Folding mechanism of reduced Cytochrome c: equilibrium and kinetic properties in the presence of carbon monoxide.
Folding mechanism of reduced Cytochrome c: equilibrium and kinetic properties in the presence of carbon monoxide.
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DOI：
10.1016/j.jmb.2008.08.025
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发表时间：
2008-11-07
影响因子：
5.6
通讯作者：
Roder, Heinrich
中科院分区：
文献类型：
--
作者：
Latypov, Ramil F.;Maki, Kosuke;Cheng, Hong;Luck, Stanley D.;Roder, Heinrich
尽管结构相似，但铁和亚铁形式的细胞色素c（cyt c）在其配体结合特性、稳定性、折叠和动力学方面存在很大差异。还原的血红素铁仅在促进天然甲硫氨酸-铁键的弱化或破坏的不稳定条件下结合双原子配体如CO。这使得CO成为一种有用的构象探针，用于检测在内源性配体的情况下不能观察到的部分结构化状态。血红素吸光度，圆二色性和NMR被用来表征变性剂诱导的展开平衡的Fe 2+细胞色素C在存在和不存在的CO。除了本机状态（N），不结合CO，和展开的CO-复合物（U-CO），结构上不同的CO结合形式（M-CO）积累到高水平（约75%的人口）在中间盐酸胍浓度。不同构象的探针的展开过渡的比较表明，M-CO是一个紧凑的状态，包含一个本地样的螺旋核心和区域的本地无序的部分包含本地Met 80配体和相邻的循环。动力学测量的CO结合和解离的天然，部分变性和完全展开的条件下表明，一个状态，M，这是结构上类似于M-CO的填充，即使在没有CO。CO配体的结合能降低这种高能量状态的自由能到这样的程度，它积累，即使在温和的变性平衡条件下。在这项研究中获得的热力学和动力学参数提供了一个完全自洽的描述连接的展开/CO-结合平衡的减少细胞色素C。
Despite close structural similarity, the ferric and ferrous forms of cytochrome c (cyt c) differ greatly in terms of their ligand binding properties, stability, folding and dynamics. The reduced heme iron binds diatomic ligands such as CO only under destabilizing conditions that promote weakening or disruption of the native methionine-iron linkage. This makes CO a useful conformational probe for detecting partially structured states that cannot be observed in the absence of endogenous ligands. Heme absorbance, circular dichroism and NMR were used to characterize the denaturant-induced unfolding equilibrium of Fe2+ cyt c in the presence and absence of CO. In addition to the native state (N), which does not bind CO, and the unfolded CO-complex (U-CO), a structurally distinct CO-bound form (M-CO) accumulates to high levels (~75% of the population) at intermediate guanidine hydrochloride concentrations. Comparison of the unfolding transition for different conformational probes reveals that M-CO is a compact state containing a native-like helical core and regions of local disorder in the segment containing the native Met80 ligand and adjacent loops. Kinetic measurements of CO binding and dissociation under native, partially denaturing and fully unfolded conditions indicate that a state, M, that is structurally analogous to M-CO is populated even in the absence of CO. The binding energy of the CO ligand lowers the free energy of this high-energy state to such an extent that it accumulates even under mildly denaturing equilibrium conditions. The thermodynamic and kinetic parameters obtained in this study provide a fully self-consistent description of the linked unfolding/CO-binding equilibria of reduced cyt c.
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