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 cyt b5）和线粒体外膜（OM cyt b5）中。 虽然Mc细胞色素b5已被确定从一些哺乳动物的来源，唯一的OM细胞色素b5已被明确确定的日期来自大鼠肝脏。 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，大鼠OM细胞色素b5具有比Mc细胞色素b5低得多的还原电位和高得多的化学和热变性稳定性。此外，大鼠OM细胞色素b5中的氯化血红素在生理温度下被动力学捕获。从氨基酸序列比对，晶体结构比较，分子动力学模拟，几个氨基酸残基可以被确定为大鼠OM细胞色素b5的不寻常的生物物理特性的潜在决定因素。 因此，将进行一项系统性研究，其中大鼠OM蛋白中的这些残基被牛Mc亚型中的相应残基取代。 进行这些研究的预期是降低大鼠OM cyt b5的氯化血红素释放的稳定性和动力学障碍。 将进行一项补充研究，其中牛Mc cyt b5的稳定性将通过纳入大鼠OM蛋白中发现的稳定特征而增加。 同样重要的是要确定大鼠OM细胞色素b5的生物物理特性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所共有的。因此，将合成编码人睾丸细胞色素b5的基因，该蛋白质很可能是大鼠OM细胞色素b5的人类似物，将其置于适合于高水平表达的载体中并表征其生物物理性质。

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.