Nanosecond step-scan FTIR spectroscopy of hemoglobin: Ligand recombination and protein conformational changes
Nanosecond step-scan FTIR spectroscopy of hemoglobin: Ligand recombination and protein conformational changes
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DOI：
10.1021/bi961522n
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发表时间：
1996-10-08
期刊：
影响因子：
2.9
通讯作者：
Spiro, TG
中科院分区：
文献类型：
--
作者：
Hu, XH;Frei, H;Spiro, TG
采用纳秒级时间分辨率的步进扫描傅里叶变换红外光谱技术研究了碳一氧血红蛋白（HbCO）的光循环反应。在1951 cm（-1）处的强CO伸缩带可作为结扎状态的方便监测器。双生子和二阶相的CO重组发生的速率是在良好的协议与以前的可见光吸收测量，显示的分子机制是不受干扰的高蛋白浓度（6.7 mM的血红素）所需的足够的蛋白质信号。虽然光解程度（43%）不足以驱动R->T四级跃迁，但蛋白质TRIR（时间分辨红外）差带（1250-1700 cm（-1））仍然揭示了有趣的三级动力学。大多数的频带在非常早期的时候，可能之前的成对重组阶段（τ = 50纳秒）充分发展。然而，一些带出现得更慢，时间常数为0.4 μ s，反映了三级运动，这与之前通过紫外共振拉曼光谱检测到的四级运动完全光解的HbCO。TRIP带的弛豫比CO再结合（有效时间常数为160 μ s）快（τ =类似于90 μ s）或慢（τ =类似于250 μ s），这表明CO再结合中三级过程的分布或链的不等价性。
Step-scan FTIR spectroscopy with nanosecond time resolution is applied to the photocycle of carbonmonoxy hemoglobin (HbCO). The strong CO stretching band at 1951 cm(-1) serves as a convenient monitor of the state of ligation. Both geminate and second-order phases of CO recombination occur at rates which are in excellent agreement with previous visible absorption measurements, showing the molecular mechanisms to be unperturbed by the high protein concentrations (6.7 mM in heme) required for adequate protein signals. While the extent of photolysis (43%) was insufficient to drive the R-->T quaternary transition, the protein TRIR (time-resolved infrared) difference bands (1250-1700 cm(-1)) nevertheless reveal interesting tertiary dynamics. Most of the bands are fully developed at very early times, possibly preceding the geminate recombination phase (tau = 50 ns). Some bands arise more slowly, however, with a time constant of 0.4 mu s, reflecting a tertiary motion which is coincident with a quaternary motion previously detected by ultraviolet resonance Raman spectroscopy of fully photolyzed HbCO. Relaxation of the TRIP bands is either faster (tau = similar to 90 mu s) or slower (tau = similar to 250 mu s) than CO rebinding (effective time constant of 160 mu s), ring either a distribution of tertiary processes or a chain inequivalence in CO rebinding.