Peterson E S, Friedman J M
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biochemistry. 1998 Mar 31;37(13):4346-57. doi: 10.1021/bi9708693.
The highly conserved tryptophan at position beta37 occupies a key locus at the hinge region within the alpha1beta2 interface of the mammalian hemoglobins. This residue is thought to play an important role in mediating the heme-heme interaction associated with the cooperative binding of oxygen; however, its explicit function is unclear. In this study, the proximal heme environments of several beta37 mutants of adult human hemoglobin (HbA) are probed using visible (Soret band enhanced) resonance Raman spectroscopy. In the equilibrium deoxy derivatives of these mutants, a systematic variation in proximal strain, as reflected in the iron-proximal histidine (F8) stretching frequency, nu(Fe-His), is seen upon mutation of the beta37 residue. The variation in proximal strain correlates with both the ligand binding rates [Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335] and conformational changes observed at the FG corner through X-ray crystallography [Kavanaugh et al. (1998) Biochemistry 37, 4358-4373]. The results from the deoxy samples indicate a plasticity of the tertiary structure within the T quaternary state. The correlation between the X-ray data and the Raman supports the idea that the proximal strain at the heme within the T state can be modulated by a combination of forces including those arising from the hinge region of the alpha1beta2 interface, from the binding of allosteric effectors, and from the degree of iron displacement from the heme plane. Each of these contributors appears to operate through a shifting of the F helix either away from or toward the FG corner. The Raman spectra obtained from the 10 ns CO photoproduct of the beta37 mutant Hb's indicate that these mutants contain an altered coupling between the R state alpha1beta2 interface and the proximal heme environment. This altered coupling could be due to either dissociation of the ligated mutant tetramers into dimers or the formation of an R state tetramer with significantly weakened hydrogen bonds and van der Waals contacts between the alpha1 and beta2 subunits at the interface. In either case, the results reveal a clear-cut structural basis for the quaternary enhancement effect in which the normal R state quaternary structure produces a higher affinity ligand binding site than that which occurs in the corresponding dimeric form of the protein. The normal R state interface is shown to be important for stabilizing a favorable ligand binding environment that persists long enough after laser photolysis to enhance the geminate rebinding process within the photoproduct. The addition of IHP to the solution of mutant COHb proteins results in photoproduct spectra that are all identical and are consistent with the ligand-bound derivatives having either a T state structure or a very strained and anomalous R state structure.
β37位高度保守的色氨酸在哺乳动物血红蛋白α1β2界面的铰链区占据关键位点。该残基被认为在介导与氧的协同结合相关的血红素 - 血红素相互作用中起重要作用；然而，其确切功能尚不清楚。在本研究中，使用可见（Soret带增强）共振拉曼光谱探测了成人血红蛋白（HbA）几种β37突变体的近端血红素环境。在这些突变体的平衡脱氧衍生物中，当β37残基发生突变时，可观察到近端应变的系统变化，这反映在铁 - 近端组氨酸（F8）拉伸频率ν（Fe - His）上。近端应变的变化与配体结合速率[Kwiatkowski等人（1998年）《生物化学》37卷，4325 - 4335页]以及通过X射线晶体学在FG转角处观察到的构象变化[Kavanaugh等人（1998年）《生物化学》37卷，4358 - 4373页]相关。脱氧样品的结果表明T四级状态内三级结构具有可塑性。X射线数据与拉曼光谱之间的相关性支持了这样一种观点，即T状态下血红素的近端应变可通过多种力的组合来调节，这些力包括来自α1β2界面铰链区、变构效应剂结合以及铁从血红素平面位移程度所产生的力。这些因素中的每一个似乎都是通过F螺旋远离或朝向FG转角的移动来起作用的。从β37突变体Hb的10 ns CO光产物获得的拉曼光谱表明，这些突变体在R状态α1β2界面和近端血红素环境之间存在改变的耦合。这种改变的耦合可能是由于连接的突变体四聚体解离成二聚体，或者是形成了一种R状态四聚体，其在界面处α1和β2亚基之间的氢键和范德华接触显著减弱。无论哪种情况，结果都揭示了四级增强效应的明确结构基础，即正常的R状态四级结构产生的配体结合位点亲和力高于相应蛋白质二聚体形式中的位点。正常的R状态界面对于稳定有利的配体结合环境很重要，该环境在激光光解后持续足够长的时间，以增强光产物内的双分子复合过程。向突变体COHb蛋白溶液中加入IHP会导致光产物光谱完全相同，并且与具有T状态结构或非常紧张且异常的R状态结构的配体结合衍生物一致。
