Bonaventura Celia, Henkens Robert, Friedman Joel, Siburt Claire J Parker, Kraiter Daniel, Crumbliss Alvin L
Duke University Marine Laboratory, Beaufort, NC 28516, USA.
Biochim Biophys Acta. 2011 Oct;1814(10):1261-8. doi: 10.1016/j.bbapap.2011.06.012. Epub 2011 Jul 8.
The structural basis of the extreme pH dependence of oxygen binding to Root effect Hbs is a long-standing puzzle in the field of protein chemistry. A previously unappreciated role of steric factors in the Root effect was revealed by a comparison of pH effects on oxygenation and oxidation processes in human Hb relative to Spot (Leiostomus xanthurus) and Carp (Cyprinodon carpio) Hbs. The Root effect confers five-fold increased pH sensitivity to oxygenation of Spot and Carp Hbs relative to Hb A(0) in the absence of anionic effectors, and even larger relative elevations of pH sensitivity of oxygenation in the presence of 0.2M phosphate. Remarkably, the Root effect was not evident in the oxidation of the Root effect Hbs. This finding rules out pH-dependent alterations in the thermodynamic properties of the heme iron, measured in the anaerobic oxidation reaction, as the basis of the Root effect. The alternative explanation supported by these results is that the elevated pH sensitivity of oxygenation of Root effect Hbs is attributable to globin-dependent steric effects that alter oxygen affinity by constraining conformational fluidity, but which have little influence on electron exchange via the heme edge. This elegant mode of allosteric control can regulate oxygen affinity within a given quaternary state, in addition to modifying the T-R equilibrium. Evolution of Hb sequences that result in proton-linked steric barriers to heme oxygenation could provide a general mechanism to account for the appearance of the Root effect in the structurally diverse Hbs of many species.
氧与根效应血红蛋白结合对极端pH的依赖性的结构基础，一直是蛋白质化学领域长期存在的难题。通过比较pH对人血红蛋白相对于斑点（黄尾拟牙鲷）和鲤鱼（鲤齿鲤）血红蛋白的氧合和氧化过程的影响，揭示了空间因素在根效应中一个以前未被重视的作用。在没有阴离子效应剂的情况下，根效应使斑点和鲤鱼血红蛋白的氧合对pH的敏感性比血红蛋白A(0)增加了五倍，在存在0.2M磷酸盐的情况下，氧合对pH的敏感性相对升高甚至更大。值得注意的是，根效应在根效应血红蛋白的氧化过程中并不明显。这一发现排除了在厌氧氧化反应中测量的血红素铁热力学性质的pH依赖性变化作为根效应的基础。这些结果支持的另一种解释是，根效应血红蛋白氧合对pH的敏感性升高归因于球蛋白依赖性空间效应，这种效应通过限制构象流动性来改变氧亲和力，但对通过血红素边缘的电子交换影响很小。这种优雅的变构控制模式除了改变T-R平衡外，还可以在给定的四级状态内调节氧亲和力。导致质子连接的血红素氧合空间屏障的血红蛋白序列的进化，可能提供一种普遍机制来解释许多物种结构多样的血红蛋白中根效应的出现。
