Hauksson J B, La Mar G N, Pande U, Pandey R K, Parish D W, Singh J P, Smith K M
Department of Chemistry, University of California, Davis 95616.
Biochim Biophys Acta. 1990 Nov 15;1041(2):186-94. doi: 10.1016/0167-4838(90)90064-m.
The products of the incorporation of various protohemin type-isomers into the heme pocket of sperm whale myoglobin were investigated by 1H-NMR in the met-cyano complexes, both immediately after reconstitution as well as at equilibrium. The type-isomers studied include those involving all possible interchanges of the two substituents on a given pyrrole. The protohemin-III and -XIII isomers, with true 2-fold symmetry, yielded only homogeneous products. Protohemins-XI, -XIV both exhibited two species after reconstitution, with one disappearing with time. Protohemin-I was the only asymmetric hemin that failed to exhibit two isomers initially. The orientation of the hemin within the pocket was established by nuclear Overhauser detected dipolar connectivities among heme substituents and between heme substituents and assigned heme pocket residues. At equilibrium, the heme orientations were dominated by the asymmetric propionate rather than vinyl dispositions on the hemin, with a clear preference for placing a propionate at the 8- vs. 5-methyl position of native myoglobin. For protohemin-XI, the propionates were found in the unexpected positions of the 7-propionate and 2-vinyl groups of native myoglobin, indicating that propionates can occupy positions well within the hydrophobic interior. The alternate heme orientation for the metastable intermediates detected for protohemin-XI and -XIV involved rotational isomerism about the alpha,gamma-meso axes bisecting the vinyl positions, but these two axes are at right angles to each other in the protein matrix. The fact that protohemin-XIV, but not protohemin-I, exhibits a reversed orientation as a reconstitution intermediate provides direct evidence that vinyl contacts, as well as propionate links, modulate the relative stabilities of the initial encounter complexes between hemin and apomyoglobin. The heme cavity molecular/electronic structure was found largely unperturbed for the complexes of the various protohemin type-isomers.
通过1H-NMR研究了各种原血红素类型异构体掺入抹香鲸肌红蛋白血红素口袋中的产物，研究对象为高铁氰基配合物，包括重构后立即以及达到平衡时的情况。所研究的类型异构体包括涉及给定吡咯上两个取代基所有可能互换的异构体。具有真正二重对称性的原血红素-III和-XIII异构体仅产生均一产物。原血红素-XI和-XIV在重构后均表现出两种物种，其中一种随时间消失。原血红素-I是唯一最初未表现出两种异构体的不对称血红素。血红素在口袋内的取向通过核Overhauser检测到的血红素取代基之间以及血红素取代基与指定的血红素口袋残基之间的偶极连接来确定。在平衡时，血红素的取向主要由不对称丙酸酯而非血红素上的乙烯基位置决定，明显倾向于将丙酸酯置于天然肌红蛋白的8-甲基与5-甲基位置。对于原血红素-XI，发现丙酸酯处于天然肌红蛋白的7-丙酸酯和2-乙烯基的意外位置，这表明丙酸酯可以占据疏水内部的位置。在原血红素-XI和-XIV中检测到的亚稳中间体的交替血红素取向涉及围绕平分乙烯基位置的α,γ-中位轴的旋转异构，但在蛋白质基质中这两个轴相互垂直。原血红素-XIV而非原血红素-I作为重构中间体表现出反向取向这一事实提供了直接证据，即乙烯基接触以及丙酸酯连接调节了血红素与脱辅基肌红蛋白之间初始相遇复合物的相对稳定性。发现各种原血红素类型异构体的配合物的血红素腔分子/电子结构在很大程度上未受干扰。
