Collaborative Research: Biochemical and Biophysical Characterization of Cytochrome b5 from Outer Mitochondrial Membrane

膜锚定的哺乳动物细胞色素b5位于内质网(微粒体或Mc Cytb5)和线粒体的外膜(OM Cytb5)。虽然Mc细胞色素b5已经从许多哺乳动物来源中被鉴定出来，但到目前为止唯一被确认为阳性的OM cyt b5来源于大鼠肝脏。大鼠OMCytb5和Mc细胞色素b5的三维结构非常相似。然而，与Mc细胞色素b5相比，大鼠OM细胞色素b5的还原潜力要低得多，对化学和热变性的稳定性要高得多。此外，大鼠OM cyt b5中的氯化血红素在生理温度下被动力学捕获。根据氨基酸序列比对、晶体结构比较和分子动力学模拟，几个氨基酸残基可以被确定为大鼠OM cyt b5异常生物物理性质的潜在决定因素。因此，将进行一项系统的研究，将大鼠OM蛋白中的这些残基替换为牛Mc亚型中的相应残基。这些研究将在降低大鼠OM cytb5释放氯化血红素的稳定性和动力学障碍的情况下进行。将进行一项补充研究，通过结合在大鼠OM蛋白中发现的稳定功能来提高牛Mc Cyt b5的稳定性。同样重要的是要确定大鼠OM细胞色素b5的生物物理性质是否仅限于这种蛋白质，或者更确切地说，是线粒体细胞色素b5共同的。因此，人类睾丸细胞色素b5的编码基因将被合成，并将其置于适合高水平表达的载体中，并对其生物物理性质进行表征。

Membrane-anchored, mammalian cytochromes b5 are located in the endoplasmic reticulum (microsomal or Mc cyt b5) and the outer membranes of mitochondria (OM cyt b5). Although Mc cytochromes b5 have been identified from a number of mammalian sources, the only OM cyt b5 that has been positively identified to date originates from rat liver. The three-dimensional structures of rat OM cyt b5 and the Mc cytochromes b5 are very similar. Nonetheless, rat OM cyt b5 has a much lower reduction potential and much higher stability toward chemical and thermal denaturation than the Mc cytochromes b5. Furthermore, hemin in rat OM cyt b5 is kinetically trapped at physiological temperatures. From amino acid sequence alignments, crystal structure comparisons, and molecular dynamics simulations, several amino acid residues can be identified as potential determinants of the unusual biophysical properties of rat OM cyt b5. Consequently, a systematic study will be conducted in which these residues in the rat OM protein are replaced with the corresponding residues in the bovine Mc isoform. These studies will be performed with the expectation of decreasing the stability and kinetic barriers for hemin release of rat OM cyt b5. A complementary study will be carried out in which the stability of bovine Mc cyt b5 will be increased by incorporating the stabilizing features found in the rat OM protein. It is also important to establish whether the biophysical properties of rat OM cyt b5 are restricted to this protein, or rather are common to mitochondrial cytochromes b5. Consequently, the gene coding for human testis cyt b5, a protein very likely to be the human analogue of rat OM cyt b5, will be synthesized, placed in a vector suitable for high level expression and characterized for its biophysical properties.