Huang Y, Ackers G K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1995 May 16;34(19):6316-27. doi: 10.1021/bi00019a009.
Subunit assembly reactions of hemoglobin's 10 ligation microstates have been studied as a function of temperature to evaluate the enthalpic and entropic components of cooperativity. It is found that the cooperative enthalpies and cooperative entropies distribute in close agreement with predictions of the symmetry rule mechanism (Ackers et al., 1992) previously deduced from the free energy distribution, in combination with structure-sensitive probes (Doyle & Ackers, 1992; LiCata et al., 1993; Daugherty et al., 1994). Principal findings of the present study are as follows: (1) In unligated hemoglobin (quaternary T), dimer-tetramer assembly is driven by a large negative enthalpy, whereas in the fully ligated (quaternary R) species, the driving force for quaternary assembly is entropic. For the eight intermediate ligation species, the switchover from enthalpic to entropic control follows precisely the symmetry rule predictions; i.e., switching from enthalpic to entropic control occurs at each of the six steps that create ligated heme sites on both sides of the dimer-dimer interface. The combinatorial distribution found previously with free energies does not therefore arise from coincidental enthalpy-entropy compensation that masks a more fundamental distribution. (2) The free energy of tertiary constraint delta Gtc, which pays for intradimer cooperativity prior to quaternary switching, contains large enthalpic and entropic components delta Htc and delta Stc. Like delta Gtc, these terms vanish at the second binding step within the T tetramer. It is found that delta Gtc arises from a net enthalpic dominance over an almost equally large T delta Stc. (3) The stepwise enthalpies correlate with stepwise values of Bohr protons and Bohr free energies (Daugherty et al., 1994) throughout the cascade of 16 stepwise reactions; the correlated clusters of these values follow predictions of the symmetry rule mechanism. (4) These results obtained with cyanomethemoglobin are consistent with the corresponding data on oxygenated hemoglobin which has been resolved at each stage of oxygenation.
血红蛋白的10种配位微观状态的亚基组装反应已作为温度的函数进行了研究，以评估协同作用的焓和熵成分。研究发现，协同焓和协同熵的分布与先前从自由能分布结合结构敏感探针推导出来的对称规则机制（Ackers等人，1992年）的预测非常一致（Doyle和Ackers，1992年；LiCata等人，1993年；Daugherty等人，1994年）。本研究的主要发现如下：（1）在未配位的血红蛋白（四级结构T）中，二聚体-四聚体组装由一个很大的负焓驱动，而在完全配位的（四级结构R）物种中，四级组装的驱动力是熵。对于八种中间配位物种，从焓控制到熵控制的转换恰好遵循对称规则预测；即，在二聚体-二聚体界面两侧产生配位血红素位点的六个步骤中的每一步都发生从焓控制到熵控制的转换。因此，先前发现的自由能的组合分布并非源于掩盖更基本分布的偶然焓-熵补偿。（2）三级约束自由能ΔGtc，它在四级转换之前为二聚体内的协同作用付出代价，包含很大的焓和熵成分ΔHtc和ΔStc。与ΔGtc一样，这些项在T四聚体的第二个结合步骤中消失。研究发现，ΔGtc源于焓对几乎同样大的TΔStc的净主导。（3）在16个逐步反应的整个级联过程中，逐步焓与玻尔质子和玻尔自由能的逐步值相关（Daugherty等人，1994年）；这些值的相关簇遵循对称规则机制的预测。（4）用氰化高铁血红蛋白获得的这些结果与在氧合的每个阶段都已解析的氧合血红蛋白的相应数据一致。
