我们表征了来自嗜热嗜氢性嗜热嗜氢细胞色素c'-α（PhCP-α）的热稳定细胞色素c'-α，以了解蛋白质热稳定性如何影响NO结合。电子顺磁性和共振拉曼光谱揭示了PhCP-α5cNO产物的形成，具有时间分辨（停流）UV-可见光吸收，表明涉及6cNO中间体。相对于AxCP-α，PhCP-α中6cNO和5cNO形成的速率分别低11倍和13倍，分别。值得注意的是,在存在和不存在NO的情况下，PhCP-α的X射线晶体结构表明，近端5cNO产物的缓慢形成是由构象刚性引起的：Arg-132残基（与近端His配体相邻）通过Arg-75和Glu-135之间的盐桥（AxCP-α或嗜冷对应物中不存在的相互作用）。总的来说,我们的数据为控制血红素蛋白中NO结合的结构因素提供了新的见解，包括与真核NO传感器相关的5cNO复合物。
