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

哺乳动物细胞色素b5位于膜锚定的内质网（微粒体或Mc细胞b5）和线粒体外膜（OM细胞b5）。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，但迄今为止唯一被确定的OM细胞色素b5来自大鼠肝脏。大鼠OM细胞色素b5和Mc细胞色素b5的三维结构非常相似。尽管如此，与Mc细胞色素b5相比，大鼠OM细胞色素b5具有更低的还原电位和更高的化学变性和热变性稳定性。此外，大鼠OM细胞b5中的血红蛋白在生理温度下被动力学捕获。通过氨基酸序列比对、晶体结构比较和分子动力学模拟，可以确定几个氨基酸残基是大鼠OM cyt b5不同寻常的生物物理特性的潜在决定因素。因此，将进行一项系统的研究，其中将大鼠OM蛋白中的这些残基替换为牛Mc亚型中的相应残基。这些研究将期望降低大鼠OM cyt b5释放血红素的稳定性和动力学障碍。将进行一项补充研究，通过结合在大鼠OM蛋白中发现的稳定特征，将增加牛mccyt b5的稳定性。同样重要的是确定大鼠OM cyt b5的生物物理特性是否仅限于该蛋白，或者更确切地说，线粒体细胞色素b5是共同的。因此，编码人类睾丸细胞b5的基因将被合成，这是一种很可能是人类类似于大鼠OM细胞b5的蛋白质，将被放置在适合高水平表达的载体中，并以其生物物理特性为特征。

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.