Mulrooney Scott B, Meinhardt David R, Waskell Lucy
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Biochim Biophys Acta. 2004 Nov 1;1674(3):319-26. doi: 10.1016/j.bbagen.2004.08.001.
Cytochrome b5 (cyt b5) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an alpha-helical membrane-spanning domain. This study investigated whether there are specific side chain helix-helix packing interactions between the COOH-terminal membrane anchor of cyt b5 and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b5. Insertion of alanine into an alpha-helix causes all amino acids at its carboxyl terminus to be rotated by 100 degrees . The ability of the alanine insertion mutants of cyt b5 to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b5 to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic alpha-helix of cyt b5 does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.
细胞色素b5（cyt b5）是一种存在于真核生物内质网中的两亲性膜结合血红素蛋白。它由三个结构域组成，一个含血红素的N端胞质亲水区、一个短的柔性连接区和一个α-螺旋跨膜区。本研究在纯化的重组系统中，研究了cyt b5的COOH端膜锚与细胞色素P450（cyt P450）2B4之间是否存在特定的侧链螺旋-螺旋堆积相互作用。在cyt b5的膜锚的六个位置插入了丙氨酸。在α-螺旋中插入丙氨酸会使其羧基末端的所有氨基酸旋转100度。cyt b5的丙氨酸插入突变体与cyt P450 2B4结合的能力与野生型蛋白相似，突变体cyt b5刺激麻醉剂甲氧氟烷代谢的能力也与野生型蛋白相似。这些结果表明，cyt b5的C端疏水α-螺旋不是通过氨基酸侧链的特定立体化学匹配与cyt P450 2B4相互作用，而是通过非特异性相互作用。
