Toward engineering the stability and hemin-binding properties of microsomal cytochromes b5 into rat outer mitochondrial membrane cytochrome b5:: Examining the influence of residues 25 and 71
Toward engineering the stability and hemin-binding properties of microsomal cytochromes b5 into rat outer mitochondrial membrane cytochrome b5:: Examining the influence of residues 25 and 71
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DOI：
10.1021/bi026005l
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发表时间：
2002-10-01
期刊：
影响因子：
2.9
通讯作者：
Benson, DR
中科院分区：
文献类型：
--
作者：
Cowley, AB;Altuve, A;Benson, DR
作为将微粒体（Me）细胞色素b(5)与大鼠肝脏线粒体外膜（OM）细胞色素（cyt） b(5)结合的稳定性和血红蛋白结合特性的更大努力的一部分，我们制备了几个大鼠OM cyt b5突变体，以研究逐渐和完全消除两个扩展的疏水网络的影响，这两个网络存在于线粒体蛋白的结构中，而在哺乳动物Me细胞色素b(5)的结构中不存在。在先前的研究中发现的疏水网络之一[Altuve， a ., Silchenko， S., Lee， K.-H.]， Kuczera， K., Terzyan， S., Zhang， X., Benson， D. R.和Rivera， M.（2001）生物化学40,9469-9483)，包含Ala-18， Ile-32， Leu-36和Leu-47的侧链，而第二个疏水网络，作为本工作的一部分，包含Ile-25， ph -58， Leu-71和血红素的侧链。大鼠OM cyt b的A18S/ 125L/132L/L47R/L71S五重突变体的x射线结构(5)表明，两个疏水网络都被消除了，Me异构体的相应结构元素已经被引入。研究发现，大鼠OM突变蛋白的稳定性在野生型> 125L > A18S/I32L/L47R > L71S > A18S/I32L/ L71S > 18S/I25L/I32L/L47R/L71S中下降，这表明这两个疏水网络确实有助于大鼠OM cyt b相对于牛Me亚型的高稳定性。令人惊讶的是，大鼠OM cyt b(5)的五倍突变体比牛Me cyt b(5)更不稳定，尽管前者在pH 7.0时表现出明显较慢的血红蛋白释放和血红蛋白重定向速度。然而，在pH 5.0时，牛Me和大鼠OM五倍突变蛋白释放血红蛋白的速率相当，这表明在生理条件下，大鼠OM蛋白中的一个或两个His轴位配体更能抵抗质子化。对载脂蛋白进行的化学变性实验结果表明，含有L71S的突变体的稳定性明显低于牛Mc apocyt b(5)，这强烈表明Lcu-71在稳定大鼠OM apocyt b5中起关键作用，可能是通过与il -25和ph -58的疏水相互作用。由于牛mcapocyt b(5)中不存在类似的相互作用，它在7 - 1位置含有Ser，因此它必须采用不同的相互作用来稳定其折叠，从而突出了大鼠OM和牛Me cyt b5之间的另一个差异。在这些研究过程中，我们还发现大鼠OM cyt b5可以强烈倾向于具有单点突变的血红蛋白取向异构体A （I32L）或异构体B (L71S)，并且在某些大鼠OM突变中血红蛋白取向异构体A和B的释放可以动力学解决。
As part of a larger effort to engineer the stability and hemin-binding properties of microsomal (Me) cytochromes b(5) into rat liver outer mitochondrial membrane (OM) cytochrome (cyt) b(5), several mutants of rat OM cyt b5 were prepared to study the effect of gradual and complete elimination of two extended hydrophobic networks, which are present in the structure of the mitochondrial protein and are absent in the structure of mammalian Me cytochromes b(5). One of the hydrophobic networks, identified in a previous study [Altuve, A., Silchenko, S., Lee, K.-H., Kuczera, K., Terzyan, S., Zhang, X., Benson, D. R., and Rivera, M. (2001) Biochemistry 40, 9469-9483], encompasses the side chains of Ala-18, Ile-32, Leu-36, and Leu-47, whereas a second hydrophobic network, identified as part of this work, encompasses the side chains of Ile-25, Phe-58, Leu-71, and the heme. The X-ray structure of the A18S/ 125L/132L/L47R/L71S quintuple mutant of rat OM cyt b(5) demonstrates that both hydrophobic networks have been eliminated and that the corresponding structural elements of the Me isoform have been introduced. The stability of the rat OM mutant proteins studied was found to decrease in the order wild type > 125L > A18S/I32L/L47R > L71S > A18S/I32L/L47R/L71S > 18S/I25L/I32L/L47R/L71S, indicating that the two hydrophobic networks do indeed contribute to the high stability of rat OM cyt b(5) relative to the bovine Me isoform. Surprisingly, the quintuple mutant of rat OM cyt b(5) is less stable than bovine Me cyt b(5), even though the former exhibits significantly slower rates of hemin release and hemin reorientation at pH 7.0. However, at pH 5.0 the bovine Me and rat OM quintuple mutant proteins release hemin at comparable rates, suggesting that one or both of the His axial ligands in the rat OM protein are more resistant to protonation under physiological conditions. Results obtained from chemical denaturation experiments conducted with the apoproteins demonstrated that mutants containing L71S are significantly less stable than bovine Mc apocyt b(5), strongly suggesting that Lcu-71 plays a pivotal role in the stabilization of rat OM apocyt b5, presumably via hydrophobic interactions with Ile-25 and Phe-58. Because comparable interactions are absent in bovine Mc apocyt b(5), which contains Ser at position 7 1, it must resort to different interactions to stabilize its fold, thus highlighting yet another difference between rat OM and bovine Me cyt b5. During the course of these investigations we also discovered that rat OM cyt b5 can be made to strongly favor hemin orientational isomer A (I32L) or isomer B (L71S) with a single point mutation and that release of hemin orientational isomers A and B can be kinetically resolved in certain rat OM mutants.