Simeonov Mario, Altuve Adriana, Massiah Michael A, Wang An, Eastman Margaret A, Benson David R, Rivera Mario
Department of Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, Lawrence, Kansas 66045-7582, USA.
Biochemistry. 2005 Jul 5;44(26):9308-19. doi: 10.1021/bi050564l.
Native-state hydrogen-deuterium exchange (HDX) monitored by NMR spectroscopy has been used to compare conformational plasticity in ferric rat liver outer mitochondrial membrane cytochrome b5 (rOM b5) and ferric bovine liver microsomal cytochrome b5 (bMc b5). Analysis of the data indicated that rOM b5 is the less conformationally flexible protein on the time scale probed by the HDX experiments. The data also suggest a likely contributor to the much higher kinetic barrier for the release of hemin from OM b5s in comparison to Mc b5s, a characteristic that may be to a large extent the source of their divergent functional properties. Specifically, the data indicate that conformational mobility within helices alpha4 and alpha5, which flank the loop harboring axial ligand His63, is considerably more restricted in rOM b5 than in bMc b5. The lower conformational flexibility of alpha4 and alpha5 in rOM b5 can reasonably be attributed to more extensive hydrophobic packing in that region of the protein, arising from two conserved side chain packing motifs in OM cytochrome b5s [Altuve, A., Wang, L., Benson, D. R., and Rivera, M. (2004) Biochem. Biophys. Res. Commun. 314, 602-609].
通过核磁共振光谱监测的天然态氢氘交换（HDX）已被用于比较铁离子态大鼠肝脏外线粒体膜细胞色素b5（rOM b5）和铁离子态牛肝脏微粒体细胞色素b5（bMc b5）的构象可塑性。数据分析表明，在HDX实验所探测的时间尺度上，rOM b5是构象灵活性较低的蛋白质。数据还表明，与Mc b5相比，OM b5中血红素释放的动力学屏障要高得多，这一特性可能在很大程度上是它们功能特性不同的原因。具体而言，数据表明，在含有轴向配体His63的环两侧的α4和α5螺旋内的构象流动性，在rOM b5中比在bMc b5中受到的限制要大得多。rOM b5中α4和α5较低的构象灵活性可以合理地归因于该蛋白质区域中更广泛的疏水堆积，这是由OM细胞色素b5中的两个保守侧链堆积基序引起的[阿尔图韦，A.，王，L.，本森，D. R.，和里维拉，M.（2004年）《生物化学与生物物理研究通讯》314，602 - 609]。
