Santucci R, Ascoli F, La Mar G N, Pandey R K, Smith K M
Department of Molecular, Cellular and Animal Biology, University of Camerino, Italy.
Biochim Biophys Acta. 1993 Jul 10;1164(2):133-7. doi: 10.1016/0167-4838(93)90239-n.
The reconstitution kinetics of horse heart myoglobin, as met-cyano derivative, with two synthetic hemins in which the 6- or the 7-propionate is replaced by a methyl group, has been investigated by circular dichroism, in order to gain information on the heme re-orientation process following the heme insertion into the globin pocket. The results obtained confirm that the preferred heme orientation places the sole propionate into the position occupied by the 6-propionate in the crystal structure, supporting the importance of the salt bridge occurring between this propionate and the basic CD3 residue of the apoprotein. Moreover, they provide new information on the correlations existing between the shape and the intensity of the dichroic bands and the heme orientation inside the reconstituted protein. Our data suggest that positive Soret CD bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'correct' orientation (as in horse heart myoglobin), and negative dichroic bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'reversed' orientation (as for example, in Glycera dibranchiata hemoglobins). Thus, negligible contribution to the CD signal in reconstituted proteins can be associated to a 'wrong' orientation of the heme group, no matter whether the orientation at the equilibrium is the 'correct' or the 'reversed' one. Finally, the results obtained indicate that the perturbations due to the heme re-orientation appear as a local phenomenon, not affecting the distant domains of the macromolecule.
为了获取关于血红素插入球蛋白口袋后血红素重新定向过程的信息，通过圆二色性研究了马心肌红蛋白作为高铁氰基衍生物与两种合成血红素的重组动力学，其中6-或7-丙酸酯被甲基取代。所得结果证实，优选的血红素取向将唯一的丙酸酯置于晶体结构中6-丙酸酯所占据的位置，支持了该丙酸酯与脱辅基蛋白的碱性CD3残基之间形成的盐桥的重要性。此外，它们提供了关于重组蛋白中二色性带的形状和强度与血红素取向之间存在的相关性的新信息。我们的数据表明，正的Soret CD带与在平衡时具有所谓“正确”取向的血红素的血红蛋白相关（如马心肌红蛋白），而负二色性带与在平衡时具有所谓“反向”取向的血红素的血红蛋白相关（例如，在双齿沙蚕血红蛋白中）。因此，无论平衡时的取向是 “正确” 还是 “反向”，重组蛋白中CD信号的可忽略不计的贡献都可能与血红素基团的 “错误” 取向相关。最后，所得结果表明，由于血红素重新定向引起的扰动表现为局部现象，不影响大分子的远端结构域。
