Investigation of laser-induced long-lived states of photolyzed MbCO.
Investigation of laser-induced long-lived states of photolyzed MbCO.
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激光诱导光解 MbCO 长寿命状态的研究。
DOI：
10.1021/bi00234a008
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Champion,PM
中科院分区：
文献类型：
--
作者：
Srajer,V;Reinisch,L;Champion,PM
物理系，东北大学，波士顿，马萨诸塞州02115 1990年12月5日接收; 1991年2月20日接收的修订版摘要：我们提出的证据，从共振拉曼和吸收测量表明，延长暴露的MbCO连续激光在低温下改变了CO的再结合动力学，并导致人口显着增加的非常长的寿命状态的光解MbCO。这种光学“泵送”过程中观察到的样品冷冻在水性缓冲液和甘油/缓冲液，并表现出幂律行为与一个非常弱的温度依赖性。CO分子从泵浦态的非指数再结合动力学与在闪光光解实验中观察到的再结合动力学的比较表明，泵浦态是不同的孪晶态，在闪光光解实验中没有观察到。因此，一个四态模型，有两个geminatestates，是牵连的MbCO。泵浦状态可能代表“分离的成对”状态，CO分子仍然在血红素口袋中，或者可能被困在一个空腔中，通过蛋白质基质的方式，与分子动力学模拟一致。显着deoxyheme relaxationfrom一个更圆顶的配置，作为与泵送过程相关的多个光解事件的结果的可能性，也探讨了。然而，在T< 180 K泵浦后所观察到的Soret带线形状和位置的微小变化往往排除了对泵浦过程的这种解释。由于产生泵浦态的产率很小（如T> 100 K时< 10~ 7），所以只有在长时间照射后才能观察到泵浦，而在闪光光解测量中甚至在多次闪光后也观察不到泵浦。在较高的温度下（T> 180 K），观察到CO分子逃逸到溶剂中。我们的数据与蛋白质的“相变”相一致，该蛋白质与周围基质相结合。蛋白质的波动淬灭低于-185 K的溶剂组成的70%甘油和低于260 K的缓冲液。我们还提出了第一个大幅度测量CO从蛋白质外部重新绑定，观察到低于200 K后，在激光照射下冷冻样品。
Department of Physics, Northeastern University, Boston, Massachusetts 02115 Received December 5, 1990; Revised Manuscript Received February 20, 1991 abstract: We present evidence from resonance Raman and absorption measurements that the extended exposure of MbCO to CW laser light at low temperatures alters the CO rebinding kinetics and leads to a significantly increased population of very long lived states of photolyzed MbCO. This optical “pumping” process is observed for samples frozen in both aqueous buffer and glycerol/buffer and exhibits power law behavior with a very weak temperature dependence. A comparison of the nonexponential rebinding kinetics of CO molecules from the pumped states with the rebinding observed in flash photolysis experiments suggests that thepumped states are distinct geminate states, not observed in flash photolysis experiments. Thus, a four-state model, with two geminatestates, is implicated for MbCO. Pumped states may represent “separated geminate pair” states with the CO molecule still in the heme pocket or possibly trapped within a cavity on its way through the protein matrix, consistent with moleculardynamics simulations. The possibility of significant deoxyheme relaxationfrom a less domed to a more domed configuration, as a result of the multiple photolysis events associated with the pumping process, is also explored. However, the small changes observed in the Soret band line shape andposition subsequent to pumping at T< 180 K tend to rule out this explanation for the pumping process. Since the yield for creating a pumped state is small (eg,< 10~ 7 for T> 100 K), pumping can be observed only after extended illumination and is absentin flash photolysis measurements, even after multiple flashes. At higher temperatures (T> 180 K), the escape of the CO molecule to the solvent is observed. Our data are consistent with a “phase transition” of the proteinthat is coupled to the surroundingmatrix. The protein fluctuations are quenched below— 185 K for a solvent composed of 70% glycerol and below~ 260 K for aqueous buffer. We also present the first large amplitude measurements of CO rebinding from the protein exterior, observed below 200 K after freezing the sample under laser illumination.