Tsuneshige A, Zhou Y X, Yonetani T
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104-6089.
J Biol Chem. 1993 Nov 5;268(31):23031-40.
To probe the molecular mechanism of allosteric function of cobaltous protoporphyrin-substituted hemoglobin (CoHb), a series of alpha, alpha-cross-linked symmetric and asymmetric Fe-Co hybrid hemoglobins, which contain (1Co porphyrin/3Fe porphyrins), (2Co porphyrins/2Fe porphyrins), and (3Co porphyrins/1Fe porphyrin) per tetramer, have been prepared. Because only Fe porphyrin-containing subunits react with CO, these Fe-Co hybrids are converted to mono-, di-, and tri-CO-ligated states in the presence of CO, respectively, and are proposed to stand as models for mono-, di-, and tri-ligated intermediates of CoHb, respectively. The oxygen binding properties of these Fe-Co hybrids were investigated by measuring oxygen binding isotherms in the presence of CO as a function of pH in the presence and absence of IHP. The ligation of CO to a beta subunit causes larger changes in the oxygen affinity and the Bohr effect than that to an alpha subunit, indicating that the ligation to a beta subunit induces larger affinity-related structural changes in cross-linked CoHb. Di- and tri-CO-ligated intermediates exhibited substantially increased oxygen affinity, reduced Bohr effect, and reduced IHP effect, indicating that they are in high affinity states. Calculation of the Adair equilibrium constants for the first and last oxygenation steps for each of these intermediates permitted the determination of the level of free energy of cooperation. The intermediately ligated species of cross-linked CoHb are distributed in multiple levels of free energy of cooperation within the free energy difference of 1.14 kcal mol-1 between deoxy and fully ligated states of cross-linked CoHb at pH 7.4. The ligation process in CoHb is determined by the number and distribution of the bound ligands, and ligation takes place through steps that require minimal free energy changes.
为了探究钴原卟啉取代血红蛋白（CoHb）变构功能的分子机制，已制备了一系列α，α-交联的对称和不对称铁-钴杂合血红蛋白，每个四聚体包含（1个钴卟啉/3个铁卟啉）、（2个钴卟啉/2个铁卟啉）和（3个钴卟啉/1个铁卟啉）。由于只有含铁血卟啉的亚基与CO反应，这些铁-钴杂合体在CO存在下分别转化为单、双和三CO连接状态，并分别被提议作为CoHb单、双和三连接中间体的模型。通过在有和没有IHP的情况下，测量作为pH函数的CO存在下的氧结合等温线，研究了这些铁-钴杂合体的氧结合特性。CO与β亚基的连接比与α亚基的连接引起氧亲和力和玻尔效应的变化更大，表明与β亚基的连接在交联的CoHb中诱导更大的与亲和力相关的结构变化。双和三CO连接中间体表现出氧亲和力显著增加、玻尔效应降低和IHP效应降低，表明它们处于高亲和力状态。计算这些中间体中每一个的第一个和最后一个氧合步骤的阿代尔平衡常数，允许确定协同自由能水平。在pH 7.4时，交联CoHb的脱氧和完全连接状态之间的自由能差为1.14 kcal mol-1，交联CoHb的中间连接物种分布在多个协同自由能水平上。CoHb中的连接过程由结合配体的数量和分布决定，连接通过需要最小自由能变化的步骤进行。
