Neya S, Funasaki N, Igarashi N, Ikezaki A, Sato T, Imai K, Tanaka N
Department of Physical Chemistry, Kyoto Pharmaceutical University, Japan.
Biochemistry. 1998 Apr 21;37(16):5487-93. doi: 10.1021/bi972632c.
Myoglobin was reconstituted with 6,7-dicarboxy-1,2,3,4,5, 8-hexamethylheme, a compact synthetic heme with the shortest acid side chains, to pursue the structural and functional consequences after intensive disruption of the heme propionate-apoglobin linkages in the native protein. The electron-withdrawing carboxylate groups directly attached to the porphyrin ring lowered the oxygen affinity by 3-fold as compared with native myoglobin. Autoxidation of the oxy derivative to the ferric protein proceeded with 1.6 x 10(-)2 min-1 at pH 7.0 and 30 degrees C. The crystallographic structure of the cyanomet myoglobin with 1.9 A resolution shows that the heme adopts a unique orientation in the protein pocket to extend the two carboxylates toward solvent sphere. The native globin fold is conserved, and the conformations of globin side chains are almost intact except for those located nearby the heme 6,7-carboxylates. The 7-carboxylate only weakly interacts with Ser92 and His97 through two mediating water molecules. The 6-carboxylate, on the other hand, forms a novel salt bridge with Arg45 owing to conformational flexibility of the guanidinium side chain. The proton NMR shows that the small heme does not fluctuate about the iron-histidine bond even at 55 degreesC, suggesting that the salt bridge between Arg45 and heme 6-carboxylate is of critical importance to recognize and fix the heme in myoglobin.
用6,7 - 二羧基 - 1,2,3,4,5,8 - 六甲基血红素对肌红蛋白进行了重构，这是一种具有最短酸性侧链的紧密合成血红素，目的是研究天然蛋白质中血红素丙酸酯 - 脱辅基球蛋白连接被强烈破坏后的结构和功能后果。与天然肌红蛋白相比，直接连接在卟啉环上的吸电子羧基使氧亲和力降低了3倍。在pH 7.0和30℃条件下，氧衍生物自氧化为高铁蛋白的反应速率为1.6×10⁻² min⁻¹。分辨率为1.9 Å的氰化高铁肌红蛋白的晶体结构表明，血红素在蛋白质口袋中采取独特的取向，使两个羧基朝向溶剂球延伸。天然球蛋白折叠得以保留，除了位于血红素6,7 - 羧酸盐附近的那些侧链外，球蛋白侧链的构象几乎完整。7 - 羧酸盐仅通过两个介导水分子与Ser92和His97发生弱相互作用。另一方面，由于胍基侧链的构象灵活性，6 - 羧酸盐与Arg45形成了一种新型盐桥。质子核磁共振显示，即使在55℃时，小血红素也不会围绕铁 - 组氨酸键波动，这表明Arg45与血红素6 - 羧酸盐之间的盐桥对于识别和固定肌红蛋白中的血红素至关重要。
