Petrich J W, Lambry J C, Kuczera K, Karplus M, Poyart C, Martin J L
Laboratoire d'Optique Appliquée, Ecole Polytechnique, ENSTA, INSERM U275.
Biochemistry. 1991 Apr 23;30(16):3975-87. doi: 10.1021/bi00230a025.
Ultrafast absorption spectroscopy is used to study heme-NO recombination at room temperature in aqueous buffer on time scales where the ligand cannot leave its cage environment. While a single barrier is observed for the cage recombination of NO with heme in the absence of globin, recombination in hemoglobin and myoglobin is nonexponential. Examination of hemoglobin with and without inositol hexaphosphate points to proximal constraints as important determinants of the geminate rebinding kinetics. Molecular dynamics simulations of myoglobin and heme-imidazole subsequent to ligand dissociation were used to investigate the transient behavior of the Fe-proximal histidine coordinate and its possible involvement in geminate recombination. The calculations, in the context of the absorption measurements, are used to formulate a distinction between nonexponential rebinding that results from multiple protein conformations (substates) present at equilibrium or from nonequilibrium relaxation of the protein triggered by a perturbation such as ligand dissociation. The importance of these two processes is expected to depend on the time scale of rebinding relative to equilibrium fluctuations and nonequilibrium relaxation. Since NO rebinding occurs on the picosecond time scale of the calculated myoglobin relaxation, a time-dependent barrier is likely to be an important factor in the observed nonexponential kinetics. The general implications of the present results for ligand binding in heme proteins and its time and temperature dependence are discussed. It appears likely that, at low temperatures, inhomogeneous protein populations play an important role and that as the temperature is raised, relaxation effects become significant as well.
超快吸收光谱用于在室温下的水性缓冲液中，在配体无法离开其笼状环境的时间尺度上研究血红素-NO的重组。虽然在没有珠蛋白的情况下，观察到NO与血红素的笼状重组存在单一屏障，但血红蛋白和肌红蛋白中的重组是非指数性的。对添加和不添加肌醇六磷酸的血红蛋白的研究表明，近端限制是双分子复合动力学的重要决定因素。配体解离后，对肌红蛋白和血红素-咪唑进行分子动力学模拟，以研究铁-近端组氨酸配位的瞬态行为及其在双分子重组中的可能作用。结合吸收测量结果，这些计算用于区分非指数性重新结合，其源于平衡时存在的多种蛋白质构象（亚状态），还是源于诸如配体解离等扰动引发的蛋白质非平衡弛豫。预计这两个过程的重要性将取决于重新结合相对于平衡波动和非平衡弛豫的时间尺度。由于NO重新结合发生在计算出的肌红蛋白弛豫的皮秒时间尺度上，时间依赖性屏障可能是观察到的非指数动力学的一个重要因素。本文讨论了这些结果对血红素蛋白中配体结合及其时间和温度依赖性的一般影响。在低温下，不均匀的蛋白质群体可能起重要作用，而随着温度升高，弛豫效应也变得显著。
