Spectroscopic evidence of the role of an axial ligand histidinate in the mechanism of adrenal cytochrome b(561).
Spectroscopic evidence of the role of an axial ligand histidinate in the mechanism of adrenal cytochrome b(561).
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轴向配体组氨酸在肾上腺细胞色素 b(561) 机制中作用的光谱证据。
DOI：
10.1021/bi301127k
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Palmer,Graham
中科院分区：
文献类型：
--
作者：
daSilva,GiordanoFZ;Shinkarev,VladimirP;Kamensky,YuryA;Palmer,Graham
肾上腺细胞色素561（AdCytb）是一个广泛存在的蛋白家族的原型，它专门为真核细胞的不同过程提供抗坏血酸提供的电子。AdCytb将细胞质抗坏血酸的氧化还原当量通过染色质颗粒的膜运输，以支持基质内去甲肾上腺素的合成。AdCytb与抗坏血酸的相互作用是AdCytb功能机制的核心，AdCytb活性部位的组氨酸被认为与细胞质抗坏血酸结合，并作为抗坏血酸氧化过程中释放的质子的受体。AdCytb含有高电位血红素和低电位血红素，但它们相对于染色质颗粒膜基质和细胞质界面的取向存在争议。使用三种光谱方法（紫外-可见吸收，近红外磁圆二色性和电子顺磁共振）的组合，我们发现作为高电位血红素的轴向配体的组氨酸残基经历了pKof ~ 8.0的去质子化，因此是与细胞质抗坏血酸相互作用的良好候选物。发现AdCytb的低电位血红素具有~ 10.5的pkf，使其不太可能在生理ph下接受质子。紫外-可见光谱显示AdCytb中有一个pkf为~ 6.5的额外质子受体基团，这是其他两种技术没有观察到的；它是否在AdCytb的机制中起作用尚不清楚。我们将这些结果整合到AdCytb还原的最新机制中，该机制基于高电位血红素在染色质颗粒膜的细胞质界面上的定位。
Adrenal cytochromeb561(AdCytb) is the prototype of a widespread protein family that specializes in delivering electrons donated by ascorbic acid for different processes in eukaryotic cells. AdCytb transports redox equivalents from cytoplasmic ascorbate across the membranes of chromaffin granules to support norepinephrine synthesis within their matrix. The interaction of AdCytb with ascorbate is central to a proposed mechanism of AdCytb’s function, and a histidine in the active site of AdCytb was suggested to bind cytoplasmic ascorbate and serve as the acceptor of the proton released during ascorbate oxidation. AdCytb contains high- and low-potential hemes but their orientation relative to the matrix and cytoplasmic interfaces of chromaffin granule membrane is disputed. Using a combination of three spectroscopic methods (UV–vis absorption, near-infrared magnetic circular dichroism, and electron paramagnetic resonance), we find that a histidine residue that serves as an axial ligand to the high-potential heme undergoes deprotonation with a pKof ∼8.0 and is thus a good candidate for interaction with cytoplasmic ascorbate. The low-potential heme of AdCytb is found to have a pKof ∼10.5, making it an unlikely candidate for accepting a proton at physiological pH. UV–vis spectroscopy reveals an additional proton acceptor group in AdCytb with a pKof ∼6.5 that is not observed by the other two techniques; whether it plays a role in the mechanism of AdCytb is unknown. We incorporate these results into an updated mechanism of AdCytb reduction predicated on the high-potential heme’s localization on the cytoplasmic interface of the chromaffin granule membrane.