Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence Spectroscopy
Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence Spectroscopy
复制标题
DOI：
10.1021/bi7025485
复制
发表时间：
2008-05-06
期刊：
影响因子：
2.9
通讯作者：
Champion, Paul M.
中科院分区：
文献类型：
--
作者：
Gruia, Flavin;Ionascu, Dan;Champion, Paul M.
采用超快激光光谱技术测量了富马Caldariomyces fumago chloroperoxidase （CPO）的低频振动相干光谱和一氧化氮再结合动力学。将CPO相干光谱与其他血红素物种的相干光谱进行比较，以衡量低频光谱的蛋白质特异性。在所有激发波长下，原生CPO的相干谱以出现在32-33 cm（-1）附近的模式为主，其相位与基态拉曼激发的振动波包一致。在常规坐标结构分解（NSD）分析的基础上，我们将这一特征分配给硫代酸结合血红素圆顶模式。探测脉冲的光谱分辨率（“失谐”探测）揭示了一个349 cm-1的模式，该模式先前使用拉曼光谱被指定为天然CPO的Fe-S拉伸模式。铁离子比铁离子表现出更大程度的光谱不均匀性，这在光学吸收光谱中有多个肩部反映出来。不同激发波长下相干光谱的变化揭示了这种非均匀性。在440 nm激发的还原CPO相干谱中，出现了接近220 cm（-1）的模式，这表明在生理相关的ph值下，铁态中存在5个组氨酸连接血红素的亚群。在与440 nm亚群的共振中，相干信号的振幅显著增加。动力学测量表明，一氧化氮与铁和亚铁CPO的结合可以描述为单指数过程，再结合时间常数分别为29.4 +/- 1和9.3 +/- 1 ps。这与先前报道的一氧化氮与辣根过氧化物酶结合的结果非常相似。
Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the low-frequency spectra. The coherence spectrum of native CPO is dominated by a mode that appears near 32-33 cm(-1) at all excitation wavelengths,with a phase that is consistent with a ground-state Raman-excited vibrational wavepacket. On the basis of a normal coordinate structural decomposition (NSD) analysis, we assign this feature to the thiolate-bound heme doming mode. Spectral resolution of the probe pulse ("detuned" detection) reveals a mode at 349 cm-1, which has been previously assigned using Raman spectroscopy to the Fe-S stretching mode of native CPO. The ferrous species displays a larger degree of spectral inhomogeneity than the ferric species, as reflected by multiple shoulders in the optical absorption spectra. The inhomogeneities are revealed by changes in the coherence spectra at different excitation wavelengths. The appearance of a mode close to 220 cm(-1) in the coherence spectrum of reduced CPO excited at 440 nm suggests that a subpopulation of five coordinated histidine-ligated hemes is present in the ferrous state at a physiologically relevant pH. A significant increase in the amplitude of the coherence signal is observed for the resonance with the 440 nm subpopulation. Kinetics measurements reveal that nitric oxide binding to ferric and ferrous CPO can be described as a single-exponential process, with rebinding time constants of 29.4 +/- 1 and 9.3 +/- 1 ps, respectively. This is very similar to results previously reported for nitric oxide binding to horseradish peroxidase.